Chemokines and Viral Infection - T. Lane (Springer, 2006) WW

303
Current Topics
in Microbiology
and Immunology
Editors
R.W. Compans, Atlanta/Georgia
M.D. Cooper, Birmingham/Alabama
T. Honjo, Kyoto H. Koprowski, Philadelphia/Pennsylvania
F. Melchers, Basel M.B.A. Oldstone, La Jolla/California
S. Olsnes, Oslo M. Potter, Bethesda/Maryland
P.K. Vogt, La Jolla/California H. Wagner, Munich
T.E. Lane (Ed.)
Chemokines
and Viral Infection
With 14 Figures and 7 Tables
123
Thomas E. Lane, Ph.D.
Associate Professor
Department of Molecular Biology and Biochemistry
Center for Immunology
3205 McGaugh Hall
University of California, Irvine
Irvine, CA 92697-3900
USA
e-mail: tlane@uci.edu
Cover Illustration by Thomas E. Lane (this volume)

Chemokines and MHV-induced demyelination (see Fig. 2 of the rst chapter)
Library of Congress Catalog Number 72-152360

ISSN 0070-217X
ISBN-10 3-540-29207-1 Springer Berlin Heidelberg New York
ISBN-13 978-3-540-29207-4 Springer Berlin Heidelberg New York
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Preface
Chemokines represent a family of over 40 small proteins that, for the most
part, are secreted into the environment and function by binding to G protein-
coupled receptors (GPCRs) that are expressed on numerous different cell
types. When initially identied close to 30 years ago, these molecules were
associated with various human inammatory diseases and it was recognized
that expression might be integral in leukocyte recruitment to inamed tis-
sue [13]. Within a relatively short period of time, early participants within the
eld determined that these proteins displayed distinct and conserved struc-
tural features and exerted potent chemotactic effects on dened lymphocyte
subsets [4]. There are now four subfamilies of chemokines identied based on
dened structural criteria relating to the positional location of conserved cys-
teine residues within the amino-terminus of the protein [4, 5]. Chemokines
are now recognized as important in numerous biological processes ranging
from maintaining the organizational integrity of secondary lymphoid tis-
sue to participating in various aspects of both innate and adaptive immune
responses following microbial infection [6, 7].
The host response to viral infection represents a well-orchestrated bal-
let consisting of numerous participants with diverse roles in defense but
with the ultimate goal of generating virus-specic lymphocytes whose job
is to control and eliminate the invading viral pathogen from infected tis-
sues. Over the years, an emerging picture has developed that indicates that
chemokines and their receptors are intimately involved in development of
effective host responses to viral pathogens. Chemokine expression is now
associated with all facets of defense against viral infection including linking
innate and adaptive immune responses. Early chemokine expression in re-
sponse to certain viruses such as murine cytomegalovirus (MCMV) is critical
in recruiting into the liver natural killer (NK) cells that control viral repli-
cation [8]. Expression of chemokines following viral infection has also been
demonstrated in tissues originally thought to be relatively immunologically
inert such as the central nervous system (CNS). For example, infection of
the CNS with either herpes simplex virus1 (HSV-1) or mouse hepatitis virus
VI Preface
(MHV) results in an orchestrated expression of chemokines whose function

is to attract antigen-educated lymphocytes into the CNS that contribute to
the control of viral replication [9, 10]. Paradoxically, chronic expression of
certain chemokines during viral persistence in CNS tissue is also associated
with immune-mediated pathology [1113]. In the face of such a robust and
effective immune response, viruses have evolved various ways to avoid or
distract the immune response thus enabling the establishment of infection.
Certain viruses have exploited the chemokine system to their benet by either
using specic chemokine receptors as coreceptors for efcient entry into host
cells (HIV) to encoding receptors with homology to chemokine receptors
(various herpes and poxviruses) that may function to subvert the immune
system [1417].
Clearly, the biological roles of chemokines in host defense and/or disease
are constantly evolving. This volume of Current Topics in Microbiology and
Immunology provides an opportunity to examine the relationship between
chemokines and viruses with regards to host defense and disease. In addition,
the potential of chemokines and their receptors as therapeutic targets for
treatment and/or prevention of disease in response to viral infection is not
overlooked.
Irvine, California, July 2005 Thomas E. Lane
References
1. Deuel TF, Keim PS, Farmer M, Henrikson RL (1977) Amino acid sequence of
human platelet factor 4. Proc Natl Acad Sci USA 74:2256
2. Luster AD, Unkeless JC, Ravetch JV (1985) Gamma-interferon transcriptionally
regulates an early-response gene containing homology to platelet proteins. Nature
315:672
3. Kaplan G, Luster AD, Hancock G (1987) The expression of a gamma interferon-
induced protein (IP-10) in delayed immune responses in human skin. J Exp Med
166:1098
4. Bagglioni M, Dewald B, Moser B (1994) Interleukin-8 and related chemotactic
cytokinesCXC and CC chemokines. Adv Immunol 55:97
5. Luster AD (1998) Chemokineschemotactic cytokines that mediate inamma-
tion. N Engl J Med 338:436
6. Cyster JG (2005) Chemokines, sphingosine-1 phosphate, and cell migration in
secondary lymphoid organs. Annu Rev Immunol 23:127
7. Luster AD (2002) The role of chemokines in linking innate and adaptive immunity.
Curr Opin Immunol 14:129
Preface VII
8. Salazar-Mather TP, Orange JS, Biron CA (1998) Early murine cytomegalovirus

(MCMV) infection induces liver natural killer (NK) cell inammation and protec-
tion through macrophage inammatory protein 1 alpha (MIP-1 alpha)-dependent
pathways. J Exp Med 187:1
9. Wickham S, Lu B, Ash J, Carr DJ (2005) Chemokine receptor deciency is associ-
ated with increased chemokine expression in the peripheral and central nervous
systems and increased resistance to herpetic encephalitis. J Neuroimmunol 162:51
10. Liu MT, Chen BP, Oertel P, Buchmeier MJ, Armstrong D, Hamilton TA, Lane TE
(2000) The T cell chemoattractant IFN-inducible protein 10 (IP-10) is essential in
host defense against viral-induced neurologic disease. J Immunol 165:2327
11. Peterson K, Errett JS, Wei T, Dimcheff DE, Ransohoff R, Kuziel WA, Evans L,
Chesebro B (2004) MCP-1 and CCR2 contribute to non-lymphocyte-mediated
brain disease by Fr98 polytropic retrovirus infection in mice: role for astrocytes
in retroviral neuropathogenesis. J Virol 78:6449
12. Liu MT, Keirstead HS, Lane TE (2001) Neutralization of the chemokine CXCL10
reduces inammatory cell invasion and demyelination and improves neurological
function in a viral model of multiple sclerosis. J Immunol 167:40914097
13. Carr DJJ, Chodosh J, Ash J, Lane TE (2003) Effect of anti-CXCL10 monoclonal
antibody on HSV-1 keratitis and retinal infection. J Virol 77:10037
14. Rucker J, Doms RW (1998) Chemokine receptors as HIV coreceptors: implications
and interactions. AIDS Res Hum Retroviruses 14 Suppl 3:S241
15. Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE, Murhpy PM,
Berger EA (1996) CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as
a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955
16. Feng Y, Broder CC, Kennedy PE, Berger EA (1996) HIV-1 entry cofactor: functional
cDNA cloning of a seven transmembrane, G protein-coupled receptor. Science
272:872
17. Casarosa P, Bakker RA, Verzijl D, Navis M, Timmerman H, Leurs R, Smit MJ
(2001) Constitutive signaling of the human cytomegalovirus-encoded chemokine
receptor US28. J Biol Chem 276:1133
List of Contents
Functional Diversity of Chemokines and Chemokine Receptors

in Response to Viral Infection of the Central Nervous System . . . . . . . . . . . . . . 1
T. E. Lane, J. L. Hardison, and K. B. Walsh
Cytokine and Chemokine Networks: Pathways to Antiviral Defense . . . . . . . . . . 29

T. P. Salazar-Mather and K. L. Hokeness
Herpes Simplex Virus and the Chemokines That Mediate the Inammation . . . . 47
D. J. J. Carr and L. Tomanek
Inuence of Proinammatory Cytokines and Chemokines

on the Neuropathogenesis of Oncornavirus
and Immunosuppressive Lentivirus Infections . . . . . . . . . . . . . . . . . . . . . . . . . 67
K. E. Peterson and B. Chesebro
HIV-1 Coreceptors and Their Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

N. Ray and R. W. Doms
A Viral Conspiracy: Hijacking the Chemokine System

Through Virally Encoded Pirated Chemokine Receptors . . . . . . . . . . . . . . . . . . 121
H. F. Vischer, C. Vink, and M. J. Smit
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

List of Contributors
(Addresses stated at the beginning of respective chapters)
Carr, D. J. J. 47 Ray, N. 97
Chesebro, B. 67
Salazar-Mather, T. P. 29
Doms, R. W. 97 Smit, M. J. 121
Hardison, J. L. 1 Tomanek, L. 47
Hokeness, K. L. 29
Vink, C. 121
Lane, T. E. 1 Vischer, H. F. 121
Peterson, K. E. 67 Walsh, K. B. 1
CTMI (2006) 303:127

c Springer-Verlag Berlin Heidelberg 2006
Functional Diversity of Chemokines and Chemokine

Receptors in Response to Viral Infection
of the Central Nervous System
T. E. Lane (u) J. L. Hardison K. B. Walsh
Department of Molecular Biology and Biochemistry, University of California, 3205
McGaugh Hall, Irvine, CA 92697-3900, USA
tlane@uci.edu
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1 Biology and Biochemistry of Coronaviridae . . . . . . . . . . . . . . . . . . . . . 2
1.2 Immunity to MHV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3 Viral Persistence and Immune-Mediated Demyelination . . . . . . . . . . . . . 4
1.4 Chemokines and Chemokine Receptors . . . . . . . . . . . . . . . . . . . . . . . . 5
2 Orchestrated Expression of Chemokines and Chemokine Receptors

Within the CNS Following Infection with MHV . . . . . . . . . . . . . . . . . . . 6
3 Chemokines, Innate Immune Response, and MHV-Infection of the CNS . 8
4 Chemokines and Chemokine Receptors

and Their Role in Acute Viral-Induced Encephalomyelitis . . . . . . . . . . . 9
4.1 CCL3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
4.2 CXCL9 and CXCL10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4.3 CCL5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.4 CCR5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.5 CCL2 and CCR2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
5 Chemokines and Chronic Viral-Induced Demyelination . . . . . . . . . . . . 14
6 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Abstract Encounters with neurotropic viruses result in varied outcomes ranging from
encephalitis, paralytic poliomyelitis or other serious consequences to relatively benign
infection. One of the principal factors that control the outcome of infection is the local-
ized tissue response and subsequent immune response directed against the invading
toxic agent. It is the role of the immune system to contain and control the spread of
virus infection in the central nervous system (CNS), and paradoxically, this response
may also be pathologic. Chemokines are potent proinammatory molecules whose
expression within virally infected tissues is often associated with protection and/or
2 T. E. Lane et al.
pathology which correlates with migration and accumulation of immune cells. Indeed,
studies with a neurotropic murine coronavirus, mouse hepatitis virus (MHV), have
provided important insight into the functional roles of chemokines and chemokine
receptors in participating in various aspects of host defense as well as disease de-
velopment within the CNS. This chapter will highlight recent discoveries that have
provided insight into the diverse biologic roles of chemokines and their receptors in
coordinating immune responses following viral infection of the CNS.
1
Introduction
1.1
Biology and Biochemistry of Coronaviridae
Coronaviruses are classied on the basis of several fundamental characteris-

tics, including nucleic acid type, a lipid envelope, and their distinctive mor-
phology [42, 64, 79]. All members have characteristic petal-shaped proteins
extending from the virion surface. Coronaviruses infect numerous verte-
brate hosts including humans, chickens, pigs, and mice, causing a wide va-
riety of disorders involving a number of different organ systems; however,
there are specic tropisms for the CNS, lungs, gastrointestinal tract, and
liver [42, 64, 79]. Receptor use among the varied coronaviruses is restricted to
several well-dened proteins. Human coronavirus infections result in acute
enteritis as well as 15% of common colds indistinguishable from those caused
by other viruses [42, 64, 79]. More recently, a human coronavirus has been
indicated to be the etiologic agent for severe acute respiratory syndrome
(SARS). SARS is a potentially lethal disease and is recognized as a health
threat internationally [43].
The rst murine coronavirus strain (mouse hepatitis virus, MHV), was
isolated in 1949 [12]. MHV is a pathogen of wild mice, and natural infection is
due to horizontal transmission, resulting in acute hepatitis with death in young
animals and a variable course of persistent gastrointestinal tract infection in
adults [79]. MHV is not an endemic mouse virus, but infects mouse colonies
sporadically. It is very closely related to some human coronaviruses both
at the genomic and protein levels. For example, human sera often contain
antibody reactive to MHV. Therefore, characterizing the immune response
to murine coronaviruses may provide important insight to mechanisms of
control and elimination which may have important implications with regards
to understanding the immune response to human coronaviruses such as the
SARS coronavirus.
Functional Diversity of Chemokines and Chemokine Receptors 3
Coronavirus genomes are single-stranded positive-polarity RNA mole-

cules, larger than the size of any other known stable RNA, ranging from
27 kb for the avian infectious bronchitis virus, to 31 kb for murine coro-
naviruses [50]. Genomic RNA is infectious, contains a cap structure at the
5 -end and poly(A) at the 3 -end. The genome is organized into seven or eight
genes, each containing one or more open reading frames (ORF) separated
by intergenic sequences that contain the signals for the initiation of tran-
scription of the subgenomic viral messenger (m)RNA species. Upon entry,
the viral RNA encodes an RNA polymerase that transcribes the genome into
a negative-stranded RNA [50]. The latter serves as templates for positive-
sensed genomic RNA and subgenomic mRNAs. Important viral structural
proteins include the envelope glycoproteins (S) that bind to receptors on cell
membranes [42, 64, 79]. Analysis of monoclonal antibody neutralization es-
cape variants demonstrated that the viral S protein controls cellular tropism
in vivo and the role of the S protein in tropism has recently been conrmed
using stable recombinant viruses in which all genes except the S protein gene
were held constant [9, 82].
1.2
Immunity to MHV Infection
The protective immune response to MHV infection is characterized predomi-

nantly by cell-mediated immunity during acute infection. A number of unique
aspects of CNS viral infection have been described by analysis of the interac-
tions between MHV and the immune response. Antibody, although protective
if administered prior to infection, is not present in the serum of infected mice
until after the vast majority of virus has been cleared from the CNS [56, 84].
Following infection, neutrophils, macrophages, and NK cells are rapidly re-
cruited into the CNS, followed by T cells and B cells [104]. Inammation is
accompanied by a progressive loss of bloodbrain barrier (BBB) integrity that
is apparent as early as 4 days post-infection. The initial inux of innate effec-
tors is important in facilitating T cell inltration, as well as regulating viral
replication [104]. However, the ability to survive MHV infection appears to
be predominantly due to an effective T cell-mediated response [103]. Recent
data have conrmed that cell-mediated immunity is critical during acute in-
fection [53, 55, 74, 76, 92]; however, the ability to prevent viral recrudescence
is associated with the continued presence of plasma cells in the CNS secreting
neutralizing antibody [56, 84].
The major effectors of anti-viral immunity are virus-specic CD8+ T cells.
Cytotoxic T lymphocyte (CTL) induction following MHV infection of the
CNS has been shown to require CD4+ T cell help [92]. Although the pre-
4 T. E. Lane et al.
cise mechanism or mechanisms by which CD4+ T cells assist CD8+ T cells

have yet to be completely determined, recent studies have demonstrated that
CD4+ T cells are important in preventing apoptosis of CTL entering the
CNS parenchyma [92]. In addition, the quality of the CTL response is CD4+
T cell-dependent [92]. An important concept derived from analysis of MHV
infection is that although CD8+ T cells are the most prominent effectors for vi-
ral clearance during the acute infection, the mechanisms which control virus
replication differ with the type of CNS cell infected. Cytolysis is important
for the control of viral replication in microglia/macrophages and astrocytes
while interferon (IFN)- is the critical effector responsible for control of virus
replication in oligodendroglia [73]. The demonstration that CD8+ CTL sup-
presses viral replication by two separate effector mechanisms, which function
within the CNS in a cell type-specic manner, is an important new concept.
1.3
Viral Persistence and Immune-Mediated Demyelination
Viral persistence in white matter tracts results in a chronic demyelinating

disease in which foci of demyelination are associated with areas of viral
RNA/antigen [51]. Clinically, mice develop loss of tail tone and a partial
to complete hind-limb paralysis. As a result of the clinical and histologic
similarities between MHV-induced demyelination and the human demyeli-
nating disease multiple sclerosis (MS), the MHV system is considered a rel-
evant model for studying the underlying immunopathologic mechanisms
contributing to immune-mediated demyelinating diseases [51]. A variety of
different mechanisms have been postulated to contribute to MHV-induced
demyelination. Several studies suggest that MHV-induced demyelination in-
volves immunopathologic responses against viral antigens expressed in in-
fected tissues [30, 31, 37, 47]. Although virus-specic antibody is considered
important in suppressing viral recrudescence [84, 85], it may also have a role
in promoting demyelination [48]. MHV infection of immunosuppressed or
immunodecient mice results in high titers of virus within the CNS and death
but not robust demyelination [53, 105]. Adoptive transfer of MHV-immune
splenocytes results in demyelination to the infected recipients, suggesting
a role for immune cells in amplifying demyelination [30, 31]. Additional
evidence for T cells in contributing to demyelination is provided by Wu et
al. [105] who demonstrated that both CD4+ and CD8+ T cells are important
in mediating myelin destruction. In support of this are studies derived from
our laboratory demonstrating that adoptive transfer of MHV-specic CD4+
or CD8+ T cells to MHV-infected RAG1/ mice results in demyelination [30,
31]. However, demyelination was more severe in recipients of CD4+ T cell
compared to CD8+ T cell recipients, and this supports a more important

role for CD4+ T cells in amplifying demyelination in this model. Indeed, we
have demonstrated that MHV-infected CD4/ mice displayed a signicant
reduction in the severity of demyelination compared to CD8/ and immuno-
competent wildtype mice, suggesting an important role for CD4+ T cells in
amplifying the severity of white matter destruction [53].
While T cells are generally considered important in driving demyelination
in mice persistently infected with MHV, the mechanisms by which these cells
participate in disease may vary and depend upon various factors including
the ability to secrete interferon (IFN)- [80, 81]. While conventional CD4 and
CD8 T cells are generally viewed as the primary T cell type important
in disease, T cells have also been shown to participate in demyelination
in MHV-infected athymic mice [16]. In addition, we and others have found
that macrophages/microglia are also important in contributing to demyeli-
nation [29, 32, 53, 59, 105]. The collective evidence points to a role for in-
ammatory T cells in contributing to macrophage/microglial inltration and
activation which ultimately results in myelin destruction. Current evidence
suggests that demyelination in MHV-infected mice is not the result of epitope
spreading and induction of an immune response against neuroantigens as
has recently been reported to occur during Theilers virus-induced demyeli-
nation [69]. However, adoptive transfer of T cells from MHV-infected rats to
nave recipients results in demyelination [100]. Whether a similar response
occurs in MHV-infected mice and what the contributions are to demyelination
is not clear at this time.
1.4
Chemokines and Chemokine Receptors
Chemokines represent a family of low molecular weight (717 kDa) proinam-
matory cytokines that are divided into four subfamilies based on structural
and functional criteria [14, 60, 94]. The two major subfamilies are the CXC
and CC chemokines. The CXC subfamily is structurally characterized by two
conserved cysteine residues that are separated by an amino acid, while the
CC subfamily is structurally characterized by conserved cysteine residues
adjacent to one another. Lymphotactin, the sole member of the C family, is
chemotactic for T cells [44]. The CX3 C chemokine, fractalkine, is unique in
that it is expressed on the surface of cells as well as being secreted into the
surrounding environment [5].
Chemokines have been shown to selectively attract distinct leukocyte pop-
ulations during periods of inammation in various disease models. The CXC
chemokines function primarily in attracting neutrophils, yet have a lim-
ited effect on T lymphocytes and monocytes [14, 60, 94]. However, there
6 T. E. Lane et al.
are exceptions to this rule in that CXC chemokines that lack the glutamic
acid-leucine-arginine (ELR) motif on the amino terminus are chemotac-
tic for T cells. For example, the non-ELR chemokine CXCL10 is a potent
chemoattractant for activated T cells and NK cells and functions by binding
to CXCR3 expressed on the surface of these cells [40, 83, 102, 106]. How-
ever, CXCL10 does not exert a chemotactic effect on neutrophils [19]. The
CC chemokines are thought to attract T cells, monocytes, and macrophages,
but not neutrophils [14, 60, 94]. The CC chemokine ligand 5 (CCL5) is able
to attract both T cells and macrophages by binding to one of several CC
chemokine receptors including CCR1 and CCR5 [14, 60, 94]. Furthermore,
there is increasing evidence that chemokines, such as CCL3, inuence other
immune system activities including TH 1/TH 2 development and T cell prolif-
eration [46, 95]. Chemokines function by binding to seven-transmembrane-
spanning G protein-coupled receptors. The chemokine receptors are divided
into those that preferentially bind CXC and CC chemokines. In addition, CC
and CXC chemokine receptors are capable of binding more than one CC
or CXC chemokine, respectively. A variety of cell types including lympho-
cytes and macrophages, as well as resident cells of the CNS such as neurons,
astrocytes, and microglia, express chemokine receptors [60, 94].
2
Orchestrated Expression of Chemokines and Chemokine Receptors
Within the CNS Following Infection with MHV
Instillation of MHV into the CNS of susceptible mice results in a well-

orchestrated expression of chemokine genes, and the expression pattern cor-
relates with the level of inammation and disease [52]. Early (~13 days)
following infection, transcripts for CXCL10 and CCL3 are detected within the
CNS, suggesting an important role in initiation of immune responses (see
following section; Table 1). By day 6 post-infection (p.i.), virus has spread
throughout the brain parenchyma, and a robust inammatory response, char-
acterized primarily by CD4+ and CD8+ T cells and macrophages, is established
within the brain. Chemokines expressed at this time include CXCL9, CXCL10,
CCL2, CCL3, CCL4, CCL5, and CCL7 (MIP-2) (Table 1). Analysis of chemokine
receptor expression by both RNAse protection assay (RPA), immunostaining,
and ow cytometry reveals that CCR1, CCR2, CCR5, and CXCR3 are the
prominent receptors expressed within the CNS at various stages of disease
(Table 2).
Chemokine transcripts are detected almost exclusively in areas in which
virus is present, indicating a localized response to infection and subsequent
Table 1 Chemokine gene expression following MHV infection of the CNS
Days post infection Chemokine Function (cells attracted) Reference(s)
13 CXCL10 NK cells 97
CCL3 Dendritic cells 96
7 and 12 CCL2 Macrophage 39, 52
CCL3 Dendritic cells, T cells 95, 96
CCL4 52
CCL5 T cells, macrophage 52, 53
CXCL9 T cells 58
CXCL10 T cells 18, 57
21 CXCL10 CD4+ T cells 59
CCL5 T cells, macrophages 32
Table 2 Chemokine receptors expressed within the CNS of MHV-infected mice
Days post infection Receptor Chemokine receptor Reference(s)

expression
13 CCR2 T cells, macrophages 13, 39

7 and 12 CCR2 T cells, macrophages 13, 39
CCR5 T cells, macrophages 29, 30
CXCR3 T cells 57
>21 CXCR3 T cells 59
CCR5 T cells, macrophage 29
spread of the virus throughout the parenchyma. In situ hybridization indi-

cates that astrocytes are the primary cellular source for many chemokines
during the acute stage of disease [52]. Infection of primary cultures of mouse
astrocytes with MHV and evaluating chemokine gene expression by RPA
provide additional support for astrocytes as an important cellular source of
chemokines in this model [52]. Moreover, viral replication appears to be a nec-
essary prerequisite for inducing chemokine expression, as infection of astro-
cytes with inactivated virus results in a muted chemokine expression prole.
Additional analysis revealed that both infected and noninfected astrocytes are
capable of secreting chemokines following instillation of virus into the brain,
indicating that viral infection is not required for chemokine gene synthesis by
target cells. These data indicate that a factor or factors (possibly type I interfer-
ons) derived from infected cells are capable of functioning in both an autocrine
and paracrine manner and regulate chemokine gene expression in response
8 T. E. Lane et al.
to viral infection. Other cell types that may also secrete chemokines following
MHV infection include resident microglia/inammatory macrophages as well
as neurons [52, 75].
By day 12 p.i., MHV-infected mice that have survived the acute stage of dis-
ease develop an immune-mediated demyelinating disease. Mice have cleared
infectious virus (as determined by plaque assay) by 12 days, yet viral RNA
and protein can be detected within white matter tracts for months after in-
fection. As the level of CNS inltration subsides following reduction of viral
burden there is a corollary reduction in the expression of chemokine tran-
scripts. Analysis of chemokine message expression within the brains and
spinal cords of MHV-infected mice during the demyelinating phase of disease
(days 12 and onward) indicates that CXCL10 and CCL5 are the two prominent
chemokines expressed [52]. In situ hybridization for chemokine transcripts
indicated expression was limited primarily to areas of viral persistence within
white matter tracts undergoing active demyelination [52]. Similar to what
was found during acute disease, astrocytes were determined to be the cel-
lular source of CXCL10 at this stage of disease whereas inammatory cells,
presumably CD4+ T lymphocytes, expressed CCL5. More recent data now
indicate that MHV-infected astrocytes treated with IFN- can also express
CCL5 mRNA transcripts and protein (T.E. Lane, unpublished observations).
Chemokine receptors expressed during chronic demyelination include CXCR3
and CCR5, which are capable of binding CXCL10 and CCL5, respectively. In-
deed, we have recently determined that the majority (~90%) of inltrating
virus-specic CD4+ and CD8+ T cells express CXCR3 (T.E. Lane, unpublished
observations).
3
Chemokines, Innate Immune Response, and MHV-Infection of the CNS
The presence of dendritic cells (DCs) within the CNS has been debated for
quite some time. However, a series of recent studies clearly indicates that
during induction of an autoimmune demyelinating disease, there exists the
presence of cell types within the brain that clearly have characteristics of
DCs [34, 65]. In addition, emerging evidence points to a previously unappre-
ciated role for chemokines in activating and inducing the migration of differ-
ing populations of DCs in response to microbial infection of the CNS [22, 23].
These cells may be important in initiation and/or maintenance of disease by
participating in the activation of T cells. Given the potential importance of
this population of cells with regards to linking innate and adaptive immune
responses following viral infection of the CNS, we investigated whether DC-
like cells were present within the CNS in response to MHV infection. In brief,
our ndings clearly indicate that a DC-like population of cells is detectable
within the CNS as early as day 2 p.i. with MHV [96]. The activation/maturation
of these cells as well as the ability to accumulate within the draining cervi-
cal lymph node (CLN) appeared to be dictated by localized expression of
CCL3 [96]. Moreover, the ability of cultured DCs to secrete cytokines asso-
ciated with the development of a TH 1 response such as interleukin (IL)-12
was profoundly altered in the absence of CCL3 [96]. The importance of CCL3
signaling and the evolution of an effective T cell response was further con-
rmed by the demonstration that in the absence of CCL3 signaling, robust
anti-viral effector responses, e.g., cytokine production and CTL activity, were
dramatically compromised following MHV infection of CCL3/ mice [95, 96].
Collectively, these studies highlight a previously unappreciated role for the
importance of chemokine signaling and DC maturation/activation following
MHV infection of the CNS. Moreover, these studies demonstrate that gener-
ation of effective T cell responses relies upon CCL3 signaling to successfully
combat MHV infection.
4
Chemokines and Chemokine Receptors
and Their Role in Acute Viral-Induced Encephalomyelitis
4.1
CCL3
CCL3 is a chemoattractant for both T cells and macrophages and has been
implicated in host defense following infection with a wide variety of microbial
pathogens. Mice decient in CCL3 production exhibit increased susceptibility
to disease following infection with paramyxovirus [17], inuenza virus [15],
and coxsackievirus, as well as other microbial pathogens [67, 72]. In all cases,
alterations in an effective host response correlated with a paucity in leukocyte
accumulation at sites of infection. Although originally thought to participate
in defense by attracting effector cells to infected tissue, recent reports also sug-
gest that CCL3 expression is important in coordinating a TH 1 response [46].
Numerous studies now indicate that DCs are capable of expressing various
chemokines including CCL3 [21, 66, 77, 78]. Moreover, DC precursors express
the CCL3 receptors CCR1 and CCR5 and are capable of responding to CCL3
in vivo and in vitro resulting in both mobilization and maturation [24, 108].
Indeed, Flesch and colleagues have demonstrated an important role for CCL3
in DC-dependent priming of CTL to viral antigens [24].
10 T. E. Lane et al.
Using CCL3/ mice, we have demonstrated a role for CCL3 in regulating

trafcking as well as antiviral effector functions following MHV infection of
the CNS [95]. Specically, our experiments revealed an important role for
CCL3 signaling in tailoring T cell responses that allowed for egress out of
draining cervical lymph nodes and trafcking into the CNS. Although gen-
eration of antigen-specic CD8+ T cells was not impaired following MHV
infection of CCL3/ mice, a signicant percentage of CD8+ T cells retained
expression of lymph-node homing receptors CD62L (L-selectin) and the CC
chemokine receptor 7 (CCR7) and did not display a dramatic increase in
mRNA transcripts for either CXCR3 or CCR5, two receptors which are im-
portant in allowing MHV-specic T cells access to the CNS [95]. Moreover,
adoptive transfer of CCL3/ CD8+ T cells into MHV-infected RAG1/ mice
(which express CCL3 following MHV infection) resulted in homing back to
secondary lymphoid organs, suggesting that lack of CCL3 imprinted on these
cells carries an inability to remodulate surface tissue homing receptors. Anal-
ysis of antiviral effector functions also revealed that CCL3/ CD8+ T cells
displayed overall muted cytolytic activity as well as expression of IFN- when
compared to CCL3+/+ CD8+ T cells [95]. Collectively, these studies highlight
that, in addition to chemotactic function, chemokines inuence specic lym-
phocyte responses and ultimately effector functions that are required for
optimal host defense against microbial pathogens.
4.2
CXCL9 and CXCL10
CXCL9 and CXCL10 attract activated T lymphocytes following binding to

CXCR3. Analysis of CXCL9 and CXCL10 mRNA expression within the CNS
of MHV-infected mice revealed that CXCL10 was clearly detectable by day 1
p.i. and was prominently expressed at days 7, 12, and 35 p.i. [52]. In contrast,
CXCL9 transcripts were only detected at days 7 and 12 p.i. [58]. These data
suggested that both CXCL9 and CXCL10 might be important in host defense
by attracting antiviral T lymphocytes into the CNS. In support of this is
the observation that administration of neutralizing antibodies specic for
either CXCL9 or CXCL10 to MHV-infected mice during the acute stage of
disease results in a dramatic increase in mortality [57, 58]. Additionally, this
treatment also resulted in a signicant decrease in numbers of CD4+ and
CD8+ T lymphocyte inltrating into the CNS which correlated with decreased
expression of IFN- and increased levels of virus [57, 58]. MHV infection of
CXCL10/ mice supported and extended our previous work on antibody-
mediated neutralization of CXCL10 in that MHV-infected CXCL10/ mice
display reduced T cell inltration into the CNS accompanied by reduced IFN-
secretion and increased viral burden [18]. Therefore, the collective evidence
points to pivotal roles for both CXCL9 and CXCL10 as important sentinel
molecules in promoting a protective response following MHV infection of the
CNS by attracting T cells into the CNS that participate in elimination of virus.
4.3
CCL5
CCL5 is a T cell and macrophage chemoattractant that has been shown to

inuence leukocyte migration during periods of inammation. Upon MHV
infection of the CNS of mice, CCL5 transcripts and protein are readily detected
within the brain [52]. Initial studies in which CD4/ or CD8/ mice were
infected with MHV indicated an overall reduction in CCL5 mRNA transcripts
within the brains of CD4/ mice, suggesting that CD4+ T cells were either
a primary cellular source for CCL5 and/or inuenced the expression of CCL5
by resident and inammatory cells [53]. We now know that both inammatory
CD4+ T cells as well as astrocytes are capable of expressing CCL5 following
instillation of MHV into the CNS [32, 53]. Furthermore, treatment with neu-
tralizing anti-CCL5 antisera results in diminished T cell and macrophage
accumulation within the CNS, suggesting that in this model CCL5 is capable
of regulating trafcking of these two populations of cells [32].
4.4
CCR5
CCR5 is a member of the CC chemokine receptor family that is expressed on

various hematopoietic cells including lymphocytes and macrophages [86].
Chemokines that are capable of binding to CCR5 include CCL3, CCL4, and
CCL5 [7, 68, 86]. Recent studies have clearly indicated that CCR5 expression
correlates with leukocyte trafcking to sites of inammation as well as
regulating the immune response following microbial infection. For example,
mice decient in CCR5 (CCR5/ ) exhibit altered T cell activity and impaired
macrophage function [88, 109]. Furthermore, macrophage trafcking in
response to antigen is impaired in CCR5/ mice, indicating that CCR5 is
required for migration of this population of cells [45]. Given that both T cells
and macrophages express CCR5 following MHV infection of the CNS and
these cells clearly inuence outcome in response to infection, we have dened
the contributions of CCR5 to both host defense and disease in response to
MHV infection. Using an adoptive transfer model in which virus-expanded
T cells are transferred into MHV-infected RAG1/ mice, we have been able to
examine how CCR5 expression inuences trafcking of T cells into the CNS.
Transfer of CCR5+/+ -derived CD4+ T cells to MHV-infected RAG1/ mice
resulted in CD4+ T cell entry into the CNS and a reduction in viral titers within
the brain [30]. These mice also displayed robust demyelination correlating
with macrophage accumulation within the CNS. Conversely, CD4+ T cells
from CCR5/ mice displayed an impaired ability to trafc into the CNS
of MHV-infected RAG1/ recipients, which correlated with increased viral
titers, diminished macrophage accumulation, and limited demyelination.
Analysis of chemokine receptor mRNA expression by M133147-expanded
CCR5/ -derived CD4+ T cells revealed reduced expression of CCR1, CCR2,
and CXCR3, indicating that CCR5 signaling is important in increased
expression of these receptors which aid in trafcking of CD4+ T cells into the
CNS. Collectively these results demonstrate that CCR5 signaling is important
to migration of CD4+ T cells to the CNS following MHV infection.
With regards to the role of CCR5 in CD8+ T cell trafcking, comparable
numbers of virus-specic CD8+ T cells derived from immunized CCR5+/+
or CCR5/ mice were present within the CNS of MHV-infected RAG1/
mice following adoptive transfer, indicating that CCR5 is not required for
trafcking of these cells into the CNS [30]. RAG1/ recipients of CCR5/ -
derived CD8+ T cells exhibited a modest yet signicant (p0.05) reduction
in viral burden within the brain that correlated with increased cytolytic
activity and IFN- expression. Histologic analysis of RAG1/ recipients of
either CCR5+/+ or CCR5/ -derived CD8+ T cells revealed only focal areas
of demyelination with no signicant differences in white matter destruction.
These data indicate that CCR5 signaling on virus-specic CD8+ T cells
modulates antiviral activities but is not essential for entry into the CNS.
Finally, MHV infection of CCR5/ mice resulted in a dramatic reduction
in macrophage (dened as CD45high F4/80+ dual-positive cells) accumulation
within the brains, and this correlated with a signicant reduction in the
severity of demyelination compared to CCR5+/+ mice. Collectively, these data
suggest that ligand binding, e.g., CCL5 and/or CCL3, and signaling via CCR5
results in macrophage migration and inltration into the CNS. However, we
have previously demonstrated that CCL3 is expressed only at low levels during
acute disease and is not detectable during chronic demyelination, whereas
robust expression of CCL5 is detected during both phases of disease, and this
suggests that CCL5 is the primary CCR5 signaling chemokine in this model.
This is supported by earlier studies that showed an important role for CCL5 in
attracting macrophages into the CNS following MHV infection [53]. There-
fore, the data presented in this study suggest that one mechanism by which
CCL5 contributes to demyelination is via attracting macrophages into the CNS
through CCR5-mediated signaling pathways. Additional evidence supporting
this is provided by the observation that even in the presence of increased
CCL5 expression at day 12 p.i., demyelination is reduced in CCR5/ mice.
4.5
CCL2 and CCR2
CCL2 is capable of regulating the pathobiology of various inammatory

diseases including MS and atherosclerosis [1, 8, 28, 33, 35, 61]. In addi-
tion to its potent chemoattractant effect on monocytes and macrophages,
CCL2 also inuences TH 2 polarization in response to certain antigenic chal-
lenge [36, 41, 46, 99]. The inuence of CCL2 on T cell polarization may be due
to the fact that CCL2 is constitutively expressed within secondary lymphoid
tissue and would be capable of affecting cellular responses following exposure
to antigen [36]. Thus, available evidence indicates that expression of CCL2 is
capable of inuencing both innate as well as adaptive immune responses by
regulating monocyte and T cell responses, respectively.
Analysis of chemokine receptor expression following MHV infection re-
veals that CCR2 is expressed by endogenous cells of the CNS as well as by
inammatory T cells and macrophages, indicating a role for these receptors
in regulating both the immune response and disease development [13, 31].
Indeed, MHV-infection of CCR2/ mice resulted in a dramatic increase in
mortality and enhanced viral recovery from the brain that correlated with re-
duced T cell and macrophage entry into the CNS compared to viral infection
of CCR2+/+ mice [13].
MHV infection of CCL2/ mice does not result in a similar disease pheno-
type as observed in CCR2/ mice. This was somewhat surprising as CCR2 is
currently the only known functional receptor for CCL2. Specically, CCL2/
mice were able to clear virus from the brain in a similar time frame as wild-
type mice, and this correlated with the ability to generate antigen-specic
T cells [39]. The deciency in CCR2/ mice to clear virus from the brain is
not the result of an inherent inability to generate an effective adaptive im-
mune response to virus, as CCR2/ mice had a similar frequency of antigen-
presenting cells (APC) and virus-specic T cells present within draining CLN
compared to either CCL2/ or wildtype mice. Our ndings from MHV in-
fection of CCL2/ mice indicated that while CCL2 does inuence leukocyte
migration into the CNS in response to viral infection, CCR2 is clearly more
inuential in directing T cell trafcking into the CNS. In support of the role
for CCL2 in promoting leukocyte migration into the CNS of MHV-infected
mice are recent studies by Perlman and colleagues demonstrating that local-
ized CCL2 expression within the CNS promotes macrophage inltration [47].
These data highlight the possibility that ligand(s) other than CCL2 are impor-
tant in signaling through the CCR2 receptor. Alternatively, it is possible that
CCR2 signaling by either endothelial cells and/or astrocytes regulates the per-
meability of the BBB, as recently suggested by Stamatovic and colleagues [91].
5
Chemokines and Chronic Viral-Induced Demyelination
Expression of chemokines has been associated with demyelinating plaque

lesions present in MS patients [3, 4, 26, 27]. Elevated levels of chemokines,
notably CXCL10, were found in the cerebral spinal uid (CSF) of MS pa-
tients during periods of clinical attack [25, 89]. Indeed, the concentration
of CXCL10 within the CSF of MS patients correlated with numbers of in-
ammatory cells and the severity of clinical disease [2, 89, 90]. Moreover,
when CXCL10 levels decreased, there was a corresponding decrease in in-
ammation and disease severity [89]. Astrocyte expression of CXCL10 has
been reported in active plaque lesions present in MS patients, and the ma-
jority of T cells inltrating into the CNS of MS patients express the CXCL10
receptor, CXCR3. Collectively, these studies highlight a potentially important
role for CXCL10 in the pathogenesis of demyelinating diseases such as MS
by attracting CXCR3-expressing T cells into the CNS and support targeting
chemokines and their receptors for therapeutic intervention in the treatment
of MS [10, 54, 70, 90].
Studies from animal models of MS support this notion by demonstrating
that blocking of CXCL10 often results in diminished disease severity accom-
panied by a marked reduction in neuroinammation. For example, several
recent reports indicate that treatment with anti-CXCL10 neutralizing antibod-
ies resulted in delayed disease onset and diminished neuroinammation in
mice with the autoimmune demyelinating disease experimental autoimmune
encephalomyelitis (EAE) [20]. These studies support the idea that localized
expression of CXCL10 within the CNS amplies disease severity by attracting
CXCR3-expressing T cells into the CNS. Once present, these cells enhance
neuroinammation by secreting additional chemokines as well as cytokines
that can activate resident glia cells. Importantly, these studies also impli-
cate CXCL10 as a potential therapeutic target and suggest that alternative
CXCR3 ligands, e.g., CXCL9 and CXCL11, do not exert a prominent effect on
T cell inltration into the CNS. However, the role of CXCL10 in contribut-
ing to neurologic disease in EAE has been questioned by results indicating
that CXCL10 may actually exert a protective effect in mice with EAE [49, 71].
Antibody-mediated neutralization following induction of EAE in rats resulted
in increased disease severity, and this was associated with smaller draining
lymph nodes and increased numbers of CD4+ T cells inltrating into the
CNS [71]. In addition, CXCL10/ mice exhibited increased clinical disease
severity following immunization with myelin peptides, and this correlated
with diminished lymph node sizes although T cell inltration into the CNS
was not dramatically altered when compared to wildtype mice [49]. In these
particular EAE models in which mice are immunized peripherally with anti-
gen, CXCL10 expression within secondary lymphoid tissue is considered im-
portant in dictating disease outcome by serving to retain lymphocytes and
tailoring T cell responses. Moreover, these ndings highlight the different
roles of CXCL10 in regulating cellular immune responses in different models
of neuroinammation and emphasize the need for a better understanding of
how signaling by this chemokine regulates inammation and disease.
As indicated, we have determined that MHV infection of the CNS results
in an orchestrated expression of chemokine and chemokine receptor genes
that are regulated, in large part, by the viral burden. Similar to MS patients,
CXCL10 is expressed primarily by astrocytes in areas undergoing demyeli-
nation, suggesting an important role in the pathogenesis of demyelination
by attracting CXCR3-expressing T cells into the CNS [52, 59]. Indeed, our
laboratory was the rst to demonstrate that treatment of mice with estab-
lished demyelination and paralysis with anti-CXCL10 neutralizing antibody
resulted in a signicant reduction in CD4+ but not CD8+ T cells present
within the CNS, and this correlated with improved motor skills and a re-
duction in the severity of demyelination [59]. Moreover, the dramatic regain
of movement in anti-CXCL10-treated mice corresponded with more than
80% of previously demyelinated axons undergoing remyelination, indicat-
ing that removal of CXCL10 promoted an environment capable of remyeli-
nation. In addition to reduced numbers of CD4+ T cells within the CNS,
there was a paucity of macrophage inltration into the CNS of anti-CXCL10-
treated mice that correlated with a dramatic reduction in the levels of the
macrophage-chemoattractant CCL5. These data were consistent with previ-
ous studies indicating that CD4+ T cells were the major source for CCL5
in MHV-infected mice undergoing demyelination [53, 59]. The inuence of
CXCL10 in contributing to T cell responses was also examined. T cells isolated
from secondary lymphoid tissue of mice treated with anti-CXCL10 displayed
muted expression of IFN- in response to viral antigen when compared to T
cells isolated from control mice, suggesting that CXCL10 also serves to inu-
ence T cell effector functions during chronic disease (T.E. Lane, unpublished
observations).
We have previously determined that CCL5 mRNA transcripts and protein
are present within the CNS of MHV-infected mice during chronic demyelina-
tion, indicating a potentially important role for this chemokine in promoting
inammation [52, 53]. In order to assess the functional role of CCL5 in par-
ticipating in viral-induced immune-mediated demyelination, MHV-infected
mice were treated via intraperitoneal (i.p.) injection with anti-CCL5 mono-
clonal antibody (mAb) following onset of clinical disease and demyelination.
Such treatment resulted in a signicant (p0.05) reduction in the severity of
clinical disease compared to mice treated with an isotype (IgG1 )-matched an-
tibody [32]. Upon removal of anti-CCL5 treatment, clinical disease returned
to mice such that there was no difference between the two experimental
groups of mice. Immunophenotyping the cellular inltrate of mice treated
with anti-CCL5 revealed reduced T cell and macrophage inltration into the
CNS that is consistent with our earlier studies that CCL5 attracts these cells
into the CNS of mice with chronic demyelination. Further, analysis of the
severity of demyelination in experimental groups of mice indicated that anti-
CCL5 treatment resulted in a signicant (p<0.05) reduction in the severity of
demyelination compared to control-treated mice.
A picture is slowly evolving from our experiments designed to test the func-
tional contributions of CXCL10 and CCL5 to chronic demyelination within
MHV-infected mice. Antibody targeting of the T cell chemoattractant CXCL10
in MHV-infected mice selectively affects CD4+ T cell accumulation within the
CNS accompanied by improved motor skills and a reduction in the severity
of demyelination [59]. In contrast, CCL5 is capable of attracting both CD4+
and CD8+ T cells into the CNS. It is also important to emphasize that our
data on CCL5 and CXCL10 inhibition with regards to T cell and macrophage
trafcking are corollary and it is possible that alternative scenarios exist.
For example, studies by Bergmann and colleagues suggest that during per-
sistent MHV infection there is limited to no trafcking of T cells from the
periphery into the CNS. Rather, upon entry during acute encephalomyelitis
a certain percentage of CD4+ and CD8+ T cells is retained and participate in
disease [62, 93]. In this instance, CXCL10 expression would not be functioning
as a T cell chemoattractant but rather to inuence specic biologic functions
of T cells as well as potentiating the retention of T cells within the CNS. In
support of this, it is possible that CXCL10 serves to enhance CD4+ T cell
proliferation, as several recent studies indicate that CXCL10 is important in
contributing to T cell proliferation [18, 71, 101].
It is unlikely that CXCL10 contributes to T cell survival, as CXCL10/ mice
do not display any abnormalities with regards to T cell half-life nor do we see
any increase in numbers of apoptotic T cells following anti-CXCL10 treatment.
In addition, Narumi et al. [71] speculate that CXCL10 actually serves to retain
CXCR3+ T cells within tissues and this inuences disease severity. Therefore,
the selective reduction in CD4+ T cells within the CNS of MHV-infected mice
may not be the result of impaired trafcking. Rather, either CD4+ T cells are
not undergoing a steady-state turnover or are actually migrating out of the
CNS in the absence of signals specifying their retention.
In addition, recent studies indicate an important role for CXCL10 in im-
parting effector functions to T cells. For example, Salomon and colleagues
demonstrated that anti-CXCL10 treatment improved joint swelling in a rodent
model of arthritis and this correlated in part with an altered TH 1/TH 2 balance,
suggesting that CXCL10 expression promotes and maintains a TH 1 state in
T cells in this model [87].
Similarly, we have shown that MHV-infection of CXCL10/ mice results
in diminished IFN- expression by virus-specic T cells, supporting the idea
that CXCL10 expression serves to maintain a TH 1-like state in T cells [18]
(T.E. Lane, unpublished observations). CCL5 signaling also modulates cy-
tokine production by T cells following antigenic challenge. In support of this
is our demonstration that inhibition of CCL5 signaling results in enhanced
IFN- expression by virus-specic T cells, supporting the idea that CCL5 ex-
pression serves to regulate a TH 1-like state in T cells [32]. Moreover, ablation
of CCL5 signaling also modies the cytolytic activity of MHV-specic CD8+
T cells [30].
6
Perspectives
This chapter highlights mechanisms by which chemokines participate in

both host defense and disease progression in response to MHV infection of
the CNS. An overview of the potential functional role for select chemokines in
linking innate and adaptive immune responses in response to viral infection
of the CNS is provided in Fig. 1. In brief, following MHV infection there
is robust expression of chemokines by infected astrocytes including CCL3
that contribute to the maturation/activation of local DCs, which ultimately
enables migration to draining cervical lymph nodes. Activated DCs present
antigen to T cells as well as secrete chemokines such as CCL3 and CXCL10
that enhance polarization to a TH 1 response. In turn, MHV-specic T cells
express chemokine receptors including CXCR3 and CCR5 that enable them to
trafc into the CNS as a result of localized expression of ligands CXCL9 and
CXCL10 (ligands for CXCR3) as well as CCL5 (ligand for CCR5). In addition,
our contention is that expression of CCR2 by endothelial cells of the BBB is
also important in increasing the permeability of this structure.
With regards to chronic disease, MHV persistence within the CNS results
in chronic expression of CXCL10 and CCL5 which together contribute to the
maintenance of a chronic inammatory disease by attracting both T cells
and macrophages (Fig. 2). Local secretion of CXCL10 and CCL5 may also
contribute to demyelination by enhancing specic T cell effector functions
including (1) secretion of IFN- that activates local inammatory macrophage
and resident microglia, as well as directly damaging oligodendrocytes and (2)
increasing CTL activity by CD8+ T cells.
Fig. 1AH Chemokines and innate/adaptive immune response following MHV in-
fection of the CNS. Instillation of MHV into the CNS of susceptible mice results in
infection of astrocytes that are an important source of chemokines including CXCL10,
CCL5, and CCL3 (A). In addition, immature DC-like cells may also be susceptible to
infection and secrete CCL3 (B) that functions in a paracrine and autocrine manner
to bind to CCR1 expressed on immature DC-like cells. As a result of CCL3 signaling
and MHV infection, the DC-like cells undergo maturation and activation (C) resulting
in a remodulation of the plasma membrane characterized by decreased expression of
CCR1 accompanied by increased expression of CCR7 as well as major histocompat-
ibility complex (MHC) class I and II. CCR7-expressing, activated DCs home to the
draining cervical lymph node (D). Upon entry, activated DCs express a variety of sol-
uble factors including CCL3 and CXCL10 (E) that activate and enhance polarization of
virus-specic T cells to a TH 1 phenotype (F). Activated T cells exit the lymph node via
the efferent lymph (G), enter the blood stream, and migrate to the CNS via expression
of the chemokine receptors CXCR3 and CCR5 (H)
Fig. 2AF Chemokines and MHV-induced demyelination. Persistent MHV infection

within astrocytes leads to chronic CXCL10 and CCL5 expression (A) that serves to
recruit CXCR3+ and CCR5+ T cells into the CNS (B). In addition, activated CD4+ T cells
secrete CCL5 that enhances macrophage migration into the CNS (C). We believe that
CXCL10 may also inuence T cell effector functions within the CNS, including CTL
activity (D) and IFN- secretion (E), leading to macrophage activation. Both IFN-
production and CTL activity may enhance tissue destruction as well as macrophage
activation that amplies myelin destruction (F)
Clearly, these observations indicate that chemokine signaling is an integral

component involved in eliciting protective immunity in response to viral in-
fection of the CNS. Conversely, our studies also indicate that chronic localized
secretion of select chemokines ultimately amplies disease severity through
maintaining inammation within the CNS. Importantly, studies derived from
the MHV system demonstrate that antibody targeting of select chemokines
offers a powerful approach towards delineating the functional contributions
of these molecules in a model of immune-mediated demyelination. Further,

these studies highlight the relevancy of such an approach in treating human
neuroinammatory and demyelinating diseases such as MS.
Acknowledgements The authors wish to thank Craig Walsh for helpful discussion.
This work was supported by National Institutes of Health grants NS41249, NS18146,
and National Multiple Sclerosis Society Grant 3278 to T.E.L. J.L.H. is supported by
postdoctoral fellowship 1652 from the National Multiple Sclerosis Society.
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CTMI (2006) 303:2946

Cytokine and Chemokine Networks:

Pathways to Antiviral Defense
T. P. Salazar-Mather (u) K. L. Hokeness
Department of Molecular Microbiology and Immunology, Division of Biology
and Medicine, Brown University, 69 Brown Street, Box G-B6,
Providence, RI 02912, USA
Thais_Mather@brown.edu
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2 NK Cells and Antiviral Defenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

2.1 NK Cell Responses in MCMV Infection . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.2 NK Cell Inammatory Responses in Liver During MCMV Infection . . . . . 32
3 CCL3/MIP-1: Primary Mediator of NK Cell Inammation . . . . . . . . . . 34

3.1 MIP-1 and Antiviral Defenses: The Cytokine and Chemokine Networks . 34
3.2 The Type 1 Interferons (IFN-/) Association . . . . . . . . . . . . . . . . . . . . 35
3.3 The CXCL9/Mig Association . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
4 CCL2/MCP-1: The First Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

4.1 Resident Macrophages: MCP-1 Producers . . . . . . . . . . . . . . . . . . . . . . . 37
5 MCP-1 and Macrophage Recruitment . . . . . . . . . . . . . . . . . . . . . . . . . . 38

5.1 MCP-1 and Antiviral Defenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Abstract The complex interplays between cytokines and chemokines are emerging
as key communication signals in the shaping of innate and adaptive immune re-
sponses against foreign pathogens, including viruses. In particular, the virus-induced
expression of cytokine and chemokine proles drives the recruitment and activation
of immune effector cells to sites of tissue infection. Under the conditions of infection
with murine cytomegalovirus (MCMV), a herpesvirus with pathogenic potential, early
immune functions are essential in the control of virus replication and virus-induced
pathology. The coordinated MCMV-induced cytokine and chemokine responses pro-
mote effective natural killer (NK) cell recruitment and function, and ultimately MCMV
clearance. The studies highlighted in this chapter illustrate in vivo pathways mediated
by innate cytokines in regulating chemokine responses that are vital for localized
antiviral defenses.
30 T. P. Salazar-Mather K. L. Hokeness
Abbreviations
MCMV Murine cytomegalovirus
NK Natural killer
MIP-1 Macrophage inammatory protein 1
Mig Monokine induced by interferon-
MCP-1 Monocyte chemoattractant protein-1
rIFN- Recombinant interferon-
1
Introduction
The host response to microbial pathogens necessitates the integrated action

of both the innate and adaptive arms of the immune system. Innate immu-
nity is largely dependent on granulocytes, macrophages, dendritic cells, and
natural killer (NK) cells, whereas adaptive immune responses require T and
B lymphocytes. It is becoming increasingly evident that an effective immune
response requires crosstalk among the various immune cells for linkage of
innate and adaptive immunity. This intercellular network of communication
signals is mediated in part by cytokine and chemokine responses generated
against infectious agents, including viruses.
Cytokines constitute a group of low molecular weight soluble proteins that
modulate various immune functions upon induction by various stimuli, such
as bacterial or viral components [1012, 32, 92]. Virtually all cells produce
these proteins, including leukocytes and infected or activated nonimmune
cells such as broblasts, endothelial cells, and a variety of tissue parenchy-
mal cells [10, 11]. Cytokines control the magnitude and kinetics of immune
responses by inducing or inhibiting the activation, proliferation, and/or dif-
ferentiation of various target cells, and they also regulate the production of
antibodies and other cytokines including chemokines [1, 11, 54].
Chemokines are small heparin-binding secreted cytokines that are func-
tionally appearing to be more diverse than used to be thought. Housekeeping
chemokines are generally constitutively expressed under physiological con-
ditions, and they function in development and homeostasis [20, 56, 73, 81,
95, 104]. Inducible inammatory chemokines function in the directed traf-
cking and localization of effector leukocytes within tissue compartments
during inammation, infection, and trauma [5, 58, 73, 82, 91, 95, 104]. There
is considerable evidence highlighting the importance of chemokine-mediated
inammatory responses to pathogens as being crucial to survival during in-
fections [18, 5355, 57, 77, 78, 80, 83]. Additionally, it has become evident that
inltrating leukocytes may sometimes play a role in disease pathogenesis [18,
Cytokine and Chemokine Networks: Pathways to Antiviral Defense 31
54, 55, 83, 98]. Nevertheless, as chemokines are central to the routing of key
immune cells, they have emerged as key players in host defense mechanisms
[5458, 62, 80, 95, 104]. These proteins mediate their biological effects through
selective interactions with seven-transmembrane G protein-coupled recep-
tors on the surface of target cells [7, 9, 59, 74, 90]. Inammatory chemokines
are selective for a broad range of receptors. Nonetheless, in vivo studies have
demonstrated specic functions for various chemokines [18, 19, 21, 25, 27,
37, 58, 80, 93].
Protective immunity to viruses is dependent on the activation and inter-
play between cytokines and chemokines to enhance or regulate innate or
adaptive (or both) effector functions. Studies have demonstrated that the cy-
tokine milieu induced by pathogens may determine the cellular constituents
that get activated, and thus the nature of the immune response, by selectively
inducing specic chemokines in infected tissue compartments [18, 37, 55,
57, 77, 78, 80]. The murine cytomegalovirus (MCMV) model of infection in
the liver has been used to demonstrate the coordinated effects of activated
cytokine and chemokine responses on the recruitment of NK cells to sites of
infection to maintain early control of virus replication. This chapter presents
a brief overview of the role of NK cells in antiviral defense, the molecular
mechanisms regulating the innate cytokine and chemokine networks pro-
moting NK cell inammation into liver, and the conceivable role of these
pathways in promoting downstream adaptive responses for effective antiviral
defense.
2
NK Cells and Antiviral Defenses
NK cells are innate effector cells that respond quickly to a variety of pathogens
before the onset of adaptive immunity [11, 96, 103]. These cells originate from
bone marrow precursors and predominate in peripheral blood and spleen.
However, they can be induced to trafc into other compartments including the
liver during infection [77, 99]. Classical NK cells are CD3 and do not express
rearranged antigen-specic receptors [11, 42, 71, 97, 103]. Instead, it is now
widely accepted that NK cells express a complex repertoire of activating and
major histocompatibility complex (MHC) class I-specic inhibitory receptors
on their surfaces that interact with ligands on target cells [23, 101103]. En-
gagement of activation receptors with ligands on infected cells permits NK
cell-mediated killing and cytokine production, while the inhibitory recep-
tors restrain NK cell activation [4, 23, 43, 101, 102]. The central role of NK
cells as mediators of antiviral defenses has long been appreciated [11, 96].
Furthermore, viruses that evade immune detection by restricting the func-

tion of MHC class I T lymphocytes are more susceptible to NK cell-mediated
protective responses [101103]. These cells are quick to respond and can be
activated to produce high levels of the cytokine interferon (IFN)-, as well
as additional antiviral and immunoregulatory cytokines [11, 12]. NK cells
have been demonstrated to mediate protection against a number of viral
infections (reviewed in references [11, 12]), most notably is the extensive
data documenting their importance in innate defenses against MCMV infec-
tion [6, 11, 15, 63, 64, 88].
2.1
NK Cell Responses in MCMV Infection
Resistance to MCMV infection is critically dependent on the activation of

NK cells for early control of viral replication in target organs such as spleen
and liver [11, 12, 14, 63, 77, 94], although T lymphocyte responses do get
activated and contribute to viral clearance late in infection [35, 39, 69, 70].
The profound antiviral effects of NK cells against acute MCMV infection have
been exemplied with a variety of experimental systems, including in vivo
depletion of NK cells [14, 15, 63, 86], infection of beige mutant mice [66, 88],
and most recently infection of mice decient in NK cell, but not NKT or
T cell, functions [50]. Together, these studies have demonstrated increased
viral titers, liver pathology, and enhanced mortality upon elimination of
functional NK cell responses. Conversely, resistance to MCMV infection has
been demonstrated when NK cells were adoptively transferred into susceptible
mice [16].
The mechanisms of NK cell-mediated defenses against MCMV infection in
vivo have not been completely elucidated, although there is evidence that
disparate NK cell responses are used in the spleen and liver for protec-
tion against MCMV infection. In the spleen, resistance seems to be mainly
through perforin-dependent mechanisms, suggesting that NK cell-mediated
cytotoxicity [94] and NK cell surface expression of an activating recep-
tor [4, 23, 43, 101, 102], found on resistant strains of mice [4, 101], is required.
In contrast, there is evidence that NK cell effector function in the liver is
mediated through production of IFN- [52, 64, 77, 78, 94].
2.2
NK Cell Inflammatory Responses in Liver During MCMV Infection
The liver is a common target organ of MCMV infection [29, 61, 65, 87], and
the rapid control of virus-induced disease is essential for survival [64, 65,
78, 80, 87]. As shown in Fig. 1a, the liver responds to MCMV infection by in-
ducing a profound accumulation of inammatory cells into the parenchyma
that peaks between 48 and 72 h after infection of C57BL/6, 129SvEV, and T- and
B cell-decient C57BL/6-SCID mice [65, 77, 79]. Studies have identied NK
cells as the major cellular constituents of the inammatory foci [3, 22, 77]. As
NK cells surround and sequester sites of MCMV antigen expression (Fig. 1b),
it is highly conceivable that the inammatory response limits the spread of
infection to neighboring hepatocytes, thus minimizing virus-induced pathol-
ogy. The precise effector mechanism used by inammatory NK cells remains
to be elucidated, although localization of IFN- within inammatory sites
([77] and Sect.3) is critical to antiviral defense.
In vivo cell trafcking studies using uorescently labeled bone marrow
cells from either C57BL/6 or C57BL/6-SCID donor mice have demonstrated
the rapid deployment and collective mobilization of NK cells between por-
tal areas and central hepatic veins in MCMV-infected recipient mice [77].
Transfer studies using bone marrow donor cells from mice depleted of NK
cell subsets using specic antibodies or from mice genetically decient in NK
cells and T lymphocytes demonstrated the accumulation of cells only in the
liver sinusoids [77]. Thus, in vivo, NK cells are induced to migrate to sites
in a pattern similar to the inammatory foci observed in histological liver
sections (see Fig. 1a).
Fig. 1ac Characterization of liver inammatory foci during MCMV infection. a and b
Parafn-embedded or frozen livers sections were prepared from C57BL/6 mice infected
with MCMV for 48 h and (a) stained with hematoxylin and eosin or (b) stained with
immune serum antibodies to MCMV. Bound antibodies were detected with NBT/BCIP,
followed by methyl green counterstain to highlight localization of inammatory nuclei.
Arrowhead denotes focus of inammatory cells. c Parafn-embedded liver sections
were prepared from C57BL/6-MIP-1 decient mice infected with MCMV for 48 h and
stained with hematoxylin and eosin. Arrow denotes cytomegalic inclusion bodies
3
CCL3/MIP-1: Primary Mediator of NK Cell Inflammation
To understand the key mechanisms governing the in vivo recruitment of
NK cells to liver during MCMV infection, it is important to appreciate the
chemokine expression prole. Of particular interest is the inammatory
chemokine CCL3 or macrophage inammatory protein-1 (MIP-1). Mice
rendered genetically decient in MIP-1 (MIP-1 knockouts) have been
shown to exhibit reduced lung inammation and delayed clearance of in-
uenza virus when compared to control animals [19]. Moreover, MIP-1 pro-
moted pulmonary inammation and antiviral defense in a model of paramyx-
ovirus infection [24]. During MCMV infection, MIP-1 messenger (m)RNA
and protein can be detected in liver at times consistent with peak NK cell
inammation (48 h after infection) [77, 78]. Results obtained from in vivo
cell trafcking studies demonstrated that the induction of NK cell inam-
mation in liver during MCMV infection was dependent on MIP-1, as NK
cell trafcking is dramatically impaired in MIP-1 knockout mice [77]. It is
notable that monocyte and macrophage trafcking to liver is not affected
in MIP-1 knockout mice, suggesting that although other chemokines are
induced in these mice, MIP-1 responses are required for NK cell recruit-
ment. Furthermore, MIP-1 knockout mice lacked detectable inammatory
foci in liver sections by histological evaluation, and displayed a profound
increase in the number of cytomegalic inclusion bodies, or MCMV-infected
cells (Fig. 1c) [77]. Flow cytometric analysis demonstrated that in the absence
of MIP-1 function the absolute numbers of NK cells become signicantly
elevated in the blood but are dramatically reduced in the liver when compared
to control mice [78]. These results imply that although NK cells get activated
in response to infection, the communication signals directing their migration
to tissue sites of virus replication are impaired in the absence of MIP-1.
Collectively, these studies dene a prominent and unique role for MIP-1 in
the initial inux of NK cells into the liver during MCMV infection.
3.1
MIP-1 and Antiviral Defenses: The Cytokine and Chemokine Networks
As discussed above, NK cells are an essential source of IFN- production

that is required for early antiviral defense in liver. During MCMV infection,
this cytokine is maximally elevated in spleen and serum within 40 h but
subsides by 48 h after viral challenge [75, 78]. It has been established that an
essential function of the MIP-1-dependent NK cell inammatory response
is to sustain IFN- production in liver beyond the systemic kinetics of
the cytokine, or further than 48 h [78]. MIP-1 knockout mice, with their
inability to mount an effective NK cell inammatory response, do not sustain

sufcient levels of IFN- in the liver. The result is a profound elevation in
spleen and liver viral titers followed by death on day 5 of MCMV infection.
Interestingly, mice genetically decient in interleukin (IL)-18 do not succumb
to MCMV infection, although they are severely compromised in systemic,
but not hepatic, IFN- responses [68]. Thus, MIP-1 functions promote viral
resistance by mediating the recruitment of NK cells and the delivery of IFN-
in a localized site of infection.
3.2
The Type 1 Interferons (IFN-/) Association
Early infection with MCMV induces production of multiple innate cytokines
including the type 1 interferons, IFN-/ [1, 11, 12]. These cytokines are
potent activators of antiviral pathways [12, 36, 46, 84, 50] as well as media-
tors of multiple immunomodulatory functions, including induction of MHC
class I expression, activation of NK cell cytotoxicity, and modulation of cy-
tokines and cytokine receptors, including regulation of other type 1 interferon
genes [12, 84]. IFN-/ expression has also been shown to affect leukocyte
trafcking [33, 38, 76, 79]. During MCMV infection, bone marrow-derived
macrophages and NK cells have been shown to migrate to secondary sites in
response to IFN-/ production [76]. Recent studies have demonstrated pro-
duction of IFN-/ in MCMV-infected livers [79]. Histological analysis of liver
sections in mice unable to respond to functions induced by IFN-/ as a result
of mutation in the receptor for the cytokines (IFN-/R knockouts) illustrated
the lack of inammatory foci but the presence of extensive virus-induced
pathology in liver [79]. Moreover, IFN-/R knockout mice had reduced lev-
els of MIP-1 protein in liver. Accordingly, IFN-/R knockout mice exhibit
profound decreases in the accumulation of NK cells in liver. Furthermore,
these mice exhibit mortality by day 5 of MCMV infection [79]. It is notable
that NK cell accumulation is not signicantly affected when uninfected MIP-
1 knockout mice are treated with recombinant IFN- (rIFN-), indicating
that IFN-/ mediate immunoregulatory events upstream of MIP-1.
In vivo cell trafcking studies demonstrated that leukocyte migration to
the liver is dependent upon the effects of IFN-/, as only donor-derived
uorescent-labeled cells from immunocompetentbut not IFN-/R
knockoutsaccumulated extensively in liver [79]. Additionally, IFN-/
mediated the recruitment of macrophage populations that were identied as
a major source of MIP-1 production [79]. Altogether, these studies identify
IFN-/ as key mediators in the production of MIP-1, and they dene
a cellular delivery mechanism driven by innate cytokines and chemokines
for regulation of NK cell inammation.
3.3
The CXCL9/Mig Association
One downstream consequence of the MIP-1-dependent NK cell in-

ammatory response is induction of the IFN--inducible chemokine
CXCL9/monokine-induced by interferon (Mig), a potent T cell chemoattrac-
tant [2, 47, 49, 100]. Mig production is severely reduced in MIP-1 knockout
mice when compared to control mice [78]. Furthermore, treatment of
immunocompetent mice with immune serum against Mig resulted in highly
signicant increases in viral titers in both spleen and liver [78]. Consistent
with results obtained using mice decient in IFN-/ and MIP-1 functions,
Mig was required for survival, as neutralization of the chemokine led to death
by day 5 of infection [78]. Studies have identied a prominent T cell response
in liver by day 5 after MCMV challenge [28, 39]. It is therefore plausible
that early expression of MIP-1 by MCMV-induced IFN-/ recruits NK
cells into the liver, and through localization of IFN- and subsequent Mig
induction, these cytokine and chemokine networks provide an important
link of innate and adaptive immune responses for overall antiviral defenses
in tissue compartments. Ongoing studies in our laboratory are evaluating
this likely scenario under the conditions of MCMV infection in the liver.
4
CCL2/MCP-1: The First Link
As discussed above, IFN-/ clearly plays a role in promoting the recruit-

ment of MIP-1-producing macrophages into liver, but the primary molecular
mechanisms guiding this process have only recently been examined during
MCMV infection. Studies have identied CCL2, or monocyte chemoattractant
protein-1 (MCP-1), as a key intermediate of IFN-/ activity for regulation
of inammatory responses in liver [30]. MCP-1 production can be induced at
this site as early as 24 h after MCMV infection. Furthermore, MCP-1 produc-
tion precedes that of MIP-1 in a temporal fashion (Fig. 2) [30]. IFN-/R
knockout mice show dramatic decreases in the levels of MCP-1 protein when
compared to immunocompetent mice. In addition, the treatment of unin-
fected immunocompetent mice with rIFN- results in a signicant release of
MCP-1 protein [30]. In vitro stimulation of nave liver leukocytes obtained
from immunocompetent mice with rIFN- was shown to generate a dose-
dependent induction of MCP-1. Together, these studies clearly establish that
the induction of MCP-1 protein in liver is dependent on IFN-/-mediated
functions.
Fig. 2a, b Kinetics of MCP-1 and MIP-1 during MCMV infection in liver. Infected
intraperitoneally with 5104 plaque-forming units (pfu) MCMV were 129 mice. Livers
were harvested from uninfected (0 h) or infected mice at the indicated time points.
MCP-1 (a) and MIP-1 (b) protein levels in liver homogenates were determined by
sandwich enzyme-linked immunosorbent assay (ELISA) [30]. The levels of detection
were 0.080.2 and 0.020.05 ng/g liver for MCP-1 and MIP-1, respectively. Data are
the meansSE (n=3 mice tested individually for each time point). (Figure used with
permission from [30]. Copyright 2005. The American Association of Immunologists)
4.1
Resident Macrophages: MCP-1 Producers
In multiple models of hepatic injury, resident macrophages have been shown

to contribute to MCP-1 production [8, 44, 72]. The experiments highlighted
in Sect. 4) strongly suggest that nave liver leukocytes can be stimulated to
release MCP-1 in the presence of IFN-/. Additional studies using enriched
F4/80-positive and F4/80-negative cell populationsF4/80 being a mouse
macrophage-restricted marker [45]from uninfected immunocompetent
mice demonstrated a clear induction of MCP-1 protein following stimulation
with r-IFN [30]. A similar induction of MCP-1 protein was observed
with enriched F4/80-positive, but not F4/80-negative, cell populations from
immunocompetent mice infected with MCMV for 24 h [30]. Thus, resident
macrophages are early responders to the effects of IFN-/ and are major
producers of MCP-1 protein in liver.
5
MCP-1 and Macrophage Recruitment
It has been clearly established that MCP-1 effectively promotes the mobiliza-
tion of inammatory macrophages to sites of tissue damage [17, 26, 31, 51, 72]
by preferential binding to the chemokine receptor CCR2 [13, 34, 40, 41, 67].
Recent studies have shown a dynamic impairment in liver macrophage and
NK cell accumulation in mice decient in MCP-1 (MCP-1 knockout) or CCR2
(CCR2 knockouts)when compared to control miceduring infection with
MCMV. Furthermore, MCP-1 and CCR2 knockout mice have decreased lev-
els of both MIP-1 and IFN- proteins [30]. These results establish a central
role for MCP-1 in promoting the recruitment of macrophages and NK cells.
Moreover, they agree with previous observations that trafcking macrophages
contribute to the initial release of MIP-1, and subsequently the delivery of
NK cell-derived IFN- in the liver [79, 80]. As CCR2 knockout mice displayed
comparable responses, the results dene MCP-1 as a key factor in initiating
critical innate inammatory events.
5.1
MCP-1 and Antiviral Defenses
It is clear from previous studies that MCP-1 is an important innate chemokine

because it is uniquely necessary for monocyte and macrophage migration
and the establishment of host defense against various pathogens [13, 18,
51, 31, 34, 40, 41, 85]. During MCMV infection, MCP-1 knockouts exhibit
a marked elevation in spleen and liver viral titers by day 4 that remains
prominent into day 5 of infection [30]. Comparable results were evident in
CCR2 knockout mice. Increased viral burden was associated with increases in
the circulating levels of the liver enzyme alanine aminotransferase, indicating
liver damage. Accordingly, histological evaluation of liver sections prepared
from uninfected control, MCP-1 and CCR2 knockout mice, did not show
variability in appearance within the groups of mice (Fig. 3ac). In contrast,
on day 5 post-MCMV infection, MCP-1 or CCR2 knockout mice revealed large
areas of necrosis as well as numerous cytomegalic inclusion bodies (Fig. 3e and
f). Mortality in these mice coincided with virus-induced liver disease, as they
succumbed to infection by day 5. This marked pathology was not evident in
control mice. Instead, control mice displayed evidence of intermittent clusters
of inammatory foci, viral clearance, and were remarkably similar to the liver
sections from the uninfected mice (Fig. 3a and d). These studies dene a role
for MCP-1, through interactions with CCR2, in promoting antiviral defense
and protection from virus-induced liver disease.
Fig. 3af Characterization of MCMV-induced liver damage in MCP-1- and CCR2-

decient mice. C57BL/6 (WT) (a and d) or mice genetically decient in MCP-1
(MCP-1 ) (b and e) or CCR2 (CCR2- ) (c and f) were either uninfected (ac) or
infected with MCMV (5104 PFU) for 5 days (df). Livers were harvested, parafn
was embedded, and they were sectioned for hematoxylin and eosin staining. Bar
represents 100 m. Arrows indicate necrotic lesions. Arrows within insets indicate
cytomegalic inclusion bodies. Figure was used with permission from reference [30].
Copyright 2005. The American Association of Immunologists
6
Conclusions
The studies highlighted in this chapter dene a network of cytokine and

chemokine pathways that promote the activation, coordination, and shap-
ing of the most effective immune response directed against a virus infection
establishing itself in tissues. Specically, the response elicits the migration
of macrophages and NK cells by the selective induction of inammatory
chemokines (Fig. 4). The importance of chemokines to antiviral defense is
underscored by the exploitation of the chemokine system by various human
and mouse viruses, including herpesviruses, poxviruses, adenoviruses, and
cytomegaloviruses [48, 53, 56, 60, 89]. Future in vivo studies dissecting the
complex interactions between cytokines, chemokines, immune cell popula-
tions, and viruses will add to our understanding of immune regulation and
perhaps the development of new therapeutic strategies for defense mecha-
nisms against viral infections.
Fig. 4 Model of cytokine and chemokine interactions critical to antiviral defense

during MCMV infection in liver. (1) The expression of the cytokine IFN-/ is lo-
cally induced in response to MCMV challenge and (2) promotes the early release of
MCP-1 from resident (F4/80+) macrophage populations. (3) MCP-1 initiates the re-
cruitment of MIP-1-producing macrophages from the periphery to the liver. (4) Sub-
sequently, MIP-1 promotes the initial mobilization of NK cells from the periphery to
the liver where they surround and sequester MCMV-infected cells to form character-
istic clusters of inammatory foci. (5) These events localize production of IFN- and
(6) promote the induction of the IFN--inducible chemokine Mig, a known potent
chemoattractant of T lymphocytes. Together, these innate cytokine and chemokine
interactions provide vital antiviral defenses in liver, and conceivably play a role in
linkage of innate and adaptive immune responses
Acknowledgements Research in the authors laboratory is supported by National In-

stitute of Health Grant CA-102708.
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CTMI (2006) 303:4765

Herpes Simplex Virus and the Chemokines

That Mediate the Inflammation
D. J. J. Carr1,2 (u) L. Tomanek1
1 Department of Ophthalmology, University of Oklahoma, DMEI 415,
Health Sciences Center, 608 Stanton L. Young Blvd., Oklahoma City, OK 73104, USA
dan-carr@ouhsc.edu
2 Department of Microbiology and Immunology, University of Oklahoma,
Health Sciences Center, Oklahoma City, OK 73104, USA
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
1.1 General Properties of Herpes Simplex Viruses . . . . . . . . . . . . . . . . . . . . 48
1.2 Herpes Simplex Virus Type 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
1.3 Herpes Simplex Virus Type 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2 HSV-1 Infection of the Eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

2.1 Innate Immune Response to Ocular HSV-1 Infection . . . . . . . . . . . . . . . 50
2.2 Chemokine Expression During the Innate Immune Response
to Ocular HSV-1 Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2.3 Adaptive Immune Response to Ocular HSV-1 Infection . . . . . . . . . . . . . 55
2.4 HSV-1 Latency in the Trigeminal Ganglion . . . . . . . . . . . . . . . . . . . . . . 55
2.5 Reactivation of HSV-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3 HSV-2 Infection of the Genitalia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

3.1 Immune Response to Genital HSV-2 Infection . . . . . . . . . . . . . . . . . . . . 57
3.2 Chemokines and HSV-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4 Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Abstract Herpes simplex viruses (HSV) are highly pervasive pathogens in the human
host with a seroconversion rate upwards of 60% worldwide. HSV type 1 (HSV-1) is
associated with the disease herpetic stromal keratitis, the leading cause of infectious
corneal blindness in the industrialized world. Individuals suffering from genital herpes
associated with HSV type 2 (HSV-2) are found to be two- to threefold more susceptible
in acquiring human immunodeciency virus (HIV). The morbidity associated with
these infections is principally due to the inammatory response, the development of
lesions, and scarring. Chemokines have become an important aspect in understand-
ing the host immune response to microbial pathogens due in part to the timing of
expression. In this paper, we will explore the current understanding of chemokine
production as it relates to the orchestration of the immune response to HSV infection.
48 D. J. J. Carr L. Tomanek
Abbreviations
HSV Herpes simplex virus
PMN Polymorphonuclear cell
NK Natural killer
HSK Herpetic stromal keratitis
IL Interleukin
DC Dendritic cell
Th1 T helper 1 cell
TG Trigeminal ganglion
IFN Interferon
TNF Tumor necrosis factor
TLR Toll-like receptor
VEGF Vascular endothelial growth factor
1
Introduction
1.1
General Properties of Herpes Simplex Viruses
Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are neurotropic
viruses that are members of the subfamily -Herpesvirinae [42]. Both types
of HSV are transmissible from person to person via infectious mucosal secre-
tions that come in contact with mucosal epithelia that line surface apertures
of the body [9, 42, 57]. Herpes simplex viruses can cause a variety of diseases
including keratitis, cold sores, encephalitis, genital herpes, cutaneous herpes,
and meningitis [12, 42]. HSV-1 and HSV-2 enter the epithelium of the host
and initiate a lytic replicative cycle [18, 4042, 57, 70]. HSV enters its target
cell through a multistep process that includes envelope glycoproteins (g) that
surround the viral particle [42, 64]. The initial interaction begins with the
binding of gC and gB to heparin sulfate proteoglycans that are found on the
surface of target cells [42, 64]. After the attachment of the viral particle to
the host cell, another viral glycoprotein, gD, interacts with other host cell
surface receptors, including herpesvirus entry mediator A, which is a tumor
necrosis factor (TNF) receptor family member, and nectins, which allow for
the fusion of the virion envelope to the cells plasma membrane via gB, gD,
gH, and gL [42, 64]. Local replication commences with transcription of viral
lytic genes [18, 33, 4042, 57, 64, 70]. Following a lytic replicative cycle, the
virus enters sensory nerve endings in the basal aspect of the epithelium and
undergoes retrograde transport to associated sensory ganglia [18, 42, 64, 70].
Within the sensory ganglia, HSV undergoes a second stage of lytic infection.
Herpes Simplex Virus and the Chemokines That Mediate the Inammation 49
Depending on the extent of the infection, HSV may travel further to the cen-
tral nervous system. Following acute infection of sensory ganglia, the virus
establishes latency in a subpopulation of neurons [18, 42, 64, 70]. Periodic
reactivation from latency during periods of stress or immune suppression
results in the re-infection of the initial port of entry [18, 42, 70].
Clearance of the virus from the host is dependent on both the hosts innate
and adaptive immune responses. Polymorphonuclear cells (PMNs) are the
rst and most predominant cell type to inltrate the area of infection, releasing
a number of soluble factors including cytokines, chemokines, and tissue-
degrading enzymes including matrix metalloproteinases [6, 22, 45, 96, 97].
Likewise, natural killer (NK) cells and subsequently, macrophages and T cells
are recruited to the site of inammation. Chemokine expression has become
an interest in the scientic community as it relates to the immune response
to infectious agents and the pathology that develops from this response.
1.2
Herpes Simplex Virus Type 1
The cornea is a transparent, avascular tissue composed of a surface epithelium,

corneal stroma, and endothelium that covers the anterior portion of the
eye [70]. It is the avascular nature of the cornea that preserves the visual axis,
providing a translucent conduit for subsequent processing of an image by the
lens and retina of the eye. However, experimental evidence suggests ocular
HSV-1 can limit the visual axis through neovascularization and inltration of
leukocytes attracted to the site through the production of chemokines [103].
Experimental infection of the cornea initiates in the surface epithelium in
the outermost squamous layer of cells. HSV-1 spreads from cell to cell in
a polarized fashion to the next layer of cells, wing cells [70]. The virus is able
to travel to the sensory nerve endings that can be found in the basal aspect of
the epithelium [70].
One clinically signicant disease that is caused by HSV-1 is herpetic stro-
mal keratitis (HSK) an intense inammatory response triggered by the viral
infection of the corneal stroma [3, 45, 54]. If left untreated, the chronic inam-
matory response leads to the formation of lesions, scarring, and eventually
blindness.
1.3
Herpes Simplex Virus Type 2
HSV-2 is the causative agent of genital herpes of which approximately 500,000

new cases arise annually [33]. It has been estimated that 33% of the adult pop-
ulation is seropositive for this sexually transmitted disease, making HSV-2 the
most common sexually transmitted pathogen worldwide [33, 73, 78, 92]. Gen-
ital herpes infection can result in complications including urinary retention
and meningoencephalitis [33, 73, 78, 92]. Approximately 3,500 births in the
United States are impacted by HSV-2 infection, which can lead to fatal infant
encephalitis [18]. Even though a relatively large percentage of the population
is seropositive for HSV-2, only a small percentage is subjected to these compli-
cations. Hormones have been implicated in the susceptibility to infection in
the female host [86]. Specically, mice exposed to progesterone are rendered
more susceptible to infection [37] whereas estradiol-treated mice are found
to be resistant to infection [27]. Although the role of ovarian sex hormones in
susceptibility to genital HSV-2 infection is not completely dened, immune
suppression [27], changes in the vaginal epithelial thickness [72], and mod-
ulation of a cell membrane receptor, nectin-1- [48], may all inuence the
infectious process.
One common denominator in the recruitment process of leukocytes into
the inamed HSV-infected tissue is the expression of chemokines. Although
a necessary process in attracting immune effector cells required to control
replication and spread of the virus, chemokine expression and the ensu-
ing inammatory response has detrimental consequences to the host, espe-
cially when considering the eye. Understanding the sequential expression of
chemokines relative to ocular HSV-1 infection is pertinent to the development
of a strategy that will ultimately control local inammation and the collateral
damage without rendering increased susceptibility to the host.
2
HSV-1 Infection of the Eye
2.1
Innate Immune Response to Ocular HSV-1 Infection
After initial infection of the virus into the cornea, an innate immune re-
sponse is triggered to clear the pathogen. Toll-like receptors (TLR), a family
of pattern-recognition molecules, are known to respond to pathogens and
serve as early warning molecules that induce the expression of proinamma-
tory molecules [5]. Of the twelve TLR subtypes found in the mouse, TLR2 and
TLR9 are expressed by corneal epithelium [36]. HSV-1 stimulates TLR2 by
unknown means resulting in the activation of nuclear factor (NF)-B and pro-
duction of interleukin (IL)-6 [46]. HSV-1 which contains CpG motifs [106] is
recognized by TLR9, resulting in the expression of type I interferon (IFN) [44].
In addition to the production of type I IFNs, the infected resident cells of the
cornea as well as neighboring cells (most probably through TLR signaling and
NF-B activation) are known to release inammatory cytokines including IL-
1, IL-6, and TNF- [34, 88]. The absence or hindrance of these cytokines
has been linked to a signicant reduction in the incidence of HSK [6, 22, 101].
It is thought that IL-1 leads to the induction of IL-6 by resident corneal
cells [6] that, in turn, elicit production of macrophage inammatory protein-
1 (CCL3) and -2 (CXCL2) [22] ultimately recruiting PMNs into the infected
tissue. PMNs inltrate the stroma underlying the infected epithelial cells, con-
tributing to clearance of the virus and limiting viral dissemination within 24 h
postinfection [6, 96, 97]. PMNs are thought to be a rich source of inducible
nitric oxide synthase (iNOS) and TNF- [13], the latter of which upregu-
lates intercellular adhesion molecule (ICAM)-1 expression [69] facilitating
the adherence of leukocytes to the endothelium [89]. The administration of
monoclonal antibody to ICAM-1 [15] or use of ICAM-1-decient mice [67]
has not been found to diminish the inltration of cells or the clinical course
of herpetic disease following corneal infection. However, ICAM-1 does play
a key role in preventing herpetic encephalitis [15, 67], suggesting pathways
independent of ICAM-1 expression are involved initially in the recruitment
of cells into the cornea, whereas controlling virus spread in the central ner-
vous system involves ICAM-1 expression. After the initial inltration of neu-
trophils, macrophages and NK cells inltrate the area but PMNs remain the
predominant cell type residing in the inamed cornea up to the rst 96 h
postinfection [93].
2.2
Chemokine Expression During the Innate Immune Response
to Ocular HSV-1 Infection
Evidence for the expression of chemokines in the cornea following HSV-1 in-
fection was rst described using endpoint polymerase chain reaction (PCR)
in which KC (CXCL1), CXCL2, IFN--inducible protein 10 (CXCL10), mono-
cyte chemoattractant protein-1 (CCL2), MIP-1 (CCL4), and regulated upon
activation, normal T cell expressed (CCL5) were observed [90]. While trauma
to the cornea in the form of scarication induced the expression, continued
expression of CCL2, CCL5, and CXCL10 were noted out to 72 h postinfection,
whereas other chemokine messenger (m)RNA levels precipitously dropped in
both BALB/c and outbred ICR mice [14, 90]. Of the chemokines noted above,
CXCL1 and CXCL2 specically target neutrophils principally through the re-
ceptor CXCR2 [11, 82, 99, 104]. Neutralization of CXCL2 with antibody leads
to a reduction in polymorphonuclear neutrophil (PMN) inltration into the
cornea [54, 104]. Likewise, CXCR2 knockout mice infected with HSV-1 show
a minimal inltration of PMNs into the cornea [3]. Even with a reduction in
PMN inux, HSK still develops in the CXCR2-decient mice, which is thought
to be due to an increase in IL-6 expression driven by elevated virus titers
ultimately facilitating angiogenesis [3]. Although evidence suggests IL-6 can
drive neovascularization through vascular endothelial growth factor (VEGF)
in the cornea, the kinetics of expression of VEGF during the infectious process
in this model suggests other dynamics are involved including T cells that are
known to contribute to HSK [16, 83] and are a source of VEGF [63].
Whereas CXCL2 is thought to be induced by IL-6 [57], another CXC
chemokine, CXCL10, has been found to be the only chemokine that is consti-
tutively expressed in the cornea as determined by PCR [14, 90] and enzyme-
linked immunosorbent assay (ELISA) [10]. CXCL10 levels rapidly rise in the
cornea following HSV-1 infection, and neutralization of the chemokine dra-
matically reduces corneal edema and inltrating cells [10]. The lone receptor
for CXCL10 is CXCR3 expressed by NK cells, macrophages, dendritic cells
(DCs), and activated T cells [21, 23, 47, 77, 94]. However, CXCR3 knock-
out mice ocularly infected with HSV-1 show a transient suppression of PMN
(Gr-1+ CD11b+ Mac-3 ) recruitment into the cornea (D.J.J. Carr, unpublished
observation), calling into question the role of CXCR3 and its ligands in PMN
recruitment. However, other studies at different anatomical sites have de-
scribed PMN inltration as a result of CXCL10 expression [8, 105]. It is tempt-
ing to speculate that CXCL10 may upregulate CD11a on PMNs enhancing the
adhesion to the endothelium as has been reported for Th1 cells [2] facilitating
diapedesis into the stroma of the cornea. However, formal proof of this notion
requires additional studies.
Of the CC chemokine ligands expressed during ocular HSV-1 infection,
CCL2 is strongly expressed throughout the initial course of acute infection as
measured by PCR [14, 90]. The role of CCL2 in the development of HSK may
be peripheral to its effects on the recruitment of leukocytes into the cornea,
since the administration of neutralizing antibody to CCL2 has no effect on the
incidence of HSK in HSV-1-infected mice [99]. In contrast, the administration
of anti-CCL3 antibody signicantly reduces the severity of corneal opacity
[99]. The kinetics of CCL3 expression suggest it is not a stimulus for the
recruitment of leukocytes into the cornea until 710 days postinfection, a time
that seems to correlate with the onset of HSK [99]. Consistent with this nding,
mice decient in CCL3 expression reportedly show little cellular inltration in
the cornea throughout the time course of infection with low to undetectable
levels of T helper (Th)1 cytokines including IL-2 and IFN- [98] normally
found during acute ocular infection [93]. Ironically, the CCL3 knockout mice
clear the virus at the same time as wildtype control animals [99], which calls
into question the mechanism of virus clearance. Since there is apparently
little leukocyte inltration, including PMNs, that is known to control HSV-1

replication in the eye [97]with a paucity of CD4+ T cells or IFN- present
as well [99]it is puzzling what mechanism(s) controls the virus.
Similar to CCL2, CCL5 is expressed throughout the course of acute
HSV-1 infection [14]. CCL5, operating through its receptor CCR5, is a strong
chemoattractant for T cells and NK cells [53, 80] but also inuences PMN
recruitment [71]. It is interesting to note that while HSV-1 tends to subvert
immune activation, CCL5 is induced by HSV-1 through NF-B and IFN
regulatory factor 3 pathways [56].
The plethora of chemokines and proinammatory cytokines produced in
the cornea during the innate immune response (i.e., 05 days postinfection)
may be generated from several sources. With the exception of CXCL10, the
chemokines CXCL1, CXCL2, CXCL9, CCL3, and CCL5 are not constitutively
expressed in the cornea (Fig. 1). Analysis by confocal microscopy has found the
endothelial layer of the cornea expresses very modest amounts of the CXCL10
in uninfected mice (D.J.J. Carr, unpublished observation). Consistent with pre-
vious results [90], scarication of the cornea (a process typically employed
Fig. 1 Expression of inammatory cytokines and chemokines in the cornea of HSV-

1-infected mice. C57BL/6 female mice (n=6/timepoint) were left alone (basal) or
scaried (0) and infected with HSV-1 (McKrae strain), 1,000 plaque-forming units
(pfu)/eye. The mice were euthanized 2436 h postinfection, perfused, and the cornea
was removed and homogenized in a buffer containing a cocktail of protease inhibitors.
The supernatant was claried (10,000g, 5 min) and assayed for cytokine/chemokine
content by ELISA. Bars represents meanSEM for each analyte under measure. CCL2
basal levels were not measured
to infect mice) alone elicits a rise in CCL3, CCL5, and CXCL10 expression
(Fig. 1). Following infection, CXCL1, CCL2, CCL5, CXCL9, and CXCL10 are
induced or upregulated within 36 h. Analysis of CCL5 and CXCL10 expression
by confocal microscopy show two different patterns of expression. CCL5 is
expressed in the epithelial layers of the eye colocalizing with HSV-1 antigen
as well as within the stroma of the cornea (D.J.J. Carr, J. Ash, T. Lane, and
W. Kuziel, submitted). By comparison, CXCL10 expression chiey colocalizes
with HSV-1 antigen expression in the epithelial layers of the cornea with punc-
tate staining in the endothelium (D.J.J. Carr, unpublished observation). The
expression prole of CCL5 and CXCL10 suggests the resident population gen-
erates most if not all of the CXCL10 within the rst 24 h postinfection, whereas
CCL5 is produced principally by resident cells but may also be provided by the
inltrating PMNs that are found within the stroma 24 h postinfection (D.J.J.
Carr, J. Ash, T. Lane, and W. Kuziel, submitted). It is likely that as the infection
Fig. 2 Chemokine expression in the cornea following HSV-1 infection. Three

different scenarios can operate in the production of chemokines within the cornea
following ocular HSV-1 infection. In 1a, HSV-1 DNA CpG motifs bind to the
intracellular toll-like receptor (TLR)9 eliciting the production of CXCL10 through
NF-B activation. In 1b, HSV-1 enters the epithelial cell and, following transcription,
induces the production of IFN-, which induces CXCL10 production. In 2, HSV-1
activation of NF-B stimulates IL-1 synthesis leading to IL-6 production, resulting
in CXCL2 and CCL3 expression. These chemokines draw in PMNs and T cells. PMNs
can secrete CXCL9 and CXCL10, which can recruit additional leukocytes including
macrophages, DCs, NK cells, and T cells
spreads over the next several hours, chemokines generated including CCL2,
CCL5, CXCL1, CXCL2, CXCL9, and CXCL10 are produced by multiple sources
including the resident broblasts, epithelial, and endothelial cells as well as
inltrating PMNs, macrophages, NK cells, and DCs [11, 25, 82, 87, 100]. Col-
lectively, the initial cascade of chemokine expression is complex but may be
divided into two principal pathways involving CXCL10 and IL-6 (Fig. 2).
The delayed expression of CCL3 in the cornea is associated with a sec-
ondary wave of PMNs and some T cells into the stroma (day 10 postinfection)
[99]. Since CCL3 targets monocytes, T cells, NK cells, basophils, eosinophils,
DCs, and hematopoietic progenitors [11, 12, 82], it is currently unknown
what events transpire to recruit the subsequent wave of cells. However, CCL3
is central to the effect since neutralizing this chemokine with antibody or
suppressing expression with IL-10 reduces leukocyte recruitment into the
cornea [99].
2.3
Adaptive Immune Response to Ocular HSV-1 Infection
Following the innate response to infection, preferential recruitment of Th1

CD4+ T cells into the cornea is observed [35, 65]. Although it is currently un-
known why there is a preferential recruitment of CD4+ T cells into the cornea
of HSV-1-infected mice, the expression of CXCR3 and CCR5 on activated
T cells and the presence of CCL5 and CXCL9 in the cornea may inuence the
recruitment process [80, 81, 102]. The presence of CD4+ T cells is crucial in
controlling local virus replication and spread [7, 26] as well as the development
of HSK [25, 58]. However, bystander activation of CD4+ T cells in addition to
virus antigen stimulation may also contribute to HSK development [24]. The
continued expression of chemokines including CXCL2, CCL2, CCL3, CCL4,
CCL5, and CXCL10 in the cornea would also provide the maintenance of
leukocytes in the tissue recruited from the periphery and facilitate collateral
damage to the cornea stroma [84]. Collectively, chemokines are instrumental
in the initial trafcking of cells into the infected anterior segment of the eye
as well as the development of HSK. Blocking their expression could preserve
the visual axis, assuming local virus replication is controlled. A summary of
chemokines expressed during the acute HSV-1 ocular infection is found in
Table 1.
2.4
HSV-1 Latency in the Trigeminal Ganglion
After the successful infection of the cornea by HSV-1, a series of events occurs
that can lead to a stable latent neuronal infection in the trigeminal ganglion
Table 1 Chemokine expression in the cornea during acute HSV-1 infection
Group Name Detection Reference(s)
CXC CXCL1 RT-PCR and ELISA 90, Fig. 1

CXCL2 RT-PCR and ELISA 3, 6, 10, 22, 54, 90, 98, 104
CXCL9 ELISA 10, Fig. 1
CXCL10 RT-PCR and ELISA 10, 14, 90, Fig. 1
CC CCL2 RT-PCR and ELISA 14, 90, 98, 99, Fig. 1
CCL3 RT-PCR and ELISA 10, 22, 90, 98, 99, Fig. 1
CCL5 RT-PCR and ELISA 10, 14, 90, Fig. 1
(TG) within 12 weeks postinfection [39, 41, 49]. Following an initial round
of replication in the corneal epithelium, the virus is able to enhance its abil-
ity to access the axonal termini (via mechanisms that are not understood),
and through retrograde axonal transport it enters the neuronal cell bodies
in which another stage of lytic replication begins [49, 70]. After this brief
replication cycle in the neuronal cell bodies, the lytic cycle genes are re-
pressed and latency is established with minimal viral gene expression [49].
Infectious HSV-1 can consistently be detected in the TG out to approximately
10 days postinfection [12]. By day 30 postinfection, latency is established
as dened by the lack of detectable infectious virions [12]. Even though
infectious virions are not readily detected during latency, HSV-1 latency-
associated transcripts (LATs) can be detected in the TG, and an associated
local immune response is evident [28, 49]. With latency established, the im-
mune system continually surveys the area with CD8+ T cells as the principal
cell type that is thought to prevent reactivation [39]. Along these lines, CD8+
T cells are thought to control the infection through noncytolytic mechanisms
using cytokines such as IFN- and TNF- with minimal destruction to neu-
rons [38, 50, 51, 95].
During latent infection, real time (RT)-PCR detection of CXCR3 and CCR5
expression has been reported [12]. Although unproven, it is likely these
chemokine receptors are found on the CD8+ T cells present in the TG during
latency [29, 38]. Although ligands for CXCR3 including CXCL9 and CXCL10
have not been evaluated during latency, one ligand for CCR5, CCL5, has been
detected [28]. Exposing latently infected mice to the potent antiviral com-
pound acyclovir has been found to reduce CCL5 expression in the TG. Yet, the
continued presence of CD8 cells suggests additional signals provide a stimulus
for retainment of these effector cells within the tissue [29].
2.5
Reactivation of HSV-1
Due to a variety of environmental cues including UV light, stress, and im-

munosuppression, the virus is able to reactivate in the latently infected neu-
rons of the TG. Through antegrade transport, the virus can again be detected
in the corneal epithelium and stroma [68, 70]. The reactivation cycle can be
repeated eliciting chronic and episodic immune activation, which leads to
progressive scarring of the cornea resulting in decreased vision, glaucoma,
iritis, cataract, and necrotizing retinitis [70]. While there is experimental evi-
dence to suggest regulatory T cells may control ocular pathogenesis [91], how
these cells impact on local chemokine expression is not understood.
3
HSV-2 Infection of the Genitalia
3.1
Immune Response to Genital HSV-2 Infection
During initial infection of the mucosa of the vagina with HSV-2, the virus
begins to replicate in the epithelium, typically restricted to the epidermis or
cervicovaginal epithelium [43]. The initial host response to infection includes
the induction of type I IFNs (i.e., IFN- species) through TLR9 recognition of
HSV-2 CpG motifs [52]. The IFN-responsive pathway, double-stranded RNA-
dependent protein kinase but not 2 ,5 -oligoadenylate synthetases is essential
for resistance to infection, as mice decient in this pathway are highly suscep-
tible to HSV-2-mediated mortality (D.J.J. Carr, L. Tomanek, R.H. Silverman,
and B.R.G. Williams, manuscript in preparation). In addition to type I IFN
production, IL-12, 1L-15, IL-18, NK cells, and PMNs are important rst lines
of defense against HSV-2 replication and spread [1, 31, 59]. Current evidence
suggests the resident populations of Langerhans cells [19] do not trafc to
the inguinal/iliac lymph nodes with most migrating cells consisting of B lym-
phocytes [40]. T lymphocytes including T cells are essential components
of the adaptive immune response in controlling genital infection with HSV-
2 [55, 60, 66, 73]. CD4+ T cells produce the majority of IFN- in response to
genital HSV-2 infection [32, 61]. Neutralization of IFN- leads to an increase
in virus titer and a decrease in T cell recruitment into the vaginal tissue [61,
73]. B cell production of antibody is initiated in the draining lymph nodes and
appears to have only a modest impact on HSV-2 titers, suggesting a limited
role for B lymphocytes in the control of genital HSV-2 infection [17, 62, 75].
Manifestations of genital herpes include macules, papules, and vesicles re-
sulting in the development of ulcers in the genital region [33]. Due to these
ulcerations, other pathogens are able to enter into the vaginal mucosa. Recent
studies have shown that patients who are infected with HSV-2 have a higher
risk of contracting HIV-1 than patients who are HSV-2 seronegative with
a two- to threefold increase in susceptibility [79].
3.2
Chemokines and HSV-2
The recruitment of leukocytes into the vaginal tissue following HSV-2 infec-
tion appears to include IFN- induction of the adhesion molecules ICAM-1
and vascular cell adhesion molecule 1 [76] since neutralizing IFN- dimin-
ishes lymphocyte inltration into the infected tissue [74]. The expression of
IFN- has also been associated with CCL5 production [30] found in the vagina
following HSV-2 infection [4, 33]. The role of CCL5 expression in recruiting
leukocytes into the infected tissue has not been described. However, plasmid
DNA containing CCL5 has been found to enhance survival of HSV-2-infected
mice [85]. Manipulating local expression of selective chemokines including
CXCL2 and CCL3 using plasmid DNA suggests these chemokines may also
play a signicant role in protection for the host during genital virus infection
by facilitating CD4+ T cell immunity and elevating IFN- production by NK
cells [20]. However, there are unresolved questions that remain as to those
chemokines that initiate the inammatory cascade as well as those that are
critical for resistance to genital HSV-2.
4
Perspective
Chemokines are a signicant group of soluble factors that contribute in the

clearance of HSV-1 and HSV-2 pathogens from the host. Although necessary
for an optimal immune response to the virus, chemokines initiate a frank
inammatory response that can result in a signicant detrimental outcome
to the host as it pertains to preservation of the visual axis. This chapter high-
lights the role of chemokines as they relate to the innate and adaptive immune
response following ocular HSV-1 infection. Evidence suggests that curtailing
expression of selective chemokines during HSV-1 infection of the eye may
favor preservation of sight without consequences to controlling virus repli-
cation and spread. This observation suggests that while many chemokines
are redundant in function and/or promiscuous in binding multiple receptors,
selectivity in tissue expression of chemokines and targeting specic effector
cells by those chemokines expressed in a given tissue may ultimately dictate
the inammatory response of the host and outcome of the infection. Under-
standing this process will prove benecial in developing antiinammatory
therapies for individuals experiencing chronic HSV reactivation.
Acknowledgements The work was supported by a USPHS NIH grant, EY015566 and
a Jules and Doris Stern RPB Research Professorship to DJJC.
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CTMI (2006) 303:6795

Influence of Proinflammatory Cytokines

and Chemokines on the Neuropathogenesis
of Oncornavirus and Immunosuppressive
Lentivirus Infections
K. E. Peterson1 (u) B. Chesebro2
1 Dept. of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State
University, Baton Rouge, LA 70803, USA
kpeterson@vetmed.lsu.edu
2 Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIAID,
Hamilton, MT 59840, USA
1 Retrovirus Infections of the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

1.1 Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
1.2 Diversity of Retrovirus-Induced Neurological Diseases . . . . . . . . . . . . . . 69
1.3 Diversity of Retrovirus-Induced Pathogenic Mechanisms . . . . . . . . . . . . 71
2 Fr98 Polytropic Retrovirus Model of Neuropathogenesis . . . . . . . . . . . . 72

2.1 Use of a Mouse Model to Study Retroviral Neuropathogenesis . . . . . . . . . 72
2.2 FMCF98 and Fr98 Polytropic Murine Leukemia Viruses . . . . . . . . . . . . . 72
2.3 Mapping of Neurovirulence Determinants in the Fr98 Genome . . . . . . . . 74
2.4 Contribution of Virus Burden to Pathogenesis . . . . . . . . . . . . . . . . . . . . 75
3 Cytokines and Chemokines in Fr98-Induced Neuropathogenesis . . . . . . 75

3.1 Analysis of Cytokine and Chemokine Gene Expression . . . . . . . . . . . . . 75
3.2 Kinetics of Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.3 Studies with Knockout Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
4 Cytokines and Chemokines

in Immunosuppressive Lentivirus Pathogenesis . . . . . . . . . . . . . . . . . . 79
4.1 Correlation Between Gene Expression and Neurological Disease . . . . . . . 79
4.2 Effect of HIV Proteins on Cytokine and Chemokine Induction . . . . . . . . 80
5 Potential Effects of Chemokines

During Retrovirus Infection of the Brain . . . . . . . . . . . . . . . . . . . . . . . 80
5.1 Activation and Recruitment of Microglia and Macrophages . . . . . . . . . . 80
5.2 Lymphocyte Recruitment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
5.3 Neuronal Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
5.4 Direct Stimulation of Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
5.5 Alteration or Inhibition of Neuroprogenitor Stem Cell Migration . . . . . . 83
5.6 Astrocyte Activation and Support Functions . . . . . . . . . . . . . . . . . . . . . 84
5.7 Retrovirus Entry and Spread in the CNS . . . . . . . . . . . . . . . . . . . . . . . . 85
5.8 Retroviral Protein Stimulation of Chemokine Receptors . . . . . . . . . . . . . 85
68 K. E. Peterson B. Chesebro
6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Abstract Retroviral infection of the CNS can lead to severe debilitating neurologi-
cal diseases in humans and other animals. Four general types of pathogenic effects
with various retroviruses have been observed including: hemorrhage (TR1.3), spongi-
form encephalopathy (CasBrE, FrCasE, PVC211, NT40, Mol-ts1), demyelination with
inammatory lesions (HTLV-1, visna, CAEV), and encephalopathy with gliosis and
proinammatory chemokines and cytokines, usually with microglial giant cells and
nodules [human immunodeciency virus (HIV), feline immunodeciency virus (FIV),
simian immunodeciency virus (SIV), Fr98]. This review focuses on this fourth group
of retroviruses. In this latter group, proinammatory cytokine and chemokine upreg-
ulation accompanies the disease process, and may inuence pathogenesis by direct
effects on resident CNS cells. The review rst discusses the Fr98 murine polytropic
virus system with particular reference to the roles of cytokines and chemokines in the
pathogenic process. The Fr98 data are then compared and contrasted to the cytokine
and chemokine data in the lentivirus systems, HIV, SIV, and FIV. Finally, various mech-
anisms are presented by which tumor necrosis factor (TNF) and several chemokines
may alter the pathogenesis of retrovirus infection of the CNS.
Abbreviations
CAEV Caprine arthritis encephalitis virus
CNS Central nervous system
EIAV Equine infectious anemia virus
GFAP Glial brillary acidic protein
FIV Feline immunodeciency virus
FIV-E FIV encephalitis
HAD HIV-associated dementia
HIV Human immunodeciency virus
HTLV Human T cell leukemia virus
IP Intraperitoneal
LPS Lipopolysaccharide
Mol-ts1 Moloney Ts1
NPSC Neuroprogenitor stem cells
SIV Simian immunodeciency virus
SIV-E SIV encephalitis
Inuence of Proinammatory Cytokines and Chemokines 69
1
Retrovirus Infections of the CNS
1.1
Historical Perspective
In the initial years of the study of retroviruses the main emphasis was on the
potential of these agents to induce neoplastic transformation of various tis-
sues, especially muscle cells, broblasts, mammary glands, and bone marrow-
derived hematopoietic cells. Subsequently non-neoplastic pathogenic effects
of retroviruses were described, including anemia, arthritis, glomerulonephri-
tis, immunodeciencies, osteopetrosis, and neurological disorders. The stud-
ies of visna virus infection in sheep by Sigurdsson and colleagues in the 1950s
demonstrated a slow CNS disease lasting several years (Sigurdsson et al. 1957,
1960). This long time course led ultimately to the name lentiviruses for
the subgroupincluding visnaof retroviruses with complex genomes. In
the 1970s, Gardner and colleagues described a murine retrovirus from the
oncornavirus subgroup (with simple genomes) which induced a slow neuro-
logical disease (Gardner et al. 1973). Since that time a large variety of other
retroviruses from both subfamilies capable of inducing neurological disease
in a variety of species including humans have been described (Table 1).
1.2
Diversity of Retrovirus-Induced Neurological Diseases
Retroviruses can cause several different types of neurological disease. Based

on pathology and pathogenic effects, these can be divided into four groups
(Table 1). These include: hemorrhage (TR1.3) (Park et al. 1993, 1994); spongi-
form encephalopathy (CasBrE, FrCasE, PVC211, NT40, Moloney-ts1) (Czub
et al. 1995; Gardner 1988; Hoffman et al. 1992; Kai and Furuta 1984; Wong et
al. 1985); demyelination with inammatory lesions (HTLV-1, visna, CAEV)
(Araujo and Hall 2004; Haase 1986; Jacobson 2002; Oaks et al. 2004); and
encephalopathy associated with microgliosis, astrogliosis usually with mi-
croglial nodules, and giant cells and variable mononuclear cell inltrates [hu-
man immunodeciency virus (HIV), feline immunodeciency virus (FIV),
simian immunodeciency virus (SIV), Fr98] (Glass et al. 1993, 1995; Kolson
et al. 1998; Lackner et al. 1991; Portis et al. 1995; Robertson et al. 1997).
Some viruses, such as SIV, appear to be able to induce more than one type of
pathogenic process, depending on the viral strain used and/or the extent of
immunosuppression (Lackner et al. 1991). In most models, infection of neu-
rons is not observed and the mechanism of neuronal dysfunction is assumed
to be indirect (Patrick et al. 2002; Portis 2001). In some systems, infection of
Table 1 Diversity of central nervous system pathological processes induced by retro-

viruses
Pathogenic effects Virus Species
Hemorrhage TR1.3 Mouse

Spongiform encephalopathy CasBrE Mouse
FrCasE Mouse
Mol-ts1 Mouse
NT40 Rat
PVC211 Rat
TR1.3a Mouse
Demyelination with extensive mononuclear CAEV Goat
cell inltrates EIAVb Horse
HERVc Human
HTLV-1 Human
HTLV-2d Human
Visna Sheep
Encephalopathy, gliosis, and upregulated FIV Cat
proinammatory cytokines and chemokines, Fr98 Mouse
with variable mononuclear cell inltratese HIV Human
SIV Monkey
a TR1.3 has been reported to induce both hemorrhage and spongiform encephalopa-
thy (Murphy et al. 2004)
b Equine infectious anemia virus (EIAV) sporadically induces a brain inammatory
disease but demyelination was not noted (Oaks et al. 2004)

c HERVs, human endogenous retroviruses, may be involved in multiple sclerosis
(Christensen 2005)
d HTLV-2 is believed to be associated with some rare cases of demyelinating
encephalitis (Araujo and Hall 2004)

e The extent of mononuclear cell inltration in the lentiviral models (HIV, SIV, FIV)
can vary considerably from prominent to minimal (Anthony et al. 2005; Gardner and
Dandekar 1995; Kolson et al. 1998; Lackner et al. 1991). In the Fr98 neonatal mouse
model mononuclear inltration is minimal (Peterson et al. 2001; Robertson et al. 1997)
neurons has been noted, but this does not appear to account for the disease,
because the infected cells are not present in the damaged areas (Lynch et al.
1991). Despite the diverse pathology, most retroviruses infect the same major
target cells of microglia and macrophages in the brain (Patrick et al. 2002;
Portis 2001). Brain capillary endothelial cells are often extensively infected in
many murine models and may be in some primate systems (SIV). To a lesser
extent, infection of astrocytes (Liu et al. 2004) and oligodendrocytes (Robert-
son et al. 1997) has also been observed with some retroviruses, but these types
of infected cells may nevertheless play important roles in the pathogenesis.
1.3
Diversity of Retrovirus-Induced Pathogenic Mechanisms
Mechanisms of pathogenesis are not well understood in most of these retro-

viral systems. The simplest model appears to be the TR1.3 murine retrovirus,
where infection of brain capillary endothelial cells leads to cell fusion and
damaged and leaky blood vessels (Park et al. 1993, 1994). However, more
recent data indicate that TR1.3 can also induce spongiform encephalopathy
(Murphy et al. 2004). In some rodent spongiform encephalopathy models,
such as PVC211, there is evidence that endothelial cell infection is important
to pathogenesis probably by facilitating viral entry into the CNS (Hoffman et
al. 1992; Kai and Furuta 1984). In other rodent spongiform encephalopathy
models, such as FrCasE, Cas-Br-E, and Moloney ts1, there is growing evidence
that viral envelope protein aggregation may lead to an endoplasmic reticulum
stress response, which may in turn damage certain glial cells (Dimcheff et al.
2003, 2004; Kim et al. 2004a; Liu et al. 2004). However, the explanation for
the very restricted spatial distribution of lesions in the face of widespread
virus infection remains unclear. The demyelinating inammatory viruses all
appear to induce a very strong host inammatory response involving lym-
phocytes and macrophages, and the cytokines and chemokines produced by
inltrating cells are believed to be important in demyelination and neuronal
damage (Araujo and Hall 2004; Haase 1986; Jacobson 2002).
The immunodeciency inducing lentiviruses, HIV, SIV, and FIV, as well
as Fr98, a polytropic murine retrovirus, all induce a severe clinical CNS dis-
ease with minimal morphological neuronal damage and pathology. Multiple
histopathological changes have been associated with HIV-associated demen-
tia (HAD), including microglia nodules, astrogliosis, microgliosis, neuronal
apoptosis, myelin pallor, and multinucleated giant cell formation (Anthony
et al. 2005; Glass et al. 1995; Kolson et al. 1998). Many of these alterations
have also been seen in animal models (Johnston et al. 2002; Lackner et al.
1991; Portis et al. 1995; Power et al. 2004; Robertson et al. 1997; Williams
et al. 2001). The main pathological change-associated clinical neurological
disease is the increased presence of activated macrophages and microglia in
the brain (Anthony et al. 2005; Glass et al. 1995; Portis et al. 1995; Robertson
et al. 1997; Williams et al. 2001). Virus infection does not generally induce
extensive inltration of inammatory cells from outside the CNS (Anthony
et al. 2005; Glass et al. 1995; Power and Johnson 1995, 2001; Robertson et al.
1997); however, there is evidence for a role for proinammatory cytokines
and chemokines in the pathogenesis of these viruses. The low level of in-
ammatory inltrates in all of these models is likely related to the presence
of severe immunosuppression induced by the virus (HIV, SIV, FIV) or the
neonatal state of the host (Fr98 infection). This review will focus on the role
of proinammatory cytokines and chemokines in the neuropathogenesis of
this group of retroviruses with emphasis on the Fr98 mouse model and the
use of knockout mice to study the contribution of cytokines and chemokines
to retroviral pathogenesis.
2
Fr98 Polytropic Retrovirus Model of Neuropathogenesis
2.1
Use of a Mouse Model to Study Retroviral Neuropathogenesis
In human patients, it is difcult to distinguish cause versus effect in correlative

studies of gene upregulation. Other animal models of retrovirus infection, in-
cluding SIV infection of macaques and FIV infection of felines have this same
limitation. Mouse models of retrovirus infection offer the unique advantage
over other retrovirus systems in the ability to use knockout and transgenic
animals to denitively determine whether specic proteins are necessary for
disease development. The mouse model of polytropic retrovirus infection
also allows the comparison between non-neurovirulent and neurovirulent
retrovirus infections (Peterson et al. 2001, 2004b; Robertson et al. 1997). This
permits the determination of whether a host response is specic to pathogene-
sis or is a general response to retrovirus infection. Additionally, the availability
of inbred mouse strains, the short gestation period of mice, and the short in-
cubation time of neurological disease induction in mice compared to humans
or non-human primates allows for the kinetic and statistical analysis of the
host response genes.
2.2
FMCF98 and Fr98 Polytropic Murine Leukemia Viruses
In 1990, during studies of retrovirus-induced leukemia using Friend murine

leukemia virus (MuLV) inoculation of neonatal mice, a novel retrovirus was
isolated which appeared to cause both a neurological disease and leukemia
(Buller et al. 1990). After biological and molecular cloning, the causative
virus, FMCF98, was found to have a polytropic host range, as it was capable
of infecting cell lines from mice as well as from a variety of other species, and
it caused the typical mink cell foci (MCF) associated with other polytropic
mouse retroviruses (Portis et al. 1995). After intraperitoneal (IP) infection
of susceptible mouse strains the infection expanded rst in hematopoietic
cells of bone marrow and spleen and then progressed via blood to the brain.
Table 2 Polytropic retroviruses
Virus Viral genome CNS disease Virus Upregulation

load of cytokines
and
chemokines
FMCF98 Yes (624 weeks) ++ Not tested

FB29 No +++++ No
Fr98 Yes (23 weeks) +++ Higha
Fr54 No ++ No
SE Yes (38 weeks) +++ Higha
BE Yes (38 weeks) +++ Higha
EC Yes (38 weeks) ++ Lowb
a Genes upregulated: Tnf, Tnf, IL-1, Ccl2, Ccl3, Ccl4, Ccl5, Cxcl10
b Expression of cytokine and chemokine mRNA not statistically different as compared
to mock-infected controls in whole brain; however, some cytokines, such as TNF-,
are upregulated in the middle region of the brain including the hippocampus,
thalamus, and hypothalamus
The neurological disease consisted of ataxia, hind limb weakness, tremors,

seizures, and death occurred in nearly 80% of mice by 6 months. In genetic
studies, IRW mice were highly susceptible and C57BL/10 were highly resistant
and two uncharacterized host genes appeared to account for most of this
resistance (Buller et al. 1990). Because studies of a disease taking 68 months
were difcult, a virus with a more rapid phenotype was derived by cloning the
envelope gene of FMCF98 into the backbone of a rapidly replicating retrovirus,
FB29 (Table 2) (Portis et al. 1995). By 2030 days postinfection with this virus,
Fr98, mice had the same neurological symptoms and pathology as produced
by FMCF98. Within the brain, endothelial cells and microglia were the main
target cells of Fr98, although rare infection of oligodendrocytes was also
detected (Robertson et al. 1997). The brain pathology consisted of prominent
reactive astrogliosis and widespread white matter microglial infection with
microgliosis and occasional microglial giant cells and nodules, and minimal
vacuolation (Portis et al. 1995). Neuronal cell death was not a prominent
feature (Portis et al. 1995).
2.3
Mapping of Neurovirulence Determinants in the Fr98 Genome
Since neurological disease was not induced by most murine retroviruses, it was
of interest to determine which regions of the Fr98 genome were important for
this brain disease. Fr98 was compared to Fr54, a non-neurovirulent chimeric
polytropic retrovirus with a similar structure (Hasenkrug et al. 1996). Fr54
contained the envelope (env) gene of FMCF54 inserted into FB29, and dif-
fered from Fr98 by several amino acid residues in the polymerase (pol) and env
genes. Fr54 was able to infect the same cell types in the brain as Fr98; however,
no neurological symptoms were observed. The level of Fr54 virus infection was
slightly lower than that of Fr98, but there was no clear difference in the neu-
ropathology (Hasenkrug et al. 1996; Robertson et al. 1997). Thus the pathology
appeared to be due to retroviral infection, independent of the disease induc-
tion, suggesting perhaps that the disease symptoms and death were due to bio-
chemical abnormalities not represented by obvious morphological changes.
In order to determine which regions of the Fr98 genome were responsi-
ble for induction of the neurological disease, several chimeric recombinant
viruses using sequences of either Fr98 or Fr54 were studied (Hasenkrug et
al. 1996; Peterson et al. 2004b). These data indicated that there were two
non-overlapping regions of the Fr98 genome which inuenced neuroviru-
lence, one from SphI to EcoRI (SE), and the other from EcoRI to ClaI (EC)
(Table 2). Both these chimeras induced disease slower than Fr98, suggesting
that they acted by different but complementary mechanisms. The EC region
contained only env sequences, whereas the SE region contained both pol and
env sequences. However, only env sequences were involved in neurovirulence
because a chimera with Fr98 sequences from BbsI to EcoRI (BE), containing
only env, was similar in virulence to SE (Peterson et al. 2004b) (Table 2). The
BE region contains 11 env amino acid differences between Fr98 and Fr54, and
current studies are investigating which residues are most important to dis-
ease induction (K. Peterson and B. Chesebro, in preparation). The EC region
contained 17 env amino acid differences, and mutagenesis studies identied
changes at two positions (residues 195 and 198) as being important for disease
induction (Poulsen et al. 1998).
The mechanisms of disease induced by these two env regions are still
unclear; however, some clues were provided by previous studies. Earlier data
indicated that the SE/BE region appeared to inuence the brain viral load
and the kinetics of replication, and for these chimeras a high viral load was
required for disease. In contrast, the EC region induced disease at a lower
viral load, and thus appeared to generate neurotoxicity which did not require
high virus replication (Poulsen et al. 1998).
2.4
Contribution of Virus Burden to Pathogenesis
A role for higher viral load inuencing disease was also shown in studies
using direct brain infection by intraventricular inoculation of virus-infected
neural stem cells (Poulsen et al. 1999). In these experiments virus infection
of brain endothelial cells was minimal, whereas infection of microglial cells
was greatly increased for all viruses tested. Interestingly, with this method
even the avirulent virus Fr54 was capable of inducing neurological disease,
albeit considerably more slowly than Fr98. These data indicated that all the
polytropic retroviruses studied had potential to induce neurological disease if
expressed in the brain at a high enough level. Perhaps the role of the SE/BE env
region is to elevate viral replication to these high levels even when the standard
route of IP infection is used. In contrast, since the EC chimera induced disease
even at lower viral loads, it would appear that the critical Fr98 amino acids
at positions 195 and 198 in this chimera have a higher potential for disease
induction than the comparable Fr54 residues.
3
Cytokines and Chemokines in Fr98-Induced Neuropathogenesis
3.1
Analysis of Cytokine and Chemokine Gene Expression
Fr98-induced neurological disease is not associated with the widespread

spongiform degeneration, intracerebral hemorrhaging, or extensive leuko-
cyte inltration associated with other neurovirulent murine retroviruses.
Instead, the primary pathology associated with Fr98 infection is gliosis with
increased presence of activated microglia, macrophages, and astrocytes in
the brain (Portis et al. 1995; Robertson et al. 1997). As the pathology between
Fr54 and Fr98 infection is similar (Portis et al. 1995; Robertson et al. 1997),
the pathogenic mechanism by which Fr98 induces disease does not appear to
cause distinctive morphological alterations. Possibly this pathogenesis acts
at a molecular level through the regulation of gene expression. Molecular
analysis of gene expression by RNAse protection assay conrmed the lack of
lymphocytic inltration in the brain with no detectable increase in messen-
ger (m)RNA for T cell- or B cell-specic genes or T cell-associated cytokines
or cytokine receptors in brain tissue from Fr98-infected mice compared to
mock-, Fr54-, or FB29-infected mice (Peterson et al. 2001). Additionally, no
signicant changes in gene expression were detected in the apoptosis-related
genes, Fas, Fasl, Fadd, Tradd, Bax, Bcl-X, Bcl-2 ,and Bcl-W. Fr98 pathogenesis
was associated with a heightened proinammatory response with increased

mRNA expression of TNF(TNFSF1A,TNF), TNFSF1B(TNF, lymphotoxin
A), IL1, CCL2(MCP-1), CCL3(MIP-1), CCL4(MIP-1), CCL5(RANTES),
CXCL1(MIP-2), and CXCL10(IP-10) in the brain tissue of Fr98-infected mice
(Peterson et al. 2001). Induction of cytokines and chemokines correlated
with neurovirulence, not virus presence, as no signicant mRNA upregula-
tion of these genes was observed in FB29- or Fr54-infected mice. Protein
levels of TNF(TNF), CCL2(MCP-1), CCL3(MIP-1), CCL4(MIP-1), and
CCL5(RANTES) were also increased in brain tissue from Fr98-infected mice
(Fig. 1), indicating a correlation between the upregulation of cytokine and
chemokine mRNA and protein expression. By in situ hybridization analysis,
CCL2(MCP-1) and CXCL10(IP-10) mRNA was expressed by astrocytes, while
CCL3(MIP-1), CCL4(MIP-1), and CCL5(RANTES) mRNA was expressed
by uninfected cells, possibly microglia or macrophages (Peterson et al. 2004a;
K. Peterson and B. Chesebro, unpublished observations).
Comparison of proinammatory cytokine and chemokine expression fol-
lowing infection of the chimeric polytropic retroviruses indicated a corre-
Fig. 1 Increased protein expression of TNF and chemokines in Fr98-infected mice.

Enzyme-linked immunosorbent assay (ELISA) kits specic for the appropriate murine
protein were purchased from R&D Systems (Minneapolis, MN). Brain tissue from
Fr98-, Fr54-, or mock-infected mice were removed at 14 days postinfection, snap frozen
in liquid nitrogen, and stored at 80C until use. The samples were homogenized in
0.1 M Tris pH 7.4, 0.15 M NaCl, 0.5% NP40 with complete protease inhibitor (Roche
Applied Science, Indianapolis, IN) to a nal concentration of 30% w/v. Samples were
incubated overnight at 4C, and 50 l per sample was added to duplicate wells for each
protein-specic ELISA. Data are presented as femtograms of the protein of interest
per milligram of brain tissue and are the mean +/ standard error of 48 samples
per group. A signicant increase (p<0.05) in protein levels of brain tissue from Fr98-
infected mice was observed for all proteins analyzed
lation between the SE/BE neurovirulent determinant of the Fr98 envelope

and the induction of cytokine and chemokine mRNA expression (Peterson
et al. 2001). Increased cytokine and chemokine expression was observed in
the brain tissue of SE and BE-infected mice (Peterson et al. 2001, 2004b).
In contrast, cytokine expression in EC-infected mice was more restricted,
with localized upregulation of tumor necrosis factor (TNF) in the middle
(hippocampus, midbrain, and thalamus) region of the brain (Peterson et al.
2001, 2004b). As Fr98, SE and BE replicate in the brain at a two- to threefold
higher level than EC or Fr54 (Table 2) (Hasenkrug et al. 1996; Peterson et al.
2004b; Robertson et al. 1997), the upregulation of cytokine and chemokine
expression may be due to the increased viral burden associated with these
viruses. Alternatively, specic envelope determinants may be necessary for
the induction of these proinammatory factors.
3.2
Kinetics of Gene Expression
The correlation between increased proinammatory cytokines and chemo-

kines and retrovirus-induced neuropathogenesis suggests that these proin-
ammatory factors may induce pathogenesis or be induced as a result of
pathogenic insult. One way to dissect the cause versus effect relationship
of these events is the analysis of the timing of upregulation of cytokines
and chemokines relative to disease onset. Kinetic analysis of gene expres-
sion is difcult in HIV, SIV, and FIV studies due to limitations in tissue
sampling as well as the variation in disease progression and the incidence
of disease. However, in the Fr98 mouse model, after infection with high
virus doses, 100% of the animals develop neurological disease within 14 to
16 days postinfection (Peterson et al. 2001; Poulsen et al. 1999). An increase
in mRNA expression of TNF, CCL2(MCP-1), CCL3(MIP-1), CCL4(MIP-1),
CCL5(RANTES), CXCL1(MIP-2), and CXCL10(IP-10) was detected in pre-
clinical mice, 34 days prior to the neurological disease, suggesting that these
cytokines and chemokines might contribute to disease induction (Peterson et
al. 2001). In contrast, increased expression of TNFSF1B(TNF, lymphotoxin
A) and interleukin (IL)-1 mRNA was only consistently found in Fr98-infected
mice with clinical disease, suggesting that these cytokines may be a response
to disease rather than a cause of pathogenesis. Interestingly, mRNA expres-
sion of two additional chemokines, CCL7(MCP-3) and CCL12(MCP-5), was
upregulated 3 to 4 days prior to clinical disease development, but returned
to basal levels at the time of clinical disease (Peterson et al. 2004a). Simi-
larly, CCL12(MCP-5)-positive cells were found in brain tissue of preclinical
mice, but were difcult to detect in mice with clinical disease. Although
CCL7(MCP-3) and CCL12(MCP-5) are not upregulated at the time of disease,

they could contribute to the early stages of disease development prior to the
onset of clinical disease.
3.3
Studies with Knockout Mice
Knockout mouse and antibody blocking studies indicated a role for specic
cytokines and chemokines in Fr98-mediated pathogenesis in the brain. Mice
decient in CCR2, the primary ligand for CCL2(MCP-1) and CCL12(MCP-5),
had reduced incidence and kinetics of neurological disease following Fr98
infection, indicating that CCR2 stimulation was contributing to pathogenesis
(Peterson et al. 2004a). Antibody blocking studies indicated that the CCR2
ligand CCL2(MCP-1), but not ligands CCL7(MCP-3) or CCL12(MCP-5), con-
tributed to Fr98-induced disease. Thus, CCL2(MCP-1) stimulation of CCR2
appears to be a mechanism of Fr98-mediated neuropathogenesis. In contrast,
CCR5 was not necessary for Fr98 pathogenesis as no decrease in neurovir-
ulence was observed in CCR5-decient mice, despite increased mRNA and
protein expression of the CCR5 ligands CCL3(MIP-1), CCL4(MIP-1), and
CCL5(RANTES) (Peterson et al. 2001, 2004a; Fig. 1).
In TNF-decient mice, the rate of Fr98-induced disease progression was
signicantly delayed, demonstrating that TNF contributed to neuropatho-
genesis (Peterson et al. 2004b). Interestingly, the role of TNF in pathogenesis
varied with different chimeric polytropic retroviruses. For example, the
chimeric virus BE induced high levels of TNF mRNA in the brain, whereas
the chimeric virus EC induced only localized production of TNF mRNA
in the middle (midbrain, hippocampus, and thalamus) region of the brain.
This correlation suggested that TNF deciency might inhibit BE-induced
disease and not EC-induced disease. However, paradoxically, the opposite
was observed. TNF deciency signicantly inhibited EC-induced clinical
disease, but had no detectable effect on BE-induced disease (Peterson et al.
2004b). This result demonstrated the danger of drawing conclusions strictly
from correlative data.
EC-induced upregulation of the microglia and macrophage marker F4/80
was not observed in TNF-decient mice, suggesting that TNF may contribute
to disease by the activation or recruitment of microglia or macrophages
(Peterson et al. 2004b). In contrast, TNF was not necessary for retrovirus-
induced activation of astrocytes as measured by expression of glial brillary
acidic protein (GFAP) mRNA. Further studies analyzing the responses of
retrovirus infection in knockout mice should provide valuable information in
regards to how cytokines and chemokines contribute to neuropathogenesis.
Increased TNF mRNA expression has also been associated with the spongi-
form degeneration and neurological disease induced by the FrCasE and Mol-
ts1 murine retrovirus infections (Askovic et al. 2001; Choe et al. 1998; Peterson
et al. 2004b). However, deciencies in TNF or TNFR1 did not alter the patho-
genesis of disease induced by FrCasE and Mol-ts1, respectively (Jolicoeur et
al. 2003; Peterson et al. 2004b).
4
Cytokines and Chemokines
in Immunosuppressive Lentivirus Pathogenesis
4.1
Correlation Between Gene Expression and Neurological Disease
Similar to the upregulation of cytokines and chemokines in the Fr98

model, several studies have demonstrated expression of proinammatory
cytokines and chemokines in HAD. Increased mRNA or protein expression of
CCL2(MCP-1), CCL3(MIP-1), CCL4(MIP-1), CCL5(RANTES), CXCL10(IP-
10), TNF(TNF ), interferon (IFN)-, and IL-1 have been observed in brain
tissue or cerebrospinal uid of HIV-infected patients with dementia (Conant
et al. 1998; Kolb et al. 1999; McManus et al. 2000; Schmidtmayerova et
al. 1996; Tyor et al. 1992; Vago et al. 2001). A strong correlation was also
observed between TNF mRNA expression and clinical signs of dementia
(Glass et al. 1993), although, based on studies using TNF knockout mice
in the Fr98 system, gene expression of TNF does not always correlate with
a role of TNF in neuropathogenesis. The source of CCL2(MCP-1) expression
in HAD patients was astrocytes (Conant et al. 1998), while CCL5(RANTES)
was expressed by lymphocytes and uninfected microglia/macrophages
(Vago et al. 2001) and TNF, IFN-, and IL-1 were produced by perivascular
macrophages and endothelia (Tyor et al. 1992).
Increased cytokine and chemokine mRNA and protein expression also
correlated with encephalitis induced by SIV or FIV infection. Increased cere-
brospinal uid levels of CCL2(MCP-1) was a strong indicator of SIV-induced
encephalitis (Zink et al. 2001). Additionally, increased mRNA and/or protein
expression of CCL3(MIP-1), CCL4(MIP-1), CCL5(RANTES), CCL7(MCP-
3), CXCL10(IP-10), IL-1, IL-4, IL-6, IFN-, and TNF have been associated
with encephalitis in the SIV model (Lane et al. 1996; Orandle et al. 2002; Sas-
seville et al. 1996; Sopper et al. 1996; Sui et al. 2003), with TNF expression by
macrophages in perivascular lesions (Orandle et al. 2002). Similarly, increased
expression of TNF mRNA by microglia and astrocytes appears to be involved
in the early stages of FIV-induced encephalitis in cats (Poli et al. 1999).
4.2
Effect of HIV Proteins on Cytokine and Chemokine Induction
In the Fr98 model, particular sequences in the viral envelope protein inu-
enced the production of cytokines and chemokines, possibly through the
regulation of virus levels in the brain (Peterson et al. 2001). In the SCID-
HIVE model, increased expression of both CCL2(MCP-1) and CCL3(MIP-1)
was associated with HIV-infected monocytes in the brain (Persidsky et al.
1999). Similar induction of cytokines and chemokines such as CCL2(MCP-1),
CXCL10(IP-10), CXCL9(MIG), and IL-1 were observed in mouse brain tis-
sue following inoculation with HIV-1 provirus or a chimeric HIV virus with
an ecotropic MuLV envelope protein (Potash et al. 2005; Wang et al. 2003).
Both the Tat and Env protein of HIV appeared to contribute to the upregu-
lation of cytokines and chemokines. HIV Tat stimulation of in vitro cultures
of glial cell cultures induced the expression of several chemokines including
CCL2(MCP-1) and CCL3(MIP-1) (McManus et al. 2000), while stimulation
of astrocytes with HIV gp120 induced CXCL10(IP-10) expression (Asensio
et al. 2001). A similar induction of CXCL10(IP-10) as well as CCL2(MCP-1)
was observed in mice with transgenic expression of gp120 under the GFAP
promoter (Asensio et al. 2001). Interestingly, no clinical signs of neurological
disease were reported in these in vivo models, indicating that increased pro-
duction of chemokines in the brain at the levels achieved in these experiments
was not sufcient for the induction of neurological disease.
5
Potential Effects of Chemokines During Retrovirus Infection of the Brain
Polymorphism studies have suggested that certain alleles of TNF and
CCL2(MCP-1) correlate with increased risk of the development of dementia
in HIV-infected patients (Gonzalez et al. 2002; Quasney et al. 2001). Studies
with the Fr98 mouse model also demonstrated that CCL2(MCP-1), its
primary receptor, CCR2, and TNF contributed to retroviral pathogenesis in
the brain (Peterson et al. 2004a, b). Comparison of wildtype and knockout
mice in the Fr98 pathogenesis model as well as in vitro and in vivo studies
with HIV, SIV, and FIV have indicated several mechanisms by which these
proteins may contribute to pathogenesis.
5.1
Activation and Recruitment of Microglia and Macrophages
The lack of neurological disease in TNF-decient mice following EC retrovirus

infection was associated with a lack of increased expression of the microglia
and macrophage marker F4/80 (Peterson et al. 2004b). Thus, TNF may have
an important role in the activation and/or recruitment of brain microglia and
macrophages. Autopsy ndings from HAD patients indicated a strong corre-
lation between increased staining for activated macrophages and microglial
cells in the brain and the severity of dementia (Glass et al. 1995). Similarly, an
increase in activated macrophages and/or microglia has also been associated
with encephalitis following SIV or FIV infection (Georgsson 1994; Lackner et
al. 1991; Williams and Hickey 2002). Bloodbrain barrier (BBB) permeability
and tight junction disruption have been noted in cases of HIV and SIV
infections (Boven et al. 2000; Luabeya et al. 2000), indicating that infected and
uninfected peripheral macrophages may migrate to the brain and contribute
to retroviral pathogenesis. In the SCID-HIVE mouse model, HIV-infected
cells in the CNS were surrounded by murine macrophages, suggesting the
migration of either peripheral or brain macrophages to the site of virus in the
brain (Nukuna et al. 2004). TNF expression has been demonstrated to break
down the BBB as well as induce microglia and macrophage activation and
macrophage migration in vitro (Glabinski et al. 1998; Hurwitz et al. 1994).
Chemokines may play an important role in the migration of peripherally
activated macrophages across the BBB. The term chemokine was coined to
describe a family of chemoattractant cytokines that were involved in the
recruitment and tissue extravasation of leukocytes during inammation.
The chemokine CCL2(MCP-1) induced monocyte migration across an
endothelial cell/astrocyte co-culture model of the BBB (Eugenin and Berman
2003). Expression of CCR2, the primary receptor for CCL2(MCP-1), by
macrophages was required for CCL2(MCP-1)-induced macrophage migra-
tion across a brain endothelial layer (Dzenko et al. 2005). Interestingly, CCR2
expression was also required on the brain microvessels, as neither CCR2+
nor CCR2 macrophages could migrate across CCR2 brain endothelial cells
in response CCL2(MCP-1) stimulation (Dzenko et al. 2005). It is possible that
CCL2(MCP-1) and TNF contribute to retroviral pathogenesis by the same
mechanism, with both cytokines involved in the activation and/or migration
of microglia and macrophages in the brain.
5.2
Lymphocyte Recruitment
Expression of cytokines and chemokines may also recruit CD3 lymphocytes

to the brain (Dufour et al. 2002; Moser et al. 2004; Ransohoff 2002; Ransohoff
and Tani 1998). Although a detectable increase in CD8+ CD3+ lymphocytic
inltration is not observed in the Fr98 model, increased numbers of CD8+
lymphocytes in the CNS have been reported with both HIV-1 and SIV infec-
tions (Kim et al. 2004b; Miller et al. 2004; Petito et al. 2003). However, persis-
tent depletion of CD8+ lymphocytes correlated with increased encephalitis in
brains of SIV-infected macaques (Williams et al. 2001; Williams and Hickey
2002). Thus, rather than contributing to retroviral pathogenesis, CD8+ T cells
may suppress the recruitment and trafcking of infected macrophages in the
CNS, delaying the development of neurological symptoms.
5.3
Neuronal Apoptosis
Apoptotic neurons and neuronal dropout have been observed in brain tissue
of HAD patients and animal models of retroviral neuropathogenesis, although
there does not appear to be a direct correlation with the amount of neuronal
apoptosis and clinical neurological disease (Adle-Biassette et al. 1999; Kolson
et al. 1998). In contrast, no detectable increase in neuronal apoptosis was asso-
ciated with Fr98 infection or disease (Peterson et al. 2004b; Portis et al. 1995),
but lack of apoptosis might be due to the short disease course in this model.
This result suggests that the severe clinical symptoms observed by Fr98 infec-
tion are not the result of neuronal death, but instead more likely represent neu-
ronal dysfunction. In slower disease models, apoptosis may be the end result
of neuronal damage induced by cytokines and chemokines, but this interpre-
tation is not conclusive because these proinammatory molecules can have
opposing effects in different situations. For example, TNF has been directly
implicated in inducing apoptosis through stimulation of TNFRI, but may be
anti-apoptotic when stimulating through TNFRII (Saha and Pahan 2003).
CCL2(MCP-1) inhibited neuronal apoptosis induced by either N-methyl-d-
aspartate (NMDA) or HIV Tat in mixed glial cultures (Eugenin et al. 2003).
Similar results were also observed by the addition of CCL5(RANTES), suggest-
ing that the production of chemokines during retrovirus infection may pro-
vide protection from retrovirus-induced neuronal apoptosis and may, in some
instances, decrease the pathogenesis of retrovirus infection. In vitro studies
have shown that chemokines such as CCL5(RANTES), CXCL12(SDF1), and
CCL22(MDC) can provide support for the survival of neuronal cultures in the
absence of glial feeder cells (Meucci et al. 1998).
Retrovirus proteins including HIV-1 Tat and gp120 can induce apoptosis
when added to neurons in vitro (Catani et al. 2000; Meucci et al. 1998; Zhang et
al. 2003). HIV gp120-induced neuronal apoptosis was blocked by anti-CXCR4
or anti-CCR5 antibodies, indicating that HIV gp120 neurotoxicity may be
mediated by chemokine receptor signaling (Zhang et al. 2003). CCL3(MIP-
1), CCL4(MIP-1), CCL5(RANTES), and CXCL12(SDF1) inhibited gp120-
induced apoptosis (Catani et al. 2000; Meucci et al. 1998), suggesting that the
expression of these chemokines during retrovirus infection may block the

neurotoxic effects of gp120. Thus, the ratio of chemokine expression to virus
envelope production and the levels of cytokines or chemokines produced
may regulate the amount of neuronal apoptosis associated with retrovirus
infection.
5.4
Direct Stimulation of Neurons
Chemokines may also contribute to retrovirus neuropathogenesis through

the direct stimulation of neuronal subsets in the brain. Embryonic and adult
neurons have been reported to express a number of chemokine receptors in-
cluding CCR1, CCR2, CCR5, CXCR3, and CXCR4 (Meucci et al. 1998; Tran et al.
2004; Tran and Miller 2003). Studies with hippocampal neurons demonstrated
functional chemokine receptors as stimulation of neurons with CCL3(MIP-
1), CCL5(RANTES), CCL22(MDC), and CXCL12(SDF1) induced calcium
signaling (Meucci et al. 1998). Although CCL2(MCP-1) did not induce cal-
cium signaling in hippocampal neurons (Meucci et al. 1998), studies with
Purkinje neurons indicated that CCL2 could enhance calcium signaling fol-
lowing glutamate receptor activation (van Gassen et al. 2005). Patch-clamp
experiments of cultured hypothalamic neurons indicated that chemokines
such as CXCL12(SDF1) might be directly involved in neuronal signaling
(Guyon et al. 2005). Thus, the production of chemokines following retro-
virus infection of the CNS may alter neuronal signaling patterns, leading to
neurological disease.
5.5
Alteration or Inhibition of Neuroprogenitor Stem Cell Migration
Another mechanism by which CCL2(MCP-1)/CCR2 interactions may con-

tribute to retroviral neuropathogenesis is through the alteration of neural
progenitor stem cell (NPSC) migration in the brain or by effecting axonal
growth and NPSC survival. This might be especially relevant in congenital
HIV infection and also in the Fr98 mouse model, where mice are infected
as neonates and the brain is still undergoing development. Abnormal brain
development has not been reported in CCR1-, CCR2-, CCR3-, CCL2(MCP-1)-,
or CXCL10(IP-10)-decient mice, suggesting that these chemokines are not
necessary for the normal migration of NPSC during development (Abbadie
et al. 2003; Dufour et al. 2002; Humbles et al. 2002; Khan et al. 2001; Kuziel
et al. 1997, 2003; Lu et al. 1998). However, CCL2(MCP-1) altered the migra-
tion of NPSC in vitro (Widera et al. 2004), suggesting that overexpression
of chemokines may alter NPSC migration. Additionally, lipopolysaccharide
(LPS) was shown to inhibit NPSC migration patterns in vivo, possibly through
the increased production of proinammatory cytokines and chemokines in
the brain (Monje et al. 2003). Chemokines have been shown to contribute to
NPSC migration in vivo. Mice decient in the chemokine receptor CXCR4, the
receptor for the chemokine CXCL12(SDF1), have deformed cerebellum de-
velopment and lack neuronal migration from the external granular layer (Lu
et al. 2002). The loss of CXCL12(SDF1) also affects adult neurogenesis in the
hippocampal dentate gyrus (Bagri et al. 2002). A decrease in migrating NPSC
was detected in HIV-infected patients with dementia compared to those with-
out, indicating that NPSC migration may inuence disease (Krathwohl and
Kaiser 2004). Alteration or suppression of NPSC migration could affect the
development of the dentate gyrus and cerebellum and lead to the inhibition
of memory and developmental skills associated with HIV infection in infants
(Drapeau et al. 2003; Monje et al. 2003; Rola et al. 2004; Tran and Miller 2003).
5.6
Astrocyte Activation and Support Functions
Although most retroviruses do not productively infect astrocytes, reactive

astrocytes as characterized by increased GFAP expression is commonly as-
sociated with neuropathogenesis induced by HIV, SIV, FIV, and Fr98, as well
as other retroviruses (Kolson et al. 1998; Poli et al. 1997; Rausch et al. 1994;
Robertson et al. 1997). Astrocytes are also the common source for CCL2(MCP-
1) production during retrovirus infection and may contribute to pathogenesis
by this mechanism (Conant et al. 1998; Peterson et al. 2004a; Zink et al. 2001).
CXCL10(IP-10) production by retrovirus-stimulated astrocytes has also been
detected (Asensio et al. 2001; Kutsch et al. 2000). Astrocytes express multiple
functional chemokine receptors including CCR1, CCR2, CCR3, CCR5, CCR10,
and CXCR4 (Andjelkovic et al. 2002; Croitoru-Lamoury et al. 2003; Dorf et al.
2000; Tanabe et al. 1997). Thus, the production of chemokines by astrocytes
and other glial cells in the brain may affect the function of these cells.
Astrocytes play an important support function for neurons, by removing
potentially neurotoxic glutamate from extracellular spaces and converting it
to glutamine. Activated astrocytes express two glutamate receptors: SLC1A2
(solute carrier family 1glial high-afnity glutamate transporter member 2,
also known as GLT-1 or EAAT-2), which is found on astrocytes throughout
the brain; and SLC1A3 (solute carrier family 1glial high-afnity glutamate
transporter member 3, also known as GLAST or EAAT-1), which can also be
detected on neurons (Liberto et al. 2004). Trauma to cultured astrocytes re-
sults in increased mRNA and protein expression of both SLC1A2 and SLC1A3.
Glutamate-ammonia ligase (GLUL, also known as glutamine synthase, GLNS),
which converts glutamate to the non-neurotoxic glutamine, can also be upreg-

ulated in activated astrocytes. Altered expression of SLCA3 or other glutamate
regulatory genes has been associated with neuropathogenesis, including SIV
infection of the brain (Chretien et al. 2002, 2004; Guo et al. 2003; Li et al.
1997; Martin et al. 2000). Chemokine activation of astrocytes may affect the
regulation of the glutamine synthesis and lead to the build-up of neurotoxic
glutamate.
5.7
Retrovirus Entry and Spread in the CNS
In the Fr98 model, the lack of CCR2, CCR5, or TNF did not inuence virus
burden in the brain (Peterson et al. 2004a, b). However, Fr98 has not been re-
ported to use chemokine receptors as coreceptors for virus entry. In contrast,
HIV and SIV utilize the chemokine receptors CXCR4 and CCR5 as coreceptors
for infection of T cells and macrophages, respectively (Alkhatib et al. 1996;
Feng et al. 1996; Oberlin et al. 1996). In addition, other chemokine receptors
including CCR2b, CCR3, and CCR8 have been shown to contribute to HIV or
SIV infection and are often referred to as minor coreceptors (Gorry et al. 2001;
Margulies et al. 2001). HIV patients with a CCR5 allele containing a 32-bp dele-
tion had reduced incidence of HIV encephalitis (HIV-E), indicating that CCR5
is an important contributor to retrovirus infection of macrophages and/or mi-
croglia in vivo (van Rij et al. 1999). Chemokine expression in the brain during
HIV-1 infection could impact virus infection of brain macrophages or mi-
croglia. CCL3(MIP-1), CCL4(MIP-1), and CCL5(RANTES) are ligands for
CCR5 and are detected at increased levels in patients with HAD (Schmidt-
mayerova et al. 1996; Vago et al. 2001). The expression of these CCR5 ligands
in the brain during HIV infection could block virus envelope binding of CCR5
molecules on macrophages and thus restrict the spread of HIV infection in the
brain. Alternatively, stimulation of CCR5-positive macrophages by these lig-
ands may induce increased expression of CCR5 on the cell surface, providing
ample coreceptors for virus infection of brain macrophages.
5.8
Retroviral Protein Stimulation of Chemokine Receptors
As several retroviruses use chemokine receptors for virus entry, retroviral

proteins may also contribute to pathogenesis by signaling cells through these
chemokine receptors. For example, HIV-1 gp120-induced neuronal death can
be inhibited by blocking or downregulating CXCR4 or CCR5 (Catani et al.
2000; Zhang et al. 2003; Zheng et al. 1999). CXCR4 expression has been de-
tected on multiple cell types in the CNS including microglia, astrocytes, and
neurons (Bajetto et al. 1999; Catani et al. 2000; Flynn et al. 2003), while CCR5
appears to be expressed on glial cells (van der Meer et al. 2000; Bajetto et al.
1999). Although HIV-1 primarily infects microglia/macrophages in the brain,
HIV gp120-induced stimulation of CCR5 or CXCR4 may alter the activation
state of uninfected cells or induce apoptosis. Another retroviral protein, Tat,
is also reported to mimic chemokines. Tat acts as a chemoattractant to leuko-
cytes, can induce CCL2(MCP-1) expression by astrocytes, and can displace
the binding of a-chemokines from their receptors (Albini et al. 1998; Conant
et al. 1998). HIV Tat also binds to the chemokine receptor CCR1 with similar
afnity to the chemokine CCL7(MCP-3) (Albini et al. 1998), indicating that
high levels of Tat may mimic the production of this chemokine.
The xenotropic/polytropic receptor 1 (XPR1, RMC1) is the cellular receptor
for polytropic retrovirus infection (Tailor et al. 1999; Yang et al. 1999). Based
on the presence of an SPX domain and similarities to the yeast protein SYG1,
the XPR1 protein is predicted to be involved in G protein-associated signal
transduction and function as a phosphate sensor (Battini et al. 1999). It is pos-
sible that XPR1 is involved in signal transduction of chemokine receptors or
other signal transduction pathways that lead to the cellular activation. Further
studies that elucidate the function of XPR1 would determine if stimulation of
this receptor contributes to neuropathogenesis.
6
Conclusions
The correlation between increased proinammatory cytokines and chemo-

kines and neuropathogenesis induced by Fr98, HIV, SIV, and FIV suggests
that some proinammatory factors contribute to the disease process. Further-
more, polymorphism studies in HAD patients and knockout mouse studies
in the Fr98 mouse model indicate that the upregulation of both TNF and
CCL2(MCP-1) contributes to Fr98 and HIV-induced retroviral neuropatho-
genesis. Other proinammatory cytokines, such as IL-1 or TNFSF1b(TNF,
lymphotoxin A) may be a host reaction to retrovirus-induced damage. Proin-
ammatory cytokines and chemokines are most commonly known for their
ability to recruit lymphocytes and other immune cells to the sites of infec-
tion. However, the upregulation of cytokines and chemokines associated with
neuropathogenesis induced by HIV-1, SIV, FIV, and Fr98 retrovirus infec-
tion is not associated with substantial lymphocyte inltration, possibly due
to the immunosuppressed nature of the host. The pathogenesis of cytokines
and chemokines during retrovirus infection may be due to stimulation of resi-
dent CNS cells. Several mechanisms by which proinammatory cytokines and
chemokines may contribute to the neuropathogenesis include the activation

of perivascular macrophages and microglia, induction of neuronal apoptosis,
interference with neuronal signal transduction, alteration of neuroprogenitor
stem cell migration, and altering the activation and support functions of astro-
cytes. Further investigation is needed to determine the relative contribution
of these mechanisms to the clinical neurological disorders associated with
retrovirus infection. Additionally, it is important for possible therapeutic po-
tential to determine whether certain chemokines might slow the progression
of disease development as is suggested by some in vitro experiments.
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CTMI (2006) 303:97120

HIV-1 Coreceptors and Their Inhibitors

N. Ray (u) R. W. Doms
Department of Microbiology, University of Pennsylvania, 301A Johnson Pavilion,
Philadelphia, PA 19104, USA
nray@mail.med.upenn.edu
1 Identication of Chemokine Receptors as Coreceptors

for HIV-1 Env-Mediated Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
2 Determinants of Tropism and Chemokine Receptor Usage . . . . . . . . . . . 101
3 Impact of Nonfunctional Chemokine Receptor Alleles

on HIV-1 Resistance and Disease Progression . . . . . . . . . . . . . . . . . . . . 101
4 Coreceptor-Based Antiretroviral Therapy . . . . . . . . . . . . . . . . . . . . . . . 103

4.1 CCR5-Based Antiretroviral Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
4.1.1 TAK-779, TAK-220, and TAK-652 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.1.2 SCH-C and SCH-D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
4.1.3 UK-427857 (Maraviroc) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
4.1.4 CMPD 167 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
4.1.5 Spirodiketopiperazine-Based Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . 107
4.2 CXCR4-Based Antiretroviral Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.2.1 Small Molecule CXCR4 Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5 Resistance to Coreceptor Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
6 Impact of Chemokine Receptor Inhibitors

on Clinical Monitoring and Treatment . . . . . . . . . . . . . . . . . . . . . . . . . 109
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Abstract Entry of human immunodeciency virus (HIV) into target cells is mediated
by the viral Envelope glycoprotein (Env) and its coordinated interaction with a recep-
tor (CD4) and a coreceptor (usually the chemokine receptors CCR5 or CXCR4). This
review describes the identication of chemokine receptors as coreceptors for HIV-1
Env-mediated fusion, the determinants of chemokine receptor usage, and the impact
of nonfunctional chemokine receptor alleles on HIV-1 resistance and disease progres-
sion. Due to the important role of chemokine receptors in HIV-1 entry, inhibitors
of these coreceptors are good candidates for blocking entry and development of an-
tiretroviral therapies. We discuss the different CCR5- and CXCR4-based antiretroviral
drugs that have been developed thus far, highlighting the most promising drug can-
didates. Resistance to these coreceptor inhibitors as well as the impact of these drugs
on clinical monitoring and treatment are also discussed.
98 N. Ray R. W. Doms
1
Identification of Chemokine Receptors as Coreceptors
for HIV-1 Env-Mediated Fusion
Human immunodeciency virus (HIV) initiates infection by attaching and

subsequently fusing its viral membrane to the plasma membrane of a target
cell. As shown in Fig. 1, the process is mediated primarily by the viral Envelope
protein (Env). Env is a glycoprotein, 160 kDa in size (gp160), that is proteolyt-
ically processed into a surface subunit (gp120) and a transmembrane subunit
(gp41) by a host cell protease during transit to the cell surface. Each gp120
molecule interacts with one gp41 molecule to form a noncovalently linked
complex, and the gp120gp41 complexes then exist as trimers on the surface
of HIV virions. These trimers bind with high afnity to CD4, the primary re-
ceptor for HIV on the target cell surface. The presence of CD4 is a prerequisite
for efcient HIV infection and explains the tropism of HIV for CD4-positive
T cells and macrophages.
Three main lines of evidence derived from experiments reported in the
mid-1980s and early 1990s, however, brought into question the role of CD4
as the only receptor for HIV. First, studies with recombinant human CD4
demonstrated the ability of this molecule to render cells susceptible to Env-
mediated fusion and virus entry, but only when expressed in human cell
types [20, 61]. Further work with cell hybrids pointed toward the existence of
an essential coreceptor, specic to human cells, in the absence of which CD4
was unable to support Env-mediated fusion [4, 14]. Second, in the late 1980s,
different HIV-1 isolates were shown to display distinct tropisms for various

Fig. 1 Model of HIV entry. The Env glycoprotein of HIV mediates fusion of viral and
cellular membranes. Env is a homotrimeric protein (rst panel) in which each subunit
is composed of surface gp120 and membrane-spanning gp41 proteins. Binding to CD4
is mediated by the gp120 subunit, and CD4 binding induces conformational changes in
gp120 that result in the exposure of a conserved region that is important for coreceptor
binding (second panel). In the native trimer, this conserved region is hidden in part by
variable loops that are thought to undergo conformational changes and consequent
repositioning after CD4 binding. Following CD4 binding, gp120 binds to a seven-
transmembrane domain coreceptor (CoR; third panel). Coreceptor binding can be
inhibited by a number of CCR5 and CXCR4 inhibitors such as TAK-220, SCH-C, SCH-
D, UK-427857, CMPD 167, GSK-873140, AMD-3100, and AMD-070. The hydrophobic
fusion peptide at the N terminus of gp41 becomes exposed and inserts into the cell
membrane. Ultimately, coreceptor binding leads to the formation of a six-helix bundle
in which the helical HR2 domains of each gp41 subunit fold back and bind to the triple-
stranded HR1 domains (fourth panel), bringing the fusion peptide and transmembrane
domain of gp41, and their associated membranes, into close proximity
HIV-1 Coreceptors and Their Inhibitors 99
CD4-positive human cell types in vitro. Some isolates are able to infect CD4-
positive T cell lines but not primary macrophages, whereas others showed the
opposite tropism and were able to infect primary macrophages much better
than T cell lines ([71] and citations therein). The former were designated
T cell-line tropic (T-tropic) and the latter macrophage tropic (M-tropic).
Isolates that efciently infected both T cell lines and macrophages were called
dual-tropic. Soon after, it was also reported that viral isolates from peripheral
blood of recently infected individuals are primarily M-tropic [85, 91, 116]
and as the infection progresses to acquired immunodeciency syndrome
(AIDS), T-tropic viruses can be isolated in most patients [23, 91, 102]. Again,
cell hybrid experiments indicated that these specic tropisms were likely
resulting from the requirement for an additional coreceptor in the susceptible
cells rather than the presence of an inhibitor in the nonsusceptible cells.
Finally, the identity of the putative coreceptor became apparent from studies
with the chemokines macrophage inammatory protein (MIP)-1, MIP-1,
and RANTES (regulated on activation, normal T-expressed and secreted),
which were shown to block HIV-1 infection [21].
The rst coreceptor was identied using a functional complementary DNA
(cDNA) cloning strategy based on the ability of a cDNA library to render
a CD4-expressing murine cell susceptible for fusion with cells expressing Env
from a T-tropic strain [43]. This strategy allowed the isolation of a single
cDNA; sequence analysis revealed that the encoded protein was a member of
the superfamily of the seven-transmembrane domain G protein-coupled re-
ceptors. The protein was rst named fusin for its role in HIV fusion, and later
CXCR4 when it was shown to be a chemokine receptor for the CXC chemokines
stromal cell-derived factor (SDF)-1 and SDF-1 [11, 78]. Importantly, CXCR4
did not function as a coreceptor for M-tropic virus isolates. However, at around
the same time, the chemokine receptor CCR5 was shown to bind MIP-1,
MIP-1, and RANTES, the same chemokines that had been shown to block
infection by some HIV-1 strains [21]. Shortly thereafter, CCR5 was shown
to be the major coreceptor for M-tropic HIV-1 strains [3, 19, 32, 37, 39].
CCR5-using (R5-tropic) HIV-1 strains tend to be transmitted between in-
dividuals, whereas strains using CXCR4 (X4-tropic) emerge in a subset of
HIV-1-infected individuals at later stages of infection [25, 96]. In addition to
the two main coreceptors for HIV-1, other chemokine receptors as well as
some orphan receptors have been shown to be capable of mediating virus
fusion. These alternative coreceptors include CCR2b, CCR3, CCR8, CCR9,
CXCR6 (STRL33/Bonzo), CX3 CR1, ChemR23, GPR15 (BOB), and APJ (re-
viewed in [8, 95]). A subset of these alternative coreceptors, such as CCR3,
CCR8, and CXCR6, are used efciently by some HIV-1 isolates to infect cell
lines over-expressing these proteins [7, 86, 114]. Certain minor coreceptors
(CXCR6 and GPR15) are expressed in the placenta and colon and could con-
ceivably play a role in mother-to-child or homosexual transmission [33], while
CCR8 is expressed on thymocytes and so could play a role in virus infection
in the thymus. However, with only a handful of exceptions [93, 110, 115],
infection of human macrophages and peripheral blood mononuclear cells
(PBMCs) by HIV-1 strains is dependent upon the presence of either CCR5 or
CXCR4 in conjunction with CD4. Most of the alternative coreceptors are either
not expressed on CD4-positive cells at detectable levels, or are expressed at
levels that are below that needed for efcient virus infection. Thus, at present
there is no compelling evidence to indicate that receptors other than CCR5 or
CXCR4 are important for HIV-1 infection in vivo.
2
Determinants of Tropism and Chemokine Receptor Usage
The env genes from different HIV-1 isolates display signicant sequence het-
erogeneity in specic regions of gp120. There are ve variable regions in
gp120 (V1V5) that are separated by ve constant regions (C1C5). Studies
involving Env chimeras between M- and T-tropic isolates identied the V3
loop as the primary determinant of viral tropism [17, 18, 28, 51, 94, 101] and
coreceptor usage [19, 22, 85, 98, 99]. Although even single amino acid changes
in the V3 loop have been shown to be sufcient to alter tropism [27], many of
the mutations observed in this region that alter tropism have strain-specic
effects, and no single amino acid has emerged as crucial. In general, how-
ever, an increase in the net positive charge (i.e., more basic residues) in the
V3 loop has been demonstrated in viral isolates from HIV-positive patients
over time and correlates with conversion to T cell tropism and CXCR4 us-
age [9, 19, 22, 27, 49, 94]. Moreover, the specic nature of the V3 loop, based
on whether it is from an R5 or X4 strain, is the primary determinant of direct
Env chemokine receptor binding and Env-mediated inhibition of chemokine
and monoclonal antibody binding to chemokine receptors [22, 105, 112].
3
Impact of Nonfunctional Chemokine Receptor Alleles
on HIV-1 Resistance and Disease Progression
A mutation in the CCR5 open reading frame results in the premature trun-
cation and a consequent 32-bp deletion in the protein (CCR5 32). Although
this mutation is relatively common in the Caucasian population, with an al-
lele frequency of 1520%, it was found to be signicantly underrepresented
in the HIV-1 infected groups [30, 88], and individuals homozygous for the
mutation are only rarely infected with HIV [10, 45, 70, 77, 103]. In fact, in
a group of people at high risk, two individuals that remained uninfected de-
spite repeated exposure, were found to be homozygous for the same ccr5
mutation [60]. Lymphocytes from these individuals are resistant in vitro to
M-tropic strains but permissive for T-tropic strains of HIV-1 [79]. In addition,
HIV-1-infected individuals who are heterozygous for the ccr5 mutation have
around a 2-year delay in their progression to AIDS compared to wildtype
controls [30, 50, 69, 117]. These ndings highlight the importance of CCR5 as
a coreceptor in HIV-1 entry in humans.
In addition to the ccr5 mutation, other genetic polymorphisms have
been found in certain chemokine receptors and their corresponding ligands.
The CCR2-64I polymorphism causes a conservative valine to isoleucine mu-
tation in CCR2. This mutation does not impact initial HIV transmission.
However, individuals harboring this polymorphism progress to AIDS signif-
icantly slower (by 23 years) as compared to CCR2+/+ HIV-1 seroconvert-
ers [55, 74, 84, 97]. Moreover, seropositive individuals who carry this allele
in combination with the ccr5 allele experience an additive delay in the pro-
gression to symptomatic disease [55, 97]. The mechanism for this protective
effect is not clear, as CCR2 does not appear to function as a coreceptor for
HIV entry on primary cells, and CCR2-64I appears to have normal receptor
function [57].
Another notable coreceptor polymorphism is the CCR5 59029 G/A single-
nucleotide polymorphism. Both alleles, either G or A at position 59020 in the
CCR5 promoter, are very common across racial groups. In one study, individu-
als selected for the absence of CCR5 32 or CCR2-64I had a mean time to AIDS
for 59029 G/G that was 3.8 years longer (p=0.004) than for 59029 A/A individ-
uals [67]. In reporter gene assays, promoter fragments differing in sequence
only at 59029 G versus A had differential activity, with the 59029 A promoter
being more active than the 59029 G promoter [67], suggesting that 59029
G/G individuals might have decreased transcription and consequently lower
expression of CCR5. This is consistent with the observation that CCR5 32
heterozygotes progress to AIDS more slowly in the absence of therapy, strongly
suggesting that CCR5 levels are rate-limiting for HIV infection in vivo.
The SDF-1 3 A allele is a G to A transition at position 809 of the 3 -
untranslated region (UTR) of the messenger RNA (mRNA) encoding one of the
two chemokine ligands for CXCR4, SDF-1. In one report based on pooled se-
roconverters from three cohorts, a strong association was described between
the 3 A/3 A genotype and delayed onset of AIDS [111]. However, in several
subsequent reports, no association was found between homozygosity for the
SDF-1 3 A allele and retarded progression to disease [13, 47, 66, 68, 74, 107].
Moreover, in an international meta-analysis study, the SDF-1 3 A allele was

shown to not be predictive of disease outcome or progression [53].
Finally, individuals whose CCL3L1 (MIP1P) gene copy number is above
the population median have a reduced risk for acquiring HIV with each
stepwise increase in copy number of this gene [44]. Moreover, a gene dose
lower than the overall cohort median or the population-specic median is
associated with a dose-dependent increased risk of progressing rapidly to
AIDS or death. Since CCL3L1 is a potent ligand for CCR5, it stands to reason
that increasing CCL3L1 copy number leads to a reduction in the proportion
of CD4-positive CCR5-expressing T cells and a consequent diminution in the
risk of acquiring HIV as well as the rate of progression to AIDS.
4
Coreceptor-Based Antiretroviral Therapy
Since the discovery of chemokine receptors as coreceptors for HIV-1 entry,

a signicant focus of antiretroviral drug development has been directed to-
ward antagonists of these receptors and their interactions with HIV-1 Env.
The structures of some of the important coreceptor inhibitors discussed be-
low are shown in Fig. 2. The HIV coreceptors are especially attractive drug
targets, particularly CCR5, which is used by most primary HIV-1 strains.
The strongest evidence for the importance of CCR5 in HIV-1 transmission
and pathogenesis is the profound resistance to HIV-1 infection of individuals
encoding two copies of a nonfunctional ccr5 gene [60, 88]. The lack of seri-
ous immunological defects in humans homozygous for the inactivating 32-bp
deletion (CCR5 32) suggests that CCR5 may be a good therapeutic target.
4.1
CCR5-Based Antiretroviral Therapy
Early attempts at blocking CCR5 involved altered versions of the natural

CCR5 ligands, MIP-1, MIP-1, and RANTES. These protein-based inhibitors
blocked CCR5-mediated HIV-1 infection by competing with gp120 for the
receptor-binding site, downregulation of receptor expression, and induction
of receptor signaling events that altered cellular differentiation and suscep-
tibility to HIV-1. Thus far, the poor pharmacokinetics and bioavailability of
these drugs have prevented their development as candidates for antiretrovi-
ral therapeutics. However, the newer derivatives of RANTES (such as PSC-
RANTES) might have potential as components of microbicides, as they can
block vaginal transmission of virus in a rhesus macaque model [56].
Fig. 2 Structures of CCR5 and CXCR4 inhibitors
4.1.1
TAK-779, TAK-220, and TAK-652
Small molecule inhibitors of CCR5 have proved to have signicant potential

as anti-HIV-1 therapy. TAK-779 was the rst described small-molecule CCR5
inhibitor. This compound blocks CCR5-mediated signaling at nanomolar
concentrations without any effects on CCR5 expression [6, 40]. Accordingly,
the compound can block infection by R5- but not X4-tropic HIV-1 strains
by interfering in the interaction between gp120 and CCR5. TAK-779 can
also block binding to CCR2, which shares sequence homology with CCR5.
Therefore, TAK-779 is able to block infection by simian immunodeciency
virus (SIV)rcm , which uses CCR2 as its major coreceptor [113]. Despite its
potent antiviral capacity, further development of TAK-779 was abandoned
because it was not orally bioavailable and caused irritation at the site of
injection [52].
However, a derivative of TAK-779, termed TAK-220, is orally bioavailable
and is capable of blocking HIV-1 replication in PBMCs in the low nanomolar
range. Specically, TAK-220 can inhibit R5 HIV-1 isolates with IC50 and IC90
values of 1.1 nM and 13 nM [52, 104]. When administered orally to fasting rats
and monkeys at a dose of 5 mg/kg, the bioavailability of TAK-220 was 9.5%
and 28.9%, respectively [52]. TAK-220 also inhibits the binding of RANTES
and MIP-1 to CCR5-expressing cells, but does not block binding of MIP-
1. Owing to its potent activity and favorable pharmacokinetics, TAK-220 is
a candidate for clinical development.
Recently, another TAK-779 derivative, TAK-652, was described [5].
TAK-652 selectively inhibited R5 HIV-1 but not X4 HIV-1 replication. This
compound was able to potently inhibit the replication of six R5 HIV-1 clinical
isolates, including reverse transcriptase- and protease inhibitor-resistant
mutants, with mean IC50 and IC90 values of 0.061 nM and 0.25 nM,
respectively. In addition, all recombinant HIV-1 strains with seven different
subtype (A to G) Env proteins were equally susceptible to TAK-652 with
a mean IC50 of 1.0 nM. Single oral doses of TAK-652 (25100 mg) were safe
and well tolerated. TAK-652 showed good oral absorption, and its plasma
concentration at 24 h after administration (25 mg) was 8.8 nM.
4.1.2
SCH-C and SCH-D
SCH-C is another small, non-peptide CCR5 antagonist that inhibits ligand

binding to CCR5. SCH-C has broad and potent antiviral activity in vitro
against primary R5-using HIV-1 isolates, with mean IC50 values between
0.4 and 9 nM [100]. High doses of SCH-C are also able to slightly inhibit
ligand binding to CCR2. SCH-C can strongly inhibit replication of an R5-
using HIV-1 isolate in vivo in severe combined immunodeciency (SCID)-hu
Thy/Liv mice [100]. The compound has also shown favorable pharmacoki-
netics in rodents and primates, with an oral bioavailability of 50%60% and
a serum half-life of 56 h. Clinical data obtained from an early human trial
where SCH-C monotherapy was administered (25 mg twice daily) to HIV-
infected adults for 10 days demonstrated viral load reductions between 0.5
and 1.0 log10 [83]. However the same study found a prolongation of the QTc
interval, suggesting possible adverse cardiac effects at high doses, resulting
in cessation of further development of this compound.
SCH-D, a derivative of SCH-C, was shown to have greater potency in
vitro and in vivo [92]. In a phase I clinical trial, SCH-D monotherapy with
escalating doses of 1050 mg twice daily over 14 days, no adverse effects were
seen. A dose-dependent reduction in plasma viremia was observed, with mean
reductions of up to 1.62 log10 [92].
4.1.3
UK-427857 (Maraviroc)
Another CCR5 inhibitor, UK-427857 (recently termed maraviroc), potently

blocks cell entry of a range of lab-adapted and primary R5-tropic HIV-1 iso-
lates in vitro (IC90 <10 nM) [38]. Despite its potency against R5-tropic isolates,
the compound is specic and remains inactive against X4-tropic isolates [38].
UK-427857 blocks gp120 and chemokine ligand binding to CCR5, but does
not induce intracellular signaling or trigger receptor internalization [38].
UK-427857 is orally bioavailable, has favorable pharmacokinetics, and did
not have any serious adverse effects on healthy volunteers [2]. In a study
evaluating the effects of short-term (10 days) UK-427857 monotherapy on
HIV viral load in HIV patients, subjects received 25 mg of the drug once
a day, 100 mg twice daily, or a placebo for 10 days. The mean CCR5 receptor
saturation at the 100-mg dose was in excess of 90% and the mean decrease
in viral load was 1.42 log10 from baseline to day 11 [80]. At the 25-mg dose,
the mean receptor saturation fell to less than 80% by day 10 and the mean
decrease in viral load was 0.42 log10 [80]. The absence of any severe adverse
effects combined with the potent antiviral properties of UK-427857 make it
an attractive candidate for further development.
4.1.4
CMPD 167
CMPD 167 is a cyclopentane-based compound that can cause rapid and sig-
nicant decline in plasma viremia in rhesus macaques chronically infected
with SIV [108]. Moreover, vaginal application of gel-formulated CMPD 167
prevented vaginal SIV transmission in 2 out of 11 animals, and reduced early
viral replication in all 11 animals receiving the drug compared to control-
treated animals. These results provide a proof of principle for the use of
small-molecule CCR5 inhibitors as a component of a topical microbicide to
prevent HIV-1 sexual transmission. However, CMPD 167 is no longer being

developed as an anti-HIV-1 therapeutic agent.
4.1.5
Spirodiketopiperazine-Based Inhibitors
The rst spirodiketopiperazine (SDP) derivative, E913, was described in 2001.

E913 was active against R5-tropic HIV-1 in vitro, with IC50 values of 3060 nM
in PBMCs [64]. Subsequently, a more potent SDP derivative, AK602/GSK-
873140, was described [31, 62]. In vitro, AK602 had potent antiviral activity
against a wide range of laboratory and primary R5 HIV-1 isolates, including
multidrug-resistant strains, with IC50 values ranging from 0.1 to 0.6 nM. The
variability of this antiviral activity was low (similar to zidovudine), probably
owing to the high potency of AK602 as compared with E913 or TAK-779.
AK602 binds with high afnity to CCR5 (K d =2.91.0) and specically blocks
binding of MIP-1 even though it preserves RANTES and MIP-1 binding
and their functions, including CC chemokine-induced chemotaxis and CCR5
internalization. In a nonobese diabetic SCID mouse model, AK602 elicited
a 2.0 log10 decline in plasma viremia in R5 HIVJRFL -infected mice [63]. In
a phase I clinical trial involving multiple-dose escalation in healthy subjects,
AK602 was well tolerated and had no severe adverse effects. A phase II clinical
trial of AK602 is currently underway.
4.2
CXCR4-Based Antiretroviral Therapy
As mentioned earlier, CXCR4 is the other major coreceptor commonly in-

volved in HIV-1 infection in vivo [43]. CXCR4 is expressed on most CD4-
positive lymphocytes as well as on many cell types that lack CD4 [58]. In some
but not all individuals who progress to AIDS, viruses evolve to use CXCR4
for entry either in addition to or in place of CCR5 [25]. While this coreceptor
switch is not required for disease progression, it does result in expanded viral
tropism, since CXCR4 is more commonly expressed on CD4-positive T cells
than is CCR5 [12].
4.2.1
Small Molecule CXCR4 Inhibitors
Inhibitors of CXCR4 are also under development, but these drugs may be
more difcult to eld because CXCR4, and its ligand SDF-1, are essential for
normal development in the mouse [118]. AMD-3100 is a bicyclam compound
that disrupts the interaction of gp120 and CXCR4 and is capable of blocking
replication of CXCR4 utilizing strains in vitro and in murine models [34, 48,
90]. AMD-3100 is very specic for CXCR4 and does not interact with any other
known CXC- or CC-chemokine receptor [26]. An orally bioavailable AMD-
3100 derivative, AMD-070, is currently in clinical trials. Several other CXCR4
antagonists have been described including polyphemusin analogs T22, T134,
and T140, as well as a D-Arg peptide referred to as ALX40-4C [36, 75]. ALX40-
4C was well tolerated in a phase I clinical trial, suggesting but not proving
that CXCR4 inhibition may be tolerated in adults [35].
5
Resistance to Coreceptor Inhibitors
HIV-1 can acquire resistance to coreceptor inhibitors either by changing the

mechanism or dynamics of coreceptor engagement or by switching coreceptor
usage. The former could involve usage of the same coreceptor in a drug-bound
form, increased afnity of the virus for the coreceptor leading to competitive
removal of bound inhibitor molecules, or easier triggering of Env trimers
resulting in membrane fusion even at low coreceptor density. For R5-tropic
viruses, in particular, coreceptor switching (from R5 to X4) is a matter of
concern because the presence of X4-tropic viruses is associated with advanced
stages of disease and poor prognosis. Many of the studies addressing selection
of viruses resistant to coreceptor inhibitors have used cell lines that express
only one coreceptor [1, 29, 54, 65, 89]. Consequently, the resistant viruses
selected in these studies did not have display coreceptor switching, but rather
had alterations in how the virus bound to the original coreceptor.
In cells expressing both CCR5 and CXCR4, coreceptor switching of viruses
passaged in the presence of coreceptor antagonists has been observed [73,
106]. For instance, when an R5 virus was passaged in the presence of
AD101 (a CCR5 antagonist), the virus evolved to use CCR5 via an inhibitor-
independent mechanism [106]. Similar results were observed with SCH-C,
another CCR5 inhibitor [72, 106]. Finally, when X4 and R5X4-tropic viruses
were passaged in the presence of the CXCR4 antagonist AMD-3100, R5 viruses
expanded in the cultures [41, 46]. When CCR5 viruses persisted in the pres-
ence of AD101, the selected variants had various amino acid changes in Env,
did not use CXCR4 for entry, and were able to replicate efciently in PBMCs
in a CCR5-dependent manner [106]. Despite their newly acquired resistance,
these variants used CCR5 with reduced efciency for entry into cell lines,
implying that resistance might be associated with a loss in viral tness [106].
Another possible mechanism by which HIV-1 could evolve resistance to
CCR5 or CXCR4 inhibitors would be to acquire the ability to use alternative
coreceptors for virus entry. Switching to alternative coreceptors has not yet
been observed. However, in an experimental setting where CCR5-negative
peripheral blood lymphocytes (PBLs) were infected with an R5/X4/CXCR6-
tropic HIV-1 isolate in the presence of AMD-3100, CXCR6-positive PBLs were
preferentially infected [93]. This suggests that CXCR6 can be used as a core-
ceptor by HIV-1 on primary cells, and further that in the presence of CCR5
and CXCR4 inhibitors, variants using CXCR6 (or other minor coreceptor)
might arise in vivo in tissues that express high amounts of minor coreceptors.
It is also worth noting that SIV strains isolated from red-capped mangabeys
often use CCR2 as their major coreceptor, perhaps because many red-capped
mangabeys are CCR5-negative due to a naturally occurring polymorphism
in the CCR5 open reading frame [16]. This represents an example of novel
coreceptor use in the face of strong selective pressure. Theoretically, this could
also occur in humans, though based on the expression patterns and levels of
alternative coreceptors in humans we feel that resistance to CCR5 inhibitors in
humans will likely involve either altered use of CCR5 by virus, or by coreceptor
switching to CXCR4.
6
Impact of Chemokine Receptor Inhibitors
on Clinical Monitoring and Treatment
Accurate and timely clinical monitoring is an integral part of any effective

antiretroviral therapeutic regimen. Viral genotyping to determine specic
resistance-associated mutations is increasingly becoming part of the clini-
cal monitoring process. In the context of coreceptor antagonists, it will be
particularly important to assess the relative proportions of R5, R5X4, and
X4 viruses in individuals prior to initiating treatment. CCR5 inhibitors may
not be benecial to patients with X4 viruses and vice versa for CXCR4 in-
hibitors. Moreover, the possibility of coreceptor switching (from R5 to X4)
will have to be carefully monitored. Monitoring host factors that might im-
pact CCR5 expression could also prove useful in predicting treatment success,
since CCR5 levels can inuence the efcacy of coreceptor antagonists [81].
At present, phenotyping tests on cell lines expressing CCR5 or CXCR4 along
with CD4 are used to measure the relative levels of coreceptor use on patient
samples. However, it is not clear how results from these in vitro tests translate
to coreceptor use in vivo.
As various entry inhibitors move through clinical development, it will be
important to determine if particular combinations of entry inhibitors should
be used. Coreceptor inhibitors effectively reduce the levels of coreceptor avail-
able to virus. As a result, virus entry is either inhibited or the rate of virus
entry is slowed. As a consequence of slower fusion rates, virus becomes more
susceptible to the fusion inhibitor T-20 (enfuvirtide) [81]. Enfuvirtide binds
to a region on gp41 that becomes exposed as a result of CD4 binding, but
that is lost once coreceptor binding triggers membrane fusion [15]. Slower
coreceptor binding causes the T-20 binding site to be exposed for a longer
period of time, resulting in synergistic inhibition of virus fusion [81, 82]. This
nding provides a strong theoretical basis for using coreceptor inhibitors in
conjunction with T-20. Moreover, since T-20 can inhibit both R5- and X4-
tropic viruses, the use of T-20 in conjunction with a CCR5 inhibitor may limit
the evolution of X4-tropic virus strains.
It is also possible that particular combinations of coreceptor inhibitors
could be used together effectively, much like nucleoside and nonnucleoside
reverse transcriptase inhibitors that are used in combination. In fact, a recent
study reported greater synergism of the CCR5 inhibitor AK602/GSK-873140
with CXCR4 inhibitors (such as AMD-3100) than with other classes of anti-
HIV drugs [76]. Another recent study indicates that virus resistance to some
CCR5 inhibitors may not result in resistance to other CCR5 inhibitors. Specif-
ically, a virus resistant to UK-427857 remained sensitive to CCR5 inhibitors
with a different structure [109]. As a result, it may be possible to apply dif-
ferent classes of CCR5 inhibitors in combinationor sequentiallyshould
resistance to one inhibitor arise. In conclusion, coreceptor antagonists consti-
tute an important and valuable new class of drugs, and with careful monitoring
and synergistic use, they can be successfully incorporated into an efcacious
antiretroviral regimen.
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CTMI (2006) 303:121154


Through Virally Encoded Pirated Chemokine Receptors
H. F. Vischer1 C. Vink2 M. J. Smit1 (u)
1 Leiden/Amsterdam Center for Drug Research (LACDR), Division of Medicinal
Chemistry, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1083,

1081 HV Amsterdam, The Netherlands
smit@few.vu.nl
2 Department of Medical Microbiology, Cardiovascular Research Institute Maastricht,
University of Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands
1 Chemokine System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
2 Viral Immune Evasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
3 Herpesvirus-Encoded GPCRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

3.1 -Herpesvirinae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
3.1.1 Roseoloviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
3.1.2 Cytomegaloviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
3.2 -Herpesvirinea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
3.2.1 Rhadinoviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
3.2.2 Lymphocryptoviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
4 Poxvirus-Encoded GPCRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

4.1 Yatapoxviruses, Suipoxviruses, and Capripoxviruses . . . . . . . . . . . . . . . 143
4.2 Avipoxviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Abstract Several herpesviruses and poxviruses contain genes encoding for G protein-
coupled receptor (GPCR) proteins that are expressed on the surface of infected host
cells and/or the viral envelope. Most of these membrane-associated proteins display
highest homology to the subfamily of chemokine receptors known to play a key role in
the immune system. Virally encoded chemokine receptors have been modied through
evolutionary selection both in chemokine binding prole and signaling capacity,
ultimately resulting in immune evasion and cellular reprogramming in favor of viral
survival and replication. Insight in the role of virally encoded GPCRs during the viral
lifecycle may reveal their potential as future drug targets.
122 H. F. Vischer et al.
1
Chemokine System
The chemokines and their cognate receptors play a key role in the immune
system during homeostasis and inammation by coordinating leukocyte
migration, activation, degranulation, and differentiation. In addition, the
chemokine system is involved in organogenesis, angiogenesis, and directing
metastasis and growth of tumor cells (Murphy et al. 2000). The mammalian
chemokine system (e.g., human, mouse, and rat) constitutes of approximately
45 chemokine ligands and 20 chemokine receptors (Murphy 2002).
Chemokines are a family of small proteins that adopt a similar tertiary
folding, even in cases of low overall sequence identity (varying from 20% to
95%). They are characterized by a exible amino-terminal domain, followed
by a conserved core region consisting of a so-called N-loop, three anti-parallel
-strands, and a carboxyl-terminal -helix, stabilized by disulde bonds be-
tween four conserved cysteine residues (Mizoue et al. 1999). Four subclasses
of chemokines (i.e., CC, CXC, CX3C, and XC) have been recognized on the
basis of the number and sequential spacing of the rst two conserved cysteine
residues that are situated near the amino terminus (Zlotnik and Yoshie 2000).
In addition, chemokines can be functionally classied into inducible (inam-
matory) and homeostatic (constitutively expressed) chemokines, mediating
inammation-directed or basal (homing) leukocyte trafcking, respectively
(Proudfoot 2002).
Recruitment of specic leukocyte populations by chemokines is essentially
determined by the spatiotemporal expression of selected chemokine recep-
tors, which belong to the membrane-associated G protein-coupled receptor
(GPCR) family. Chemokine receptors are classied (i.e., CCR111, CXCR1
6, CX3CR1, and XCR1) according to their ability to bind a specic subclass
of chemokines (Murphy 2002). Chemokine receptors are not only expressed
on leukocytes but also on endothelial, smooth muscle, epithelial, stromal,
and neural cells (Onuffer and Horuk 2002). Interestingly, most inammatory
chemokines display a high level of promiscuity by binding several chemokine
receptor subtypes, and vice versa. In contrast, homeostatic chemokines are
generally more specic, each interacting with a single chemokine receptor
subtype (Proudfoot 2002). Given the prominent role of chemokine receptors
in regulating intracellular signaling in response to chemokine ligands, these
receptors are the most promising targets for immunomodulatory therapeutic
interventions (Onuffer and Horuk 2002; Gao and Metz 2003). Interestingly,
such receptors are also employed by several viruses in order to subvert the
immune system and/or redirect intracellular signaling for their own benet
(Alcami 2003).
A Viral Conspiracy: Hijacking the Chemokine System 123
2
Viral Immune Evasion
Viruses are small, infectious, parasitic pathogens that (ab)use the host cell
metabolism and consume cellular biomolecule resources for their replica-
tion. An important strategy which enables viruses to replicate efciently in
a host cell is to interfere with recognition and subsequent elimination of the
infected cell by the immune system. To this end, distinct viruses have em-
ployed different strategies. For instance, large double-stranded DNA viruses,
such as herpesviruses and poxviruses, encode viral mimics of host cytokines
and chemokines, as well as their soluble binding proteins and/or membrane-
associated receptors, to subvert the immune system (Alcami 2003). The viral
genes encoding these proteins have probably been derived from the genomes
of the viral host during evolution. Of particular interest are the viral genes that
code for membrane-associated GPCRs, as these proteins are localized at the
boundary of the extracellular and intracellular milieu, and transmit signals
from the outside to the inside of the cell. The amino acid sequences of the
virally encoded GPCRs (vGPCRs) are generally highly diverged between and
within virus subfamilies. This suggests that these GPCRs have distinct and
specialized functions that are optimized for different biological properties
of each virus. Nonetheless, the majority of vGPCRs display highest sequence
similarity to the subfamily of chemokine receptors (Fig. 1, Tables 1 and 2).
Some vGPCRs are indeed responsive to chemokines, whereas for others no
endogenous ligands have been identied and remain orphan. Importantly,
in contrast to their cellular homologs, a number of vGPCRs signal ligand-
independently (i.e., constitutively). Constitutive GPCR signaling is of ma-
jor signicance as revealed by several pathologies associated with activating
GPCR mutations (Seifert and Wenzel-Seifert 2002). This constitutive activity
of many vGPCRs, together with the current awareness that chemokines and
their receptors play prominent roles in inammatory pathologies and tumor
metastases (Proudfoot 2002), suggests that vGPCRs may be key players in
virus-associated diseases.
3
Herpesvirus-Encoded GPCRs
Herpesviruses have been isolated from a wide variety of vertebrates and are
generally characterized by their strict specicity for a single host species
(Davison 2002). Herpesviruses have been classied into three subfamilies,
the -, -, and -herpesvirinae, on the basis of their biological properties,
Fig. 1 Phylogenetic relationship between host chemokine receptors and virally en-
coded GPCRs. Deduced amino acid (reference) sequences of mouse, rat, and human
chemokine receptors and vGPCRs were retrieved from the GenBank database at NCBI
and analyzed using the ClustalW method (Gonnet series). Chemokine receptor or-
thologs of mouse, rat, and human all cluster in a single branch per subtype, and are
each presented as a single branch for clarity
Table 1 Herpesvirus-encoded GPCRs
Subfamily Genus Species vGPCR Cellular homologa %b
-Herpesvirinae Simplexvirus Human herpesvirus 1 HHV-1 - - -

Human herpesvirus 2 HHV-2 - - -
Varicellovirus Human herpesvirus 3 HHV-3 - - -
-Herpesvirinae Cytomegalovirus Cercopithecine herpesvirus 8 CeHV8 UL33 CCR10 20
(Rhesus cytomegalovirus) UL78 CXCR1 14
Rh214 CCR5 22
Rh215 CXCR1 22
Rh216 CCR1 21
Rh218 CXCR3 22
Rh220 CX3CR1 34
Simian cytomegalovirus SCMV ORF3 CCR4 22
(African green monkey ORF4 CCR3 25
cytomegalovirus) ORF5 CCR2 22
ORF6 CXCR1 21
ORF7 CXCR6 21
Pongine herpesvirus 4 PoHV4 UL33 CCR3 20
(Chimpanzee cytomegalovirus) UL78 CXCR1 13
US27 CXCR3 23
US28 CX3CR1 38
Human herpesvirus 5 HHV-5 UL33 CCR10 21
(Human cytomegalovirus) UL78 Somatostatin R3 12
US27 CXCR3 23
US28 CX3CR1 36
125
Table 1 (continued)
126
Murid herpesvirus 1 MCMV M33 CCR10 21

(Mouse cytomegalovirus) M78 Opiate R-like 1 13
Murid herpesvirus 2 RCMV R33 CCR10 23
(Rat cytomegalovirus) R78 CCR10 15
Tupaiid herpesvirus 1 TCMV T33 CCR10 23
(Tupaia herpesvirus) T78 Formyl peptide R-like 12
Roseolovirus Human herpesvirus 6a HHV-6a U12 CCR10 19
U51 Cysteinyl leukotriene R2 16
Human herpesvirus 6b HHV-6b U12 CCR10 19
U51 Cysteinyl leukotriene R2 16
Human herpesvirus 7 HHV-7 U12 CX3CR1 20
U51 CCR2 16
-Herpesvirinae Lymphocryptovirus Callitrichine herpesvirus 3 CalHV3 ORF6 CXCR5 13
CeHV15 BILF1 CXCR4 14
Human herpesvirus 4 HHV-4 BILF1 CXCR4 15
(Epstein-Barr virus)
Rhadinovirus Alcelaphine herpesvirus 1 AHV1 A5 CCR3 15
Porcine lymphotropic herpesvirus 1 PLHV1 A5 CXCR2 14
Porcine lymphotropic herpesvirus 2 PLHV2 A5 CXCR2 15
Porcine lymphotropic herpesvirus 3 PLHV3 A5 CCR10 13
Bovine herpesvirus 4 BHV4 - - -
Cercopithecine herpesvirus 17 CeHV17 ORF74 CXCR1 24
Human herpesvirus 8 HHV-8 ORF74 CXCR2 26
(KS-associated herpesvirus)
H. F. Vischer et al.
Table 1 (continued)
Macaca fuscata rhadinovirus MFRV ORF74 CXCR1 24

Murid herpesvirus 4 MuHV4 ORF74 CCR4 20
Saimiriine herpesvirus 2 HVS2 ORF74 CXCR2 24
(Herpesvirus saimiri)
Equid herpesvirus 2 EHV2 ORF E1 CCR3 51
ORF E6 CCR10 16
ORF74 CXCR5 22
ORF E1 CCR3 51
a Nearest cellular homologs are identied by basic local alignment search tool (BLAST) analysis of each vGPCR on the human protein
sequence reference database at NCBI, subsequently followed by ClustalW analysis
b Percentage amino acid identity
127
Table 2 Chordopoxvirus-encoded GPCRs
128
Genus Species vGPCR Cellular homologa %b
Avipoxvirus Fowlpox virus FPV FPV021 GPCR1 32

FPV027 GPCR1 29
FPV206 EBV-induced GPCR2 35
Canarypox virus CNPV CNPV039 GPCR1 34
CNPV045 GPCR1 70
CNPV277 EBV-induced GPCR2 36
CNPV315 Chemokine-like R1 28
Capripoxvirus Goatpox virus GTPV - - -
Sheeppox virus SPPV SSPV09 CCR8 40
Lumpy skin disease virus LSDV LSDV011 CCR8 39
Molluscipoxvirus Molluscum contagiosum virus MOCV - - -
Orthopoxvirus Camelpox virus CMPV - - -
Cowpox virus CPV - - -
Ectromelia virus ECT - - -
Monkeypox virus MPV - - -
Vaccinia virus VV - - -
Variola virus VAR - - -
Parapoxvirus Bovine papular stomatitis virus BPSV - - -
Orf virus ORFV - - -
Suipoxvirus Swinepox virus SPV SPV146 CCR8 34
Yatapoxvirus Yaba monkey tumor virus YMTV 7L, 145R CCR8, CCR8 51, 39
Yaba-like disease virus YLDV 7L, 145R CCR8, CCR8 53, 44
Unclassied Mule deer pox DPV83 gp013, gp162 CCR8, CCR4 42, 32
a Nearest cellular homologs are identied by basic local alignment search tool (BLAST) analysis of each vGPCR on the human protein
sequence reference database at NCBI, subsequently followed by ClustalW analysis

H. F. Vischer et al.
b Percentage amino acid identity

genome organization, and deduced amino acid sequence similarity between

conserved gene orthologs (McGeoch et al. 2000). About 43 genes are shared
between most members of the three herpesvirus subfamilies (Davison et al.
2002). These so-called core genes encode proteins that contribute to universal
processes such as viral DNA replication and packaging into the viral capsid.
During the course of coevolution with their host, individual herpesvirus
subtypes acquired unique genes through pirating or gene duplication. Among
these so-called accessory genes are genes that allow the virus to subvert the
host immune response.
3.1
-Herpesvirinae
The -Herpesvirinae subfamily comprises two genera, namely Roseolovirus

and Cytomegalovirus (CMV). Hitherto, four members of the Roseolovirus
genus have been isolated, three of which are found in man. In contrast, host-
specic cytomegaloviruses have been isolated from a wide variety of mammals
from the orders Rodentia (e.g., mouse and rat), Scandentia (e.g., tree shrew),
and Primates (e.g., rhesus macaque, African green monkey, chimpanzee, and
human). CMV genomes are the largest of all herpesviruses (195241 kb),
whereas genomes of roseoloviruses are somewhat smaller (153162 kb). The
genomes of roseoloviruses and CMVs, share extensive characteristics, in-
cluding position and orientation of large blocks of genes (Neipel et al. 1991;
Gompels et al. 1995; Nicholas 1996; Weir 1998).
3.1.1
Roseoloviruses
Three distinct species of Roseolovirus have been isolated from peripheral

blood of humans. Human herpesvirus (HHV)-6A was rst isolated from pe-
ripheral blood mononuclear cells derived from adults with acquired immun-
odeciency syndrom (AIDS) and displaying lymphoproliferative disorders
(Salahuddin et al. 1986). In addition, a second highly related variant of HHV-
6, sharing an overall nucleotide sequence identity of 90% (Dominguez et al.
1999), but displaying distinct biological properties, was formally recognized
and named HHV-6B. The third human roseolovirus, HHV-7, is highly related
to the HHV-6 variants with respect to genome organization as well as se-
quence, with HHV-6 and HHV-7 genes sharing deduced amino acid identities
between 22% and 75% (Nicholas 1996; Dominguez et al. 1999).
Primary infection with HHV-6B occurs between 6 and 12 months of age,
whereas infection with HHV-7 occurs at a later time, though often within
the rst 4 years of childhood (De Bolle et al. 2005). The time of HHV-6A
infection is still unknown, but is thought to occur following infection with
HHV-6B. As a consequence, roseoloviruses are ubiquitously spread in the
general adult population, usually reaching a seroprevalence of greater than
95%. Primary infection with HHV-6b or HHV-7 results in an acute febrile
illness that is in some cases followed by the appearance of a mild skin rash on
the face and trunk (i.e., exanthem subitum or roseola infantum; Yamanishi et
al. 1988; Tanaka et al. 1994). Interestingly, infection with HHV-6A is usually
asymptomatic (Dewhurst et al. 1993; Stodberg et al. 2002; Freitas et al. 2003).
Clinical complications of (primary) HHV-6 and -7 infections include febrile
seizure, but also meningoencephalitis, encephalopathy, and multiple sclerosis
(for a review see De Bolle et al. 2005). Importantly, primary HHV-7 infection
can reactivate HHV-6 (Frenkel and Wyatt 1992; Katsafanas et al. 1996; Tanaka-
Taya et al. 2000). In contrast, reactivation of HHV-6 in healthy children has
been reported to occur usually without clinical consequences (Caserta et al.
2004).
Roseoloviruses are (T-)lymphotropic and replicates most efciently in
vitro CD4+ T lymphocytes (Takahashi et al. 1989), but can also infect various
other cell types in vitro (De Bolle et al. 2005). HHV-6B and HHV-7 replicate
predominantly in salivary glands, with viral shedding into saliva being the
major route of virus transmission (Harnett et al. 1990). After primary infec-
tion, roseoloviruses persist latently in the host in monocytes and early bone
marrow progenitor cells (De Bolle et al. 2005). In healthy individuals, the
pathogenic potential of roseoloviruses is kept under control by the immune
system. However, both HHV-6 and HHV-7 can reactivate under immunosup-
pressive conditions (e.g., in AIDS patients and transplant recipients).
The genome of roseoloviruses contains two GPCR-encoding genes, i.e.,
U12 and U51. The U12 and U51 genes are situated on similar positions and
have a similar orientation as the UL33 and UL78 genes of CMVs, respectively.
The U12 and U51 genes of HHV-6 are expressed with similar late and early
kinetics (Isegawa et al. 1998; Menotti et al. 1999). Temporal expression proles
of the HHV-7-encoded GPCRs have not been reported yet, but are presumably
similar to those observed for the HHV-6- and CMV-encoded receptors.
U12 and U51 display the highest amino acid sequence identity to human
chemokine receptors. Although the shared sequence identity between these
virally encoded receptors and the cellular receptors is rather limited (<20%),
both U12 and U51 are highly responsive to a variety of CC chemokines.
HHV-6B U12 displays high binding afnity for CCL5, CCL4, and CCL2, and
lower afnity for CCL3 (Fig. 2). Moreover, these CC chemokines induce U12-
mediated increases in intracellular Ca2+ levels in stably transfected K562 cells,
via pertussis toxin-insensitive signaling pathways (Isegawa et al. 1998). In-
terestingly, the HHV-7-encoded U12 displays a different chemokine binding

prole than HHV-6B U12 and induces intracellular Ca2+ signaling in sta-
bly transfected K562 cells in response to CCL17, CCL19, CCL21, and CCL22
(Fig. 2), but not CCL1, CCL2, and CCL5 (Nakano et al. 2003; Tadagaki et al.
2005). In addition, expression of HHV-7 U12 in Jurkat cells induces chemotaxis
of these cells towards CCL19 and CCL21 (Tadagaki et al. 2005). Interestingly,
CCL17 and CCL22 are unable to attract U12-expressing Jurkat cells. Hence,
besides CCR7 and its cognate ligands, U12 expression on the surface of HHV-
YLDV
7L
HHV7
HHV7 U51
U51
HHV7
U12 HHV7
HHV7
U51
U12 8 HHV7
9 10 HHV7
7 4 U51
3 U12
1 6
19 25 27 HHV7
1 18 17 7
16 20 U12
1 15 21 4
MHV68 14 22 1
5
ORF74 homeostatic homeostatic
13 1
HVS2 &
4 1
ORF74 HHV8 12 inflammatory 2 YLDV
ORF74 7L
2 8 HHV5
MHV68 HHV8 1 1 HHV6
ORF74 ORF74 2 HHV6 US28
U51
2 2 2 U12
HHV8 2 HHV6 HHV5
ORF74 3 3 1 4 5 US28
U12
4 4 1 5 8 HHV6 HHV5 YLDV
5 U12 US28
HVS2 2 inflammatory 5 1 7L
ORF74 3
4 5
6 7 HHV6
1 1
HVS2 2 2 U12 HHV6
ORF74
7 8 3
HHV6 U51 HHV5
2 2 US28
EHV2 5 U51 HHV5
8 11
ORF74
1
9 13
3 US28 YLDV
2 HHV6 7L
3 10 23 2 U51
HHV8 HHV5
11 24 3
ORF74 3 14 26 1 US28
16 1 28 EHV2
HVS2 HHV8 3 3 HHV6
3 U51 E1
ORF74 ORF74 HHV8 6 1 3 HHV5
ORF74 HHV5 US28
HHV5 10
US28 US28
HHV5
US28
CC-chemokines CC-receptor betaherpesvirus

CXC-chemokines (ELR-) CXC-receptor
gammaherpesvirus
CXC-chemokines (ELR+) CX3C-receptor
XC-chemokines Yatapoxvirus
XC-receptor
CX3C-chemokines
Fig. 2 Chemokine binding of vGPCRs. Homeostatic as well as inammatory

chemokines bind a specic subset of herpesvirus-encoded GPCRs. Chemokines are
depicted on the inner circle. Their cognate chemokine receptors are displayed next to
them, followed by vGPCRs from the -herpesvirus, -herpesvirus, and yatapoxvirus
families that were shown to bind the respective chemokines
7-infected T lymphocytes may also support homing of these cells into lymph
nodes, which may contribute to viral dissemination.
Despite its low sequence similarity with chemokine receptors, the HHV-6-
encoded U51 displays an overlapping chemokine binding prole with HHV-6
U12. This protein binds CCL5 and CCL2 with high afnity, but is unable to bind
CCL3. In addition, HHV-6 U51 efciently binds CCL7, CCL11, CCL13, and
HHV-8-encoded viral macrophage inammatory protein (vMIP)-II (Milne
et al. 2000). Interestingly, when transfected into adherent cells (HEK293 or
143tk cells), HHV-6 U51 accumulates predominantly intracellularly in the
endoplasmic reticulum and cannot be detected on the cell surface. In contrast,
U51 is readily detectable on the cell surface of transfected T lymphocytic cell
lines as well as on HHV-6-infected cord blood mononuclear cells in vitro
(Menotti et al. 1999). Hence, expression of U51 at the cell surface appears to
be cell type-specic, and requires trafcking functions that are apparently
present in activated T cells and monocytes, but not in adherent cell types.
Interestingly, stable expression of HHV-6 U51 in epithelial cells results in
morphological alterations consisting of increased spreading and attening of
the cells and downregulation of CCL5 expression and secretion (Milne et al.
2000). The latter may contribute to immune evasion of the infected cells. The
mechanism(s) by which U51 modulates epithelial cell functioning remains to
be elucidated, but may include constitutive and/or autocrine U51-mediated
signaling as observed for other vGPCRs (Milne et al. 2000). Moreover, in view
of the impaired trafcking of U51 to the cell membrane as observed in some
adherent cell lines, U51 cell membrane expression needs to be conrmed in
epithelial cells.
Despite the low amino acid sequence identity between HHV-7 U51 and
U12, these proteins induce intracellular Ca2+ elevation in response to the
same chemokines (Tadagaki et al. 2005). In contrast, however, U51-expressing
Jurkat cells were not able to migrate towards any of the tested chemokines
(Tadagaki et al. 2005).
3.1.2
Cytomegaloviruses
Human CMV (HCMV) or HHV-5 is a widely spread virus, with a seropreva-

lence ranging from 50% to 80%, and it is able to persist lifelong in a latent
form. Primary infection of immunocompetent hosts is usually asymptomatic.
In contrast, primary infection or reactivation of the virus in immunocom-
promised hosts, such as the developing fetus, transplant recipients, or AIDS
patients, can have severe implications and be fatal (Zhou et al. 1996). Common
complications after HCMV infection include damage of liver, brain, retina,
and lung (interstitial pneumonitis; Landolfo et al. 2003). Coinfection of HCMV

with human immunodeciency virus (HIV) has been shown to accelerate pro-
gression to AIDS and dementia in HIV patients (Webster 1991; Kovacs et al.
1999). Increasing evidence suggests that HCMV may also contribute to the de-
velopment of vascular diseases, e.g., atherosclerosis, restenosis, and vascular
allograft rejection (Zhou et al. 1995; Burnett 2001).
CMV primarily infects monocytes, smooth muscle cells, and endothe-
lial and epithelial cells of the upper gastrointestinal, respiratory, or urogenital
tracts (Landolfo et al. 2003), and disseminates throughout the body by latently
infected monocytes in the blood (Streblow and Nelson 2003). Allogenic stimu-
lation of these monocytes induces differentiation into macrophages, which in
latently infected cells is accompanied by reactivation of HCMV leading to the
release of infectious virions (Streblow and Nelson 2003). CMV, like other -
and -herpesvirus subfamilies, appears to have pirated genes encoding key
regulatory cellular proteins, showing highest homology to chemokine recep-
tors (Murphy 2001; Sodhi et al. 2004b). HCMV encodes four GPCRs referred to
as US27, US28, UL33, and UL78 (Chee et al. 1990). Two GPCR-encoding genes,
i.e., UL33 and UL78, are conserved with respect to position and orientation in
the genomes of all sequenced -herpesviruses. Possibly, these genes have been
captured from an ancient host species by an ancestral -herpesvirus. Interest-
ingly, except for the intronless UL33 genes of rhesus macaque CMV (RhCMV
or cercopithecine herpesvirus 8) and rat CMV (RCMV), all UL33 genes consist
of two exons interrupted by a single intron. CMV-encoded UL33 orthologs
are fairly well conserved and display deduced amino acid sequence identities
varying from 35% to 68% (Fig. 1). In contrast, amino acid sequences have
diverged considerably between the UL78 orthologs of individual CMV species
(12%54% sequence identity), suggesting a reduced selective pressure on this
protein during the course of evolution.
Intriguingly, CMVs infecting host species of the primates (i.e., human,
chimpanzee, African green monkey, and rhesus macaque) have pirated an
additional GPCR-encoding gene cluster compared with CMVs that infect
species from the Glires (i.e., mouse and rat) or Scandentia (i.e., tree shrew)
orders. This gene cluster is located in the unique short (US) region of the
CMV genome, which is not present in nonprimate CMV genomes, and con-
sists of two adjacent genes in HCMV and chimpanzee CMV (CCMV) (US27
and US28), and ve juxtaposed genes in RhCMV and simian CMV (SCMV)
(Table 1). Interestingly, the divergence in sequence and number of genes in
this gene cluster parallels the coevolution of primate CMVs with two dif-
ferent host species families, with HCMV and CCMV infecting members of
the Hominidae family (human and chimpanzee, respectively) and RhCMV
and SCMV infecting Old World monkeys (rhesus macaque and African green
monkey, respectively). In addition, the highest amino acid sequence identities

within proteins encoded by this gene cluster as well as common chemokine
binding proles are observed between rh220, cUS28, and hUS28 (34%71%),
suggesting that these genes have emerged through duplication and rapid
diversication of an ancestral US28-like gene.
The genes encoding US28 and UL78 are expressed with early kinetics,
whereas US27 and UL33 genes are transcribed with late kinetics (Mocarski
1996). The late and early expression kinetics of UL33 and UL78, respectively,
are similar to those of their corresponding U12 and U51 counterparts in Rose-
olovirus. In addition, CMV-encoded GPCRs are constituents of the virion, with
UL33 (Margulies et al. 1996), UL78 (Oliveira and Shenk 2001), US28, rhUS28.5
(Penfold et al. 2003), and presumably US27 being expressed on the viral en-
velope. Colocalization of US28 (Fraile-Ramos et al. 2001), US27, and UL33
(Fraile-Ramos et al. 2002) with two major HCMV envelope glycoproteins, i.e.,
glycoprotein B and H, on virus-wrapping membranes of endocytotic vesi-
cles in transfected or HCMV-infected cells, indicates that these GPCRs are
incorporated in the viral envelope.
Although expression of CMV-encoded receptors seems not to be essential
for infection of permissive cells in vitro, deletion of either R33/M33 (Davis-
Poynter et al. 1997; Beisser et al. 1998) or R78/M78 (Oliveira and Shenk 2001;
Kaptein et al. 2003) has signicant impact on viral dissemination in vivo.
A reduced replication in salivary glands and a lower mortality in infected
animals is apparent in in vivo studies using recombinant RCMV and mouse
CMV (MCMV) strains, lacking the corresponding UL33 and UL78 genes (see
Vink et al. 2001 for references), underlining the importance of these receptors
in the pathogenesis of infection.
The HCMV-encoded receptor US28 is so far the best characterized HCMV-
encoded GPCR. It possesses a large spectrum chemokine-binding prole, in-
cluding binding of a number of inammatory CC as well as CX3CL chemokines
(Gao and Murphy 1994; Kuhn et al. 1995; Kledal et al. 1998; Penfold et al. 2003;
Fig. 2). This broad-spectrum binding prole suggests that US28 could act as
a chemokine scavenger and thereby aid in subversion of the immune sys-
tem (Kuhn et al. 1995; Kledal et al. 1998). CC chemokines, which are shown
to bind to US28, induce increasing levels of intracellular calcium, activa-
tion of mitogen-activated protein kinase (MAPK) and focal adhesion kinase
(FAK). Interestingly, infection of smooth muscle cells with CMV leads a US28-
dependent migration. Hence, activation of the former signaling pathways by
US28 may provide a molecular basis for the involvement of HCMV in the
progression of atherosclerosis. These effects appear to be primarily G12/13
mediated and involve activation of tyrosine kinase-linked signaling pathways
(Streblow et al. 2003).
Despite the sequence similarity to chemokine receptors and US28, US27

does not seem to interact with chemokines. Therefore, this receptor is still
classied as an orphan receptor. Interestingly, US28 is able to alter cellular
signaling in a constitutive manner when expressed in COS-7 cells and af-
ter HCMV infection (Casarosa et al. 2001; Casarosa et al. 2003a). US28 is
considered a versatile signaling device since it is able to activate multiple sig-
naling networks in a constitutively active manner via activation of effectors
and transcription factors within infected cells [i.e., InsP production, nuclear
factor (NF)-B, cAMP-response element (CRE), and nuclear factor activated
T cell (NFAT)]. US28 shows promiscuous G protein coupling, through primar-
ily Gq, Gs , and G12 proteins (Casarosa et al. 2001; Waldhoer et al. 2002; Casarosa
et al. 2003a; Minisini et al. 2003). The chemokine receptor homologs, on the
other hand, do not displayor display only limitedligand-independent
signaling and activate primarily Gi/o proteins (Offermanns 2003). Interest-
ingly, US28-mediated, constitutive signaling potentiates chemokine-induced
signaling of the Gi -coupled CCR1 (Bakker et al. 2004). Since HCMV pri-
marily infects leukocytes, smooth muscle, and endothelial cellsin which
chemokine receptors play prominent rolesHCMV-encoded receptor ex-
pression may alter ligand-induced signaling via these receptors and contribute
to the CMV-induced pathology.
US28-mediated signaling (constitutive or ligand-dependent) is accompa-
nied by G protein receptor kinases (GRK)-mediated phosphorylation of the
C-terminal tail, which is followed by a rapid constitutive, agonist-independent
endocytosis into perinuclear endosomes and recycling of the receptor US28.
The observed internalization of US28 is not dependent on the constitutive
activity but involves the C-terminal tail, which serves as a docking site for
-arrestins as well as other scaffolding proteins (Brady and Limbird 2002;
Heydorn et al. 2004; Lefkowitz and Shenoy 2005). Binding of these proteins
appears important for intracellular signaling and/or receptor trafcking. In-
terestingly, however, US28 internalization via clathrin-coated pits or lipid rafts
is independent of -arrestins but requires AP-2 adaptor complex proteins and
dynamin (Fraile-Ramos et al. 2003; Droese et al. 2004).
CC chemokines do not modulate the constitutive signaling of US28 in
the InsP, NF-B, and CRE assays (Casarosa et al. 2001; McLean et al. 2004),
while CX3CL1 acts as inverse agonist in these assays (Casarosa et al. 2001).
When US28 loses its capacity to constitutively internalize upon deletion of
its C-terminal tail, the CC chemokines, as well as CX3CL1, activate these
signaling pathways instead (Waldhoer et al. 2003). Thus, differential modu-
lation of constitutive US28 internalization kinetics and the cellular context in
which US28 is expressed determine the efcacy of chemokines acting at this
receptor.
The broad chemokine binding prole in combination with rapid and

constitutive internalization kinetics (Fraile-Ramos et al. 2001) allows US28
to sequester inammatory chemokines efciently from the environment of
HCMV-infected cells. As a consequence, the recruitment of leukocytesand
therefore the inammatory responsemay be hampered (Fig. 3; Bodaghi et
al. 1998; Billstrom et al. 1999; Randolph-Habecker et al. 2002).
Fig. 3 Suggested roles for vGPCRs. Constitutive (1) or ligand-dependent signaling (2)
of a vGPCR results in up-/downregulation of gene expression (3), including autocrine
and/or paracrine (angiogenic) factors (4) as well as cellular GPCR proteins (5). Due
to their broad-spectrum chemokine binding prole, vGPCRs may serve as chemokine
decoy receptors by internalizing bound chemokine, thereby limiting the immune re-
sponse (6). Binding of vGPCRs (US28) to membrane-associated CX3CL1 facilitates
cellcell adhesion (7). vGPCR-mediated chemotaxis in response to chemokine stim-
ulation may increase viral dissemination and other pathogenesis (8). In addition,
vGPCRs may act as HIV coreceptor (9)
The constitutive activity of US28 can be inhibited by a small nonpeptidergic

inverse agonist VUF2274 (Casarosa et al. 2003b) derived from a CCR1 antag-
onist (Hesselgesser et al. 1998). VUF2274 dose-dependently inhibits US28-
mediated constitutive activation of phospholipase C in both transfected and
HCMV-infected cells, and US28-mediated HIV entry. Importantly, VUF2274
inhibits CCL5 binding in a noncompetitive manner, thus acting as an allosteric
modulator. Although a gain in afnity is required, these inverse agonists will
serve as valuable tools to further determine the role of (constitutive activity
of) US28 in CMV infection.
For UL33, like US28, constitutive signaling has been reported in transfected
and infected cells, while no signaling has been detected for UL78 (Casarosa
et al. 2003a). Both UL33 and UL78 still remain orphan. In addition, R33 and
M33 are able to signal in a constitutively active manner (Gruijthuijsen et al.
2002; Waldhoer et al. 2002). The constitutive signaling of R33 differs from that
of UL33 in that R33 is only able to couple to Gi/o and Gq , while UL33 shows
activation of the Gq , Gi/o , and Gs classes.
Taken together, CMVs may effectively use their virally encoded receptors
to orchestrate multiple signaling networks within infected cells. Importantly,
the immediate-early (IE) promoter of HCMV, constituting the genetic switch
for progression of viral infection and reactivation, contains four consensus
CRE and four NF-B binding sites. Binding of cognate transcription factors
to these sites is required for efcient transactivation of the immediate early
promoter (Hunninghake et al. 1989; Keller et al. 2003; DeMeritt et al. 2004; Lee
et al. 2004). Moreover, NF-B is a ubiquitously expressed transcription factor
that plays a critical role in the regulation of inducible genes in immune re-
sponse and inammatory events associated with e.g., atherosclerosis (Chen et
al. 1999). NFAT is an important regulator of immune responses, developmen-
tal processes, and angiogenesis (Horsley and Pavlath 2002). It is suggestive to
propose that US28 and UL33, through constitutive activation of these tran-
scription factors, induces expression of viral IE proteins and cellular proteins,
leading to alteration of the immune response in favor of viral survival and
spreading and may contribute by this means to the onset, progression, or
enhancement of inammatory disorders. Further studies in cellular systems
more relevant to HCMV infection are required to elucidate the role of these
receptors in CMV pathology.
3.2
-Herpesvirinea
The -Herpesvirinea family is subdivided into the Lymphocryptovirus and

Rhadinovirus genera (Table 1). Although -herpesviruses have cell transform-
ing potential, their associated malignancies are mostly seen in the context of
immune suppression, such as HIV coinfection or iatrogenic immune sup-
pression, suggesting these viruses are normally controlled by the immune
system.
3.2.1
Rhadinoviruses
Hitherto, about 48 species of the Rhadinovirus genus have been isolated from
a wide variety of mammals, of which 8 genomes have been fully sequenced.
HHV-8, also known as Kaposis sarcoma-associated herpesvirus (KSHV), is
the only human rhadinovirus identied to date, and was rst discovered in
Kaposis sarcoma (KS) skin lesion of an AIDS patient (Chang et al. 1994;
Cesarman et al. 1995; Renne et al. 1996). In contrast to the ubiquitous (and
infectious) dissemination of most other herpesviruses within their natural
host populations, HHV-8 displays a rather low infectivity rate and is unevenly
distributed among geographically disparate human populations (Hayward
1999). HHV-8 seroprevalence is low (<5%) in the general population of most
European, Asian, and American countries, but can range from 10% to 40% in
some Mediterranean countries (Hayward 1999) and 40% to 100% in African
countries (Dedicoat and Newton 2003). In addition, HHV-8 seropositivity
is highly prevalent among homosexual men (Verbeek et al. 1998). HHV-8
establishes lifelong, latent infections in pre- and post-germinal center B cells
and endothelial precursor cells (Dupin et al. 1999), which is characterized by
the expression of only a limited number of viral genes (Jenner et al. 2001).
While HHV-8 infection of healthy individuals is usually without severe
pathogenic consequences, immune suppression (e.g., in AIDS or transplant
recipients) can result in impaired control of HHV-8, leading to multifocal
angioproliferative KS lesions (Sturzl et al. 1997) and/or lymphoproliferative
diseases (primary effusion lymphomas, multimeric Castlemans disease).
Like other herpesviruses, HHV-8 has captured a cellular gene from its
hosts, ORF74, which resembles chemokine receptors. The ORF74 receptor
shows signicant similarity with the human chemokine receptor CXCR2,
known to play an important role in angiogenesis, embryonic development,
wound healing, and tissue regeneration. Importantly, constitutive expression
and signaling of ORF74 induce focus formation in stably transfected NIH3T3
cells, which is accompanied by an increased production and secretion of vas-
cular endothelial growth factor (VEGF), a major angiogenesis activator (Bais
et al. 1998). Moreover, these ORF74-expressing cells form highly vascularized
tumors that resemble KS when injected into nude mice (Bais et al. 1998). Like-
wise, transgenic mice expressing HHV-8-encoded ORF74 in hematopoietic or
vascular endothelial cells develop angioproliferative KS-like lesions (Yang et

al. 2000; Guo et al. 2003; Sodhi et al. 2004c).
ORF74 can constitutively couple to G proteins of the Gq/11 , Gi/0 , and G13
classes, thereby modulating a multitude of intracellular signaling pathways,
including phospholipases, adenylyl cyclases, kinases, and small G proteins
(Arvanitakis et al. 1997; Rosenkilde et al. 1999; Couty et al. 2001; Mon-
taner et al. 2001; Shepard et al. 2001; Smit et al. 2002). Importantly, HHV-8
ORF74-mediated (constitutive) signaling is (partially) increased by angio-
genic chemokines [containing a Glu-Leu-Arg (ELR) amino acid motif in the
N-terminus]. CXCL8 (ligand for CXCR2) acts as a low-potency partial/neutral
agonist (Rosenkilde et al. 1999; Rosenkilde and Schwartz 2000; Couty et al.
2001; Smit et al. 2002; McLean et al. 2004), while the non-ELR, angiostatic
chemokines CXCL10 and CXCL12 (ligands of CXCR3 and CXCR4, respec-
tively) and HHV-8-encoded vMIP-II decrease constitutive ORF74 signaling,
thus acting as inverse agonists (Fig. 2). Importantly, constitutive signaling by
HHV-8 ORF74 as well as chemokine modulation of this constitutive activity
are prerequisites for the oncogenic potential of ORF74 in vivo (Holst et al.
2001; Sodhi et al. 2004c). ORF74-mediated modulation of intracellular signal-
ing networks leads to increased transcription of cellular gene and paracrine
factors regulating a range of cellular processes including transformation,
proliferation, and immortalization (Bais et al. 1998, 2003). HHV-8ORF74-
mediated upregulation and secretion of proangiogenic growth factors and
chemokines by lytic cells recruits neighboring cells that can be subsequently
infected by released virions (Sodhi et al. 2004a).
Examination of biopsies of KS lesions from AIDS patients revealed high
phosphorylated (activated) Akt (PKB) levels, suggesting a critical role for
this antiapoptotic serinethreonine kinase in the onset and progression of KS
pathology (Sodhi et al. 2004c). Moreover, inhibition of the PI3K/PDK1/Akt
pathway prevented proliferation and survival of ORF74-expressing endothe-
lial cells in vitro, and inhibited their tumorigenic potential upon allografting
into nude mice (Sodhi et al. 2004c). ORF74 activates Akt by stimulating PI3K
through G-subunits of both pertussis toxin-sensitive and -insensitive G pro-
teins (Montaner et al. 2001), but also via phospholipase C (PLC)-dependent
protein kinase C and p44/42 MAPK activation (Smit et al. 2002). In addition,
ORF74 activates Akt indirectly in an autocrine manner by upregulating both
the expression of VEGF (Bais et al. 1998; Sodhi et al. 2000) and its cognate
receptor KDR2 (Bais et al. 2003). Upregulation of growth factors, chemokines,
and cytokines in even a few ORF74-expressing cells drives angioproliferative
tumor formation by paracrine stimulation of neighboring cells that are la-
tently infected with HHV-8 (Montaner et al. 2003; Jensen et al. 2005). Despite
its oncogenic potential, ORF74 is not sufcient for inducing KS in immuno-
competent individuals as indicated by one case of KS in every 17,000 HHV-

8 infections. ORF74 is primarily expressed during (early) lytic replication,
which occurs in about 3% of the spindle-shaped tumor cells within KS lesions
(Kirshner et al. 1999; Sun et al. 1999), whereas the majority of cells in KS lesions
are latently infected with HHV-8 (Staskus et al. 1997). Moreover, continuous
expression of HHV-8ORF74 appears to be essential for the progression of KS
(Jensen et al. 2005). In this respect, it is puzzling how transient expression of
HHV-8ORF74 in lytic cells can cause KS. However, dysregulated expression
of ORF74 under certain circumstancessuch as HIV-1 coinfection, inam-
mation, or aborted lytic cycle progressionhas been hypothesized to result
in non-lytic (continuous) expression of ORF74 in a fraction of KS tumor cells
(Sodhi et al. 2004a). In fact, the KS incidence increases about 10,000-fold in
HIV-1-infected man, and 100,000-fold in HIV-1-infected homosexual men
(Gallo 1998; Reitz et al. 1999), whereas HHV-8-infected transplant recipients
have a 500-fold increased risk in developing KS (Cathomas 2003).
In contrast to the ability of HHV-8ORF74 to constitutively activate a mul-
titude of signaling pathways by coupling to Gq , Gi , and G12/13 proteins (Bais
et al. 1998; Munshi et al. 1999; Rosenkilde et al. 1999; Sodhi et al. 2000), the
ORF74 proteins encoded by nonhuman rhadinoviruses activate a narrower
range of G proteins in a ligand-independent manner. Herpesvirus saimiri-
encoded ORF74 (i.e., HVSORF74 or ECRF3) signals constitutively via Gi
and G12/13 proteins, but not through coupling to Gq (Rosenkilde et al. 2004).
The ORF74 protein encoded by equid herpesvirus 2 (i.e., EHV2ORF74) only
activates Gi -mediated pathways in a constitutive manner (Rosenkilde et al.
2005), whereas ORF74 of murine -herpesvirus 68 (i.e., MHV68ORF74), also
known as murine herpesvirus 4 (MuHV4), is devoid of any constitutive activity
(Verzijl et al. 2004). Both constitutive (Gi and G12/13 ) and non-constitutive (Gq )
HVSORF74-mediated signaling pathways can be stimulated by CXCL1 and
CXCL6, whereas CXCL5 and CXCL8 act as neutral antagonists (Rosenkilde
and Schwartz 2004). Interestingly, the non-ELR CXC chemokines that act
as inverse agonists on the HHV-8ORF74, do not bind the HVSORF74.
Likewise, both human and mouse CXCL1 and CXCL2 stimulates MHV68
ORF78-mediated activation PLC, NF-B, p44/p42 MAPK, and Akt, as well as
the inhibition of cAMP formation, whereas non-ELR CXC, CC, and CX3C
chemokines were ineffective (Verzijl et al. 2004). In contrast to the broad
chemokine binding prole of HHV-8, EHV2, and MHV68ORF74, only
a single chemokine (CXCL6) binds to EHV2ORF74, resulting in a further
increase of its constitutive Gi -mediated signaling (Rosenkilde et al. 2005).
Despite the apparent lack of constitutive activity, MHV68ORF74 expres-
sion in NIH3T3 cells induces focus formation by these cells (Wakeling et al.
2001). This transforming potency of MHV68ORF74 may result from consti-
tutively signaling through yet-unidentied signaling pathways (Verzijl et al.

2004). Alternatively, autocrine secretion of mouse CXCL1 (i.e., KC) by NIH3T3
cells (Bosio et al. 2002) may activate the MHV68ORF74 that is expressed on
the cell surface of these cells. In fact, MHV68ORF74-mediated signaling in
response to mouse CXCL1 enhances in vitro viral replication in permissive
NIH3T3 cells (Lee et al. 2003). In contrast, disruption of the MHV68ORF74
gene did not affect in vitro replication of MHV68 in infected NIH3T12 cells,
or in vivo replication in spleen and lungs (Moorman et al. 2003). Interestingly,
MHV68ORF74 appeared to be essential for efcient reactivation of MHV68
from latency (Lee et al. 2003; Moorman et al. 2003). Like other ORF74 genes,
MHV68ORF74 is an early lytic gene but is also expressed in latently infected
cells (Kirshner et al. 1999; Sun et al. 1999; Wakeling et al. 2001).
Interestingly, ORF74-encoding genes are absent in two rhadinoviruses:
the bovine herpesvirus 4 and alcelaphine herpesvirus 1. In contrast to
other members of the Rhadinovirus genus that are sequenced to date, the
EHV2 genome contains three additional vGPCR-encoding ORFs adjacent
to the conserved ORF74 (Telford et al. 1995). Interestingly, the hitherto
uncharacterized ORF E6 displays highest sequence identity to the A5
receptors of alcelaphine herpesvirus 1 and the lymphotropic porcine her-
pesviruses 13. Moreover, this subfamily of rhadinovirus-encoded GPCRs
is homologous to the lymphocryptovirus-encoded BILF1 receptors (see
the next section). The other two ORFs are gene duplicates that encode
the E1 protein. These ORFs are located in the terminal direct repeat
elements on both ends of the genome. E1 displays highest sequence identity
to members of the cellular CC chemokine receptor family (30%51%)
and poxvirus-encoded CC chemokine receptors (25%30%). Despite the
relatively high sequence identity with a variety of CC chemokine receptors,
only the CCR3-specic chemokine CCL11 was able to induce an E1-mediated
increase in intracellular Ca2+ levels and chemotaxis (Camarda et al. 1999;
Fig. 2).
3.2.2
Lymphocryptoviruses
In contrast to other herpesviruses, lymphocryptoviruses (LCV) have only

been isolated from higher primate species of the infraorder Simiiformes.
Hitherto, about 44 distinct LCVs have been identied (Ehlers et al. 2003).
The LCV genomes which have been sequenced include those infecting man
[i.e., HHV-4 or Epstein-Barr virus (EBV)], common marmoset [(i.e., cal-
litrichine herpesvirus 3 (CalHV3)], and rhesus macaques [i.e., cercopithecine
herpesvirus 15 (CeHV15)] have been fully sequenced (Table 1).
LCVs are ubiquitous (>90%) B lymphotropic viruses that establish life-

long, generally asymptomatic, persistent infections in memory B lympho-
cytes (Wang et al. 2001). However, the potency of LCV to transform B
lymphocytes can result in acute infectious mononucleosis, as well as ma-
lignant lymphomas, such as Hodgkins and Burkitts lymphomas, and post-
transplant/AIDS-associated lymphomas (Middeldorp et al. 2003; Thorley-
Lawson and Gross 2004). In addition, EBV has been directly associated with
nasal natural killer (NK)T cell lymphoma, nasopharyngeal and gastric car-
cinoma, oral hairy leukoplakia, and leiomyosarcoma (Middeldorp et al. 2003;
Thompson and Kurzrock 2004). Such lymphomas are thought to arise from
proliferating, infected B cells that are blocked in the transition from nave
to memory B cells, and/or are not efciently eliminated by cytotoxic T cells.
Hence, individuals with deciencies in T cell-mediated immunity (e.g., post-
transplant immunosuppression and AIDS) are in particular risk of developing
EBV-associated lymphoproliferative diseases (Rivailler et al. 2004; Thorley-
Lawson and Gross 2004).
The genome of LCVs contains one gene coding for a vGPCR, which is
transcribed in various EBV-positive tumor cells (Beisser et al. 2005). LCV-
encoded GPCRs show very limited amino acid sequence identity (<15%)
to any cellular GPCR (Table 1). Nevertheless, functional analysis revealed
that the EBV BILF1 protein is a functional membrane-associated GPCR that
constitutively activates NF-B and CRE signaling pathwaysboth implicated
in cell proliferationin a Gi ,-dependent manner (Beisser et al. 2005; Paulsen
et al. 2005). In addition, BILF1 constitutively inhibits phosphorylation of the
RNA-dependent protein kinase, which is important for antiviral responses
(Beisser et al. 2005). Hitherto, BILF1 is still considered an orphan receptor,
and information on its biological relevance is yet unknown.
4
Poxvirus-Encoded GPCRs
The Poxviridae is a family of large, brick-shaped, double-stranded DNA

viruses. A characteristic of these viruses is that they replicate in the cyto-
plasm of infected cells, independent of the host nuclear machinery. Poxvirus
infections are characterized by acute febrile illness accompanied by skin le-
sions that blister and form pockmarks. Infections are often self-limiting. Some
species of poxvirus, however, can cause life-threatening infections in certain
hosts (e.g., variola virus or smallpox infections in human). Most poxviruses
are epitheliotropic and transmitted by direct contact or via the respiratory
tract (Diven 2001). Many poxviruses are able to infect a range of host species.
Poxviruses may reside in a reservoir host in which viral infection results in

mild, subclinical conditions. However, transfer of the virus to a zoonotic host
often causes more severe pathologies (McFadden 2005).
The poxvirus family is divided into the Entomopoxvirinae and Chor-
dopoxvirinae subfamilies, which infect insects or vertebrates, respectively
(Table 2). Genome analysis and phylogenetic analysis of multiple deduced
amino acid sequences divide the Chordopox genera in four (to ve) main
subgroups (Gubser et al. 2004; see Table 2). Interestingly, the genomes of
avipoxviruses, capripoxviruses, suipoxvirus, and yatapoxviruses contain one
or more putative GPCR-encoding genes (see Table 2).
4.1
Yatapoxviruses, Suipoxviruses, and Capripoxviruses
The Yaba-like disease virus (YLDV) contains two genes, 7L and 145R, encoding
for membrane-associated proteins that display 53% and 44% amino acid
sequence identity with CCR8 (Lee et al. 2001). YLDV-encoded 7L protein,
but not 145R, displays a similar chemokine binding prole to CCR8, and
binds hCCL1, hCCL7, hCCL4, hCCL17, vMIPI, and vMIPII, but not by mCCL1
(Najarro et al. 2003). In addition, 7L couples to G proteins and induces p44/p42
MAPK phosphorylation in response to CCL1 stimulation. Protein expression
analyses of YLDV-infected cells revealed that 7L is expressed as early as
2 h postinfection and its expression increases with time. Blocking late gene
expression using a viral DNA replication inhibitor resulted in a 26% decrease
in 7L protein expression, suggesting that 7L displays both early and late gene
expression kinetics.
The mechanism by which 7L exactly interferes with the CCR8-mediated
adaptive and innate immune response has not yet been determined. How-
ever, considering the upregulation of CCL1 secretion by dendritic cells, mast
cells, and dermal endothelial cells in certain skin inammations (Gombert
et al. 2005), resulting in the recruitment of CCR8-expressing T cells and
Langerhans-type dendritic cells, it is tempting to speculate that 7L may se-
quester CCL1 from the environment of infected cells to impair the immune
response. In fact, CCR8 appears to be a vulnerable target for viral hijacking,
as several viruses specically target this receptor by mimicking its ligands
(e.g., HHV-8-encoded vMIP-I and vMIP-II, and the molluscum contagiosum
virus-encoded vMCC-1) or expressing membrane-associated CCR8 mimics.
Alternatively, 7L-mediated signaling in response to CCL1 may also activate
anti-apoptotic as well as migratory signaling pathways, as observed for CCR8
(Haque et al. 2001; Louahed et al. 2003; Spinetti et al. 2003; Haque et al. 2004),
thereby increasing cell survival and viral dissemination.
Genomes of sui- and capripoxviruses contain a single GPCR gene, of

which the deduced amino acid sequences display highest sequence identity
to CCR8 (Table 2). However, no pharmacological data are yet available for
these receptors.
4.2
Avipoxviruses
The genomes of the fowlpox and canarypox viruses of the Avipoxvirus genus
contain 3 and 4 ORFs encoding for vGPCRs. These vGPCRs share about 24%
sequence identity with some members of CXC chemokine receptor family,
but share more identity with GPCR1 and EBV-induced GPCR2. Neverthe-
less, this unique cluster of avipoxvirus-encoded GPCRs still awaits functional
characterization.
5
Conclusions
Exploitation of the chemokine receptor system through molecular mimicry

appears to be an effective means to assist viruses in evading immune surveil-
lance, thus contributing to viral dissemination and virus-induced pathology
(Fig. 3). Infection of cells and consequent expression of viral chemokine recep-
tors enables them to respond to a broad spectrum of chemokines, evading the
immune response or facilitating viral dissemination to areas with increased
chemokine expression (Figs. 2 and 3). The ability of the viral chemokine re-
ceptors to signal in a constitutively active manner via promiscuous G protein
coupling turns them into versatile signaling devices that modulate cellular sig-
naling networks, thereby reprogramming the cellular machinery to modulate
cellular function after infection.
Although many attractive roles have been attributed to this class of recep-
tors, little is known about their (patho)physiological potential. The biological
signicance of ORF74 and the members of the UL33 and UL78 family in the
pathogenesis of HHV-8 and CMV infections has been demonstrated in vivo.
Mouse models and studies using recombinant rodent CMVs that carry a dis-
rupted gene or lack the respective gene (Davis-Poynter et al. 1997; Bais et
al. 1998; Beisser et al. 1998, 1999; Yang et al. 2000; Oliveira and Shenk 2001;
Guo et al. 2003; Kaptein et al. 2003; Sodhi et al. 2004c; Streblow et al. 2005)
indicate a role for these viral receptors in pathophysiology. GPCRs consti-
tute a highly drugable class of membrane-associated proteins, accounting for
about 50% of protein targets for therapeutic interventions. In addition, the
awareness that chemokines and their cognate receptors play a prominent role
in numerous pathophysiological processes urges the quest for bioavailable
small-molecule antagonists that specically block viral chemokine receptor
functioning (Onuffer and Horuk 2002). Small nonpeptidergic compounds in-
hibiting US28 constitutive signaling can be considered as tools to investigate
the role if US28 in CMV pathology and may serve as promising therapeutics
for clinical antiviral intervention. Also for the other viral chemokine recep-
tors, however, specic (pharmacological or RNA interference) inhibitors or
antibodies targeting these viral chemokine receptors is essential to elucidate
the contribution of viral chemokine receptors to viral pathogenesis and reveal
their potential as a future drug target.
Acknowledgements H.F.V. was supported by the Technology Foundation STW, and

M.J.S. and C.V. by the Royal Netherlands Academy of Arts and Sciences.
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Subject Index
adoptive transfer 11 CD4+ T cells 4

AK602/GSK-873140 107 CD8+ lymphocytes 81, 82
alternative coreceptor 100 CD8+ T cells 3
AMD-070 108 chemokine receptor 6, 8386
AMD-3100 108 CMPD 167 106
antibody 4 coronaviruses 2
apoptosis 71, 75, 82, 83, 86, 87 coxsackievirus 9
astrocytes 4, 70, 75, 76, 7880, CXCL10 6, 76, 77, 79, 80, 83, 84
8487 Cxcl10 73
CXCL12 8284
BILF1 141, 142 CXCL9 6, 80
bloodbrain barrier 81 CXCR3 6, 83
CXCR4 82, 8486, 100
caprine arthritis encephalitis virus
(CAEV) 69, 70 dendritic cells (DCs) 8
CCL12 77, 78
CCL2 6, 7684, 86 ECRF3 see HVS-ORF74
Ccl2 73 EHV2ORF74 140
CCL22 82, 83 enfuvirtide 110
CCL3 6, 7680, 82, 83, 85 envelope 73, 74, 80, 83, 85
Ccl3 73 epitope spreading 5
CCL4 6, 7679, 82, 85 equine infectious anemia virus 70
Ccl4 73
CCL5 6, 7679, 82, 83, 85 feline immunodeciency virus (FIV)
Ccl5 73 69, 71, 72, 77, 7981, 84, 86
CCL7 6, 7779, 86 fractalkine 5
CCR1 6, 83, 84, 86 FrCasE 6971, 79
CCR10 84
CCR2 6, 78, 80, 81, 8385 T cells 5
CCR2-64I polymorphism 102
CCR5 6, 78, 8286, 100, 101 HHV-8ORF74 see ORF74
CCR5 59029 G/A single-nucleotide HIV 6971, 77, 7986
polymorphism 102 HIV Tat 80, 82, 86
CCR5 32 101, 103 HIV tropism 98
CD4 98 M-tropic 100
role as HIV receptor 98 T-tropic 100
156 Subject Index
human T cell leukemia virus (HTLV) paramyxovirus 9

69, 70 progression to AIDS 100,
HVSORF74 140 102, 103
IL-1 79 145R 143

IL-1 73 R33 134, 137
IL-1 86 R78 134
IL-1 80 RAG1| mice 4
IL-4 79 RANTES 100, 103, 105, 107
IL-6 79 derivatives of RANTES 103
inuenza virus 9 receptor US28 134
interferon (IFN)- 4, 79 resistance to coreceptor
inhibitor 108
7L 143
lymphocytes 71, 79, 81, 86 SCH-C 105
lymphotactin 5 SCH-D 106
SDF-1 100, 107
SDF-1 100, 102
M33 134, 137
secondary lymphoid organs 10
M78 134
SIV 6972, 77, 7982, 8486
macrophages 4, 70, 71, 75, 76, 78,
79, 81, 82, 8587
TAK-220 105
MHV68ORF74 140, 141 TAK-652 105
microglia 4, 70, 71, 73, 75, 76, TAK-779 104
7881, 85, 86 Theilers virus 5
MIP-1 100, 103, 105, 107 TR1.3 70, 71
MIP-1 100, 103, 105, 107 tumor necrosis factor (TNF)
Mol-ts1 70, 79 73, 7682, 85, 86
Moloney-ts1 69 tumor necrosis factor receptor I
multiple sclerosis (MS) 4 (TNFRI) 79, 82
tumor necrosis factor receptor I
neurons 69, 70, 8284, 86 (TNFRII) 82
neuroprogenitor stem cell 83 type I interferons 7
migration 87
U12 130132
oligodendrocytes 70, 73 U51 130, 132
ORF74 138, 140 UK-427857 106
angiogenesis 138, 139 UL33 130, 133, 134, 137
chemokine-induced UL78 130, 133, 134, 137
signaling 139 US27 133135
constitutive signaling 139, 140 US28 133135, 137
CXC-chemokine 139 allosteric modulator 137
expression 140, 141 CC-chemokine 134, 135
Kaposis sarcoma 138140 chemokine binding 134,
oncogene 139 136, 137
vascular endothelial growth factor chemokine-induced
(VEGF) 138, 139 signaling 135
Subject Index 157
chemotaxis 134 migration 134

constitutive signaling protein localization 134
135, 137
CX3CL-chemokine 134, 135 V3 loop 101
endocytosis 135 primary determinant of viral
expression 134 tropism 101
gene 133, 134 visna 69, 70
HIV coreceptor 137
internalization 135, 136 xenotropic/polytropic receptor 86

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303

With 14 Figures and 7 Tables

Cover Illustration by Thomas E. Lane (this volume)

Library of Congress Catalog Number 72-152360

(MHV) results in an orchestrated expression of chemokines whose function

8. Salazar-Mather TP, Orange JS, Biron CA (1998) Early murine cytomegalovirus

Functional Diversity of Chemokines and Chemokine Receptors

Cytokine and Chemokine Networks: Pathways to Antiviral Defense . . . . . . . . . . 29

Inuence of Proinammatory Cytokines and Chemokines

HIV-1 Coreceptors and Their Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

A Viral Conspiracy: Hijacking the Chemokine System

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Functional Diversity of Chemokines and Chemokine

2 Orchestrated Expression of Chemokines and Chemokine Receptors

3 Chemokines, Innate Immune Response, and MHV-Infection of the CNS . 8

4 Chemokines and Chemokine Receptors

5 Chemokines and Chronic Viral-Induced Demyelination . . . . . . . . . . . . 14

Coronaviruses are classied on the basis of several fundamental characteris-

Coronavirus genomes are single-stranded positive-polarity RNA mole-

The protective immune response to MHV infection is characterized predomi-

cise mechanism or mechanisms by which CD4+ T cells assist CD8+ T cells

Viral persistence in white matter tracts results in a chronic demyelinating

compared to CD8+ T cell recipients, and this supports a more important

Instillation of MHV into the CNS of susceptible mice results in a well-

Table 1 Chemokine gene expression following MHV infection of the CNS

Days post infection Chemokine Function (cells attracted) Reference(s)

Table 2 Chemokine receptors expressed within the CNS of MHV-infected mice

Days post infection Receptor Chemokine receptor Reference(s)

13 CCR2 T cells, macrophages 13, 39

spread of the virus throughout the parenchyma. In situ hybridization indi-

Using CCL3/ mice, we have demonstrated a role for CCL3 in regulating

CXCL9 and CXCL10 attract activated T lymphocytes following binding to

CCL5 is a T cell and macrophage chemoattractant that has been shown to

CCR5 is a member of the CC chemokine receptor family that is expressed on

CCL2 is capable of regulating the pathobiology of various inammatory

Expression of chemokines has been associated with demyelinating plaque

This chapter highlights mechanisms by which chemokines participate in

Fig. 2AF Chemokines and MHV-induced demyelination. Persistent MHV infection

Clearly, these observations indicate that chemokine signaling is an integral

of these molecules in a model of immune-mediated demyelination. Further,

60. Luster AD (1998) Chemokineschemotactic cytokines that mediate inamma-

104. Williamson JS, Sykes KC, Stohlman SA (1991) Characterization of brain-inl-

Cytokine and Chemokine Networks:

2 NK Cells and Antiviral Defenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

3 CCL3/MIP-1: Primary Mediator of NK Cell Inammation . . . . . . . . . . 34

4 CCL2/MCP-1: The First Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

5 MCP-1 and Macrophage Recruitment . . . . . . . . . . . . . . . . . . . . . . . . . . 38

The host response to microbial pathogens necessitates the integrated action

Furthermore, viruses that evade immune detection by restricting the func-

Resistance to MCMV infection is critically dependent on the activation of

As discussed above, NK cells are an essential source of IFN- production

inability to mount an effective NK cell inammatory response, do not sustain

One downstream consequence of the MIP-1-dependent NK cell in-

As discussed above, IFN-/ clearly plays a role in promoting the recruit-

In multiple models of hepatic injury, resident macrophages have been shown

It is clear from previous studies that MCP-1 is an important innate chemokine

Fig. 3af Characterization of MCMV-induced liver damage in MCP-1- and CCR2-

The studies highlighted in this chapter dene a network of cytokine and

Fig. 4 Model of cytokine and chemokine interactions critical to antiviral defense

Acknowledgements Research in the authors laboratory is supported by National In-