Distinct and separate roles for herpesvirus-conserved

UL97 kinase in cytomegalovirus DNA synthesis

and encapsidation
Dana G. Wolf*, Charmain Tan Courcelle, Mark N. Prichard†, and Edward S. Mocarski‡
Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305-5124

Communicated by I. Robert Lehman, Stanford University School of Medicine, Stanford, CA, December 7, 2000 (received for review September 12, 2000)

The human cytomegalovirus UL97 kinase, an important target of domain lead to ganciclovir resistance (7, 9–11). Recently, an
antiviral therapy, has an impact on at least two distinct phases of interesting class of potent, orally bioavailable nonnucleosidic
viral replication. Compared with wild-type virus, the UL97 deletion antiviral compounds has been developed, which includes benz-
mutant exhibits an early replication defect that reduces DNA imidazole- and sulfonamide-based derivatives (12).§ Based on
accumulation by 4- to 6-fold, as well as a late capsid maturation genetic mapping of resistance mutations, these compounds
defect responsible for most of the observed 100- to 1000-fold inhibit viral replication independently of viral DNA polymerase
reduction in replication. Block-release experiments with the anti- or DNA synthesis. 2-Bromo-5,6-dichloro-1-(␤-D-ribofuranosyl)-
viral 2-bromo-5,6-dichloro-1-(␤-D-ribofuranosyl)-benzimidazole re- benzimidazole (BDCRB) and the related compound 2,5,6-
vealed an important role for UL97 kinase in capsid assembly. trichloro-1-(␤-D-ribofuranosyl)benzimidazole block the viral
Although cleavage of concatemeric DNA intermediates to unit- DNA encapsidation process (13, 14). Both benzimidizole deriv-
length genomes remained unaffected, progeny mutant virus mat- atives are chemically related to 5,6-dichloro-2-(isopropylamino)-
uration was delayed, with accumulation of progeny at significantly 1-L-ribofuranosyl-1H-benzimidazole (also called 1263W94), an
reduced levels compared with wild type after release of this block. antiviral compound that inhibits replication by directly affecting
Transmission electron microscopy confirmed the aberrant accumu- a UL97-dependent step in replication.¶
lation of empty A-like capsids containing neither viral DNA nor an
The UL97 gene product is a member of a family of serine兾
internal scaffold structure, consistent with a failure to stably
threonine protein kinases conserved in all mammalian herpes-
package DNA in mutant virus-infected cells. The function of UL97
viruses (15) especially with regard to domains conserved in
in DNA synthesis as well as capsid assembly suggests that protein
protein kinases. Alphaherpesvirus homologs of UL97, such as
phosphorylation mediated by this herpesvirus-conserved kinase
UL13 of herpes simplex virus (HSV) and ORF47 of varicella
increases the efficiency of these two distinct phases of virus
zoster virus (VZV), have been shown to phosphorylate viral
replication.
immediate early proteins and cellular proteins (16–22) and have
been implicated in both regulation of viral gene expression
H uman cytomegalovirus (CMV), a betaherpesvirus, is a
major cause of disease in immunocompromised individuals,
including AIDS patients and recipients of blood or bone marrow
(16–22) and tissue tropism (23). From this work, the CMV UL97
kinase is predicted to phosphorylate protein substrates, and,
although autophosphorylation by recombinant UL97 protein has
allografts or of solid organ transplants (1). CMV is also a

MICROBIOLOGY
been reported (24, 25), this kinase retains the distinction of being
common cause of congenital infection leading to neurological
the member of this family that is best known for its ability to
damage and hearing loss (1).
phosphorylate nucleoside analogs (8, 9, 26). The alphaherpes-
CMV DNA synthesis proceeds in a manner similar to that of
virus UL97 homologs are nonessential for replication in cell
other herpesviruses (2), with a set of six viral DNA synthesis
culture (17, 27), yet the HSV UL13 shows a cell type-dependent
proteins, including the DNA polymerase (UL54), directing the
production of concatemeric DNA intermediates that are subse- impact on the levels of viral replication and late protein expres-
quently cleaved to genome-length units and packaged into sion (16). The stage of replication that UL97 kinase controls has
preformed capsids (2). The precise molecular events of CMV not been established. A UL97 deletion mutant (28) exhibits a
capsid assembly and DNA packaging are not yet understood, severe growth defect, with a 100- to 1000-fold reduction in virus
although these processes are thought to occur in a stepwise yield. This growth phenotype, along with the documented im-
fashion analogous to these processes in double-stranded DNA portance of the UL97 gene product as a unique target for novel
bacteriophages (3–5). Three capsid types, termed A, B, and C, antiviral therapy, prompted us to assess the nature of the block
are found in CMV-infected cells (2, 4, 5). Type B capsids
represent maturation intermediates consisting of a capsid shell
Abbreviations: CMV, cytomegalovirus; HSV, herpes simplex virus; BDCRB, 2-bromo-5,6-
with an internal scaffold core. DNA packaging into these dichloro-1-(␤-D-ribofuranosyl)benzimidazole; PFA, phosphonoformate; HF human fore-
precursors displaces the scaffold structure to yield C capsids that skin fibroblast; hpi, hours postinfection; moi, multiplicity of infection; QC-PCR, quantitative
mature by budding through nuclear membranes. Type A capsids, competitive PCR.
lacking both DNA and a scaffold core, are aberrant matur- *Present address: Department of Clinical Microbiology and Infectious Diseases, Hadassah
ation products resulting from failure to stably package viral University Hospital, Jerusalem, Israel 92210.

DNA (4, 5). †Present address: Aviron, Mountain View, CA 94043.

All antiviral drugs currently used for the treatment of systemic ‡To whom reprint requests should be addressed. E-mail: mocarski@stanford.edu.
CMV infection, including ganciclovir, foscarnet, and cidofovir, §Hallenberger, S., Trappe, J., Buerger, I., Bender, W., Eckenberg, P., Goldman, S., Haerter,
ultimately target the CMV DNA polymerase (6). Of these, M., Reefschlaeger, J. & Weber, O. Interscience Conference on Antimicrobial Agents and
Chemotherapy, September 26 –29, 1999, San Francisco, abstr. 203.
ganciclovir is the most widely used for treatment and prophylaxis ¶Davis, M. G., Talarico, C. C., Underwood, M. R., Baldanti, F. & Biron, K. K., 23rd International
of CMV disease (6, 7). Ganciclovir must be phosphorylated to Herpesvirus Workshop, August 1–7, 1998, York, United Kingdom.
a triphosphate form to inhibit the viral DNA polymerase (7). The publication costs of this article were defrayed in part by page charge payment. This
Initial phosphorylation of ganciclovir is carried out by the viral article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
UL97 kinase (8, 9) such that mutations in the UL97 kinase §1734 solely to indicate this fact.

PNAS 兩 February 13, 2001 兩 vol. 98 兩 no. 4 兩 1895–1900

to replication imposed by the deletion. These studies reveal a
biphasic phenotype, including an inhibition during the early
phase of viral DNA synthesis followed by a block in the late phase
of infection affecting capsid maturation, that establishes a role
for this kinase at distinct steps in viral replication.

Materials and Methods

Cells, Viruses, and Drug Inhibition-Release Experiments. Human
foreskin fibroblasts (HFs) were cultured and used to propagate
CMV strain AD169 (American Type Culture Collection, ATCC)
and UL97 deletion mutants RC⌬97.08 and RC⌬97.19 (28) as
previously described (29). Where appropriate, phosphonofor-
mate (PFA) (300 ␮g兾ml; Sigma) or BDCRB (20 ␮M, from L. B.
Townsend, University of Michigan) was added after a 1-h period
of virus adsorption to cells that were incubated for 96 h
postinfection (hpi). At that time cultures were washed five times
with PBS and then incubated in drug-free medium. Duplicate
samples were used for all drug block-release studies. Growth
curves were carried out in freshly plated HFs infected at a
specified multiplicity of infection (moi). Samples of infected-cell
supernatant were removed at designated time points and stored
at ⫺80°C before titration by plaque assay on HFs.

Analysis of Viral DNA. Total cellular DNA was isolated at specified

times from virus-infected (moi of 0.1–1) and mock-infected cell
monolayers. For total cellular DNA, cells were collected in 1 ml
of 1 mM EDTA, 10 mM Tris䡠HCl (pH 7.4), 0.5% SDS, and 0.5
mg兾ml proteinase K (lysis buffer), incubated at 65°C for 2 h, and
subjected to extraction with phenol and chloroform before
ethanol precipitation. For isolation of encapsidated (DNase-
resistant) DNA, cells were scraped into 1 ml of 10 mM Tris䡠HCl
(pH 7.4), 10 mM KCl, 1.5 mM MgCl2 and sonicated before the Fig. 1. Accumulation of viral DNA in cells infected with UL97 mutant
addition of DNase I (Promega) at a concentration of 50 ␮g兾ml. RC⌬97.08 (⌬97) and parental strain AD169. (A) DNA accumulation curves of
After incubation at 37°C for 2 h, SDS (to 0.5%), EDTA (to 1 DNA copies per well from QC-PCR (0, 24, 48 hpi) and blot hybridization (72, 96,
mM), and Proteinase K (to 0.5 mg兾ml) were all added before 168 hpi) after infection of HFs with an moi of 1. E, AD169; ■, RC⌬97.08. (B)
incubation at 65°C, followed by DNA extraction as described DNA blot hybridization to detect levels of viral DNA after a 96-h PFA-induced
above. To follow the accumulation of viral DNA, total cellular DNA synthesis block (D) and release (R) for a subsequent 24, 48, 72, or 96 h.
Total cellular DNA was digested with BamHI and blot hybridized to detect a
DNA was digested with BamHI, separated by electrophoresis on 2.3-kb viral BamHI fragment.
a 0.8% agarose gel, transferred to nitrocellulose, and hybridized
with radiolabeled BamHI fragment probe (AD169 nucleotides
122,699–124,902) prepared by random primed incorporation of Generation of UL97-Expressing Cell Line. U373-MG glioblastoma
[␣-32P]dCTP (Amersham Pharmacia) as described (29). After astrocytoma cells (ATCC) were transfected with plasmid
hybridization, a PhosphorImager with IMAGEQUANT software pLXSN-UL97, containing the entire UL97 gene (CMV nucle-
was used to calculate CMV DNA levels. For measurements of otides 140,460–142,716) from plasmid pMAL97 (24) inserted
small DNA copy numbers, quantitative competitive PCR into EcoRI and XhoI sites in pLXSN (32) and the neo resistance
(QC-PCR) was performed as previously described (30). To gene. Cells resistant to 400 ␮g of G418 per ml were isolated, and
detect the presence of viral genomic termini in DNA samples, individual colonies were amplified and screened for their ability
DNA was digested with EcoRI, separated as described above, to confer ganciclovir sensitivity on TK-mutant HSV-1 (33). Two
and hybridized with EcoRI terminal fragment W probe, pON227 colonies, denoted UL97–9 and UL97–21, were selected for use.
(31).
Transmission Electron Microscopy. Cells grown on coverslips in
Analysis of Viral Proteins. Rabbit polyclonal antiserum (R␣IE1- 24-well plates were infected at an moi of 0.1. When appropriate,
IE2) raised against glutathione S-transferase fusion proteins cells were fixed in 2% glutaraldehyde in phosphate buffer,
containing UL122 and UL123 was used to detect IE1 p72, IE2 postfixed with 1% aqueous uranyl acetate, dehydrated through
p86, and the late IE2 p40 proteins, and mouse monoclonal graded ethanol steps, and embedded in Poly兾Bed (Polysciences).
antibodies 1202 and 1205B (Goodwin Institute, Plantation, FL) Thin sections (70 nm) were then prepared and stained with 1%
were used to detect ppUL44 and pp65, respectively. Horseradish uranyl acetate and 1% lead citrate for electron microscopy on a
peroxidase-conjugated rabbit anti-mouse IgG (Dako) or goat Phillips CM12 microscope.
anti rabbit IgG (Vector Laboratories) was used as a secondary
antibody. Actin was detected with the use of goat polyclonal Results
anti-actin and rabbit anti-goat IgG (Santa Cruz Biotechnology). UL97 Role in DNA Synthesis. Total infected cell DNA was collected
For protein analyses, total proteins from infected (moi of 1) or from cells infected with UL97 mutants RC⌬97.08 and RC⌬97.19
mock-infected cells were harvested at the indicated times and (28) or parental CMV strain AD169 to assess the accumulation
stored in sample buffer at ⫺20°C until analyzed by immunoblot of viral DNA. Low quantities of DNA before 48 hpi necessitated
(24) that was visualized by Enhanced Chemiluminescence (Am- the use of QC-PCR up to that time point. Blot hybridization was
ersham Pharmacia). used for later time points. Mutant viruses displayed 4- to 6-fold

1896 兩 www.pnas.org Wolf et al.

 Fig. 3. DNA blot hybridization showing accumulation and cleavage of viral
DNA in cells infected with UL97 mutant RC⌬97.08 (⌬97) and parental strain
AD169 (moi of 0.1) after BDCRB drug block (D) and release (R) at the indicated
times. Total cellular DNA was digested with EcoRI and subjected to blot
analysis to detect L-S junction (upper arrow) and L terminal (lower arrow)
fragments, with the use of radiolabeled probe pON227.

readily detected in mutant virus infected cells at earlier time

points. Similar results were obtained with mutant RC⌬97.08
(data not shown). Further analysis of 35S- or [33P]orthophos-
phate-labeled total infected cell proteins at 24, 48,72, and 96 hpi
did not reveal significant differences between mutant and wild
Fig. 2. Immunoblot analysis of proteins expressed during RC⌬97.19 (⌬97) or
AD169 infection of HFs (moi of 1). The four panels (from Top to Bottom) show
type (data not shown). These observations demonstrate that
detection of IE1 and IE2 antigens with the use of R␣IE1-IE2, ppUL44 antigen synthesis of viral proteins, including replication proteins, is not
with the use of monoclonal antibody 1202, pp65 (ppUL83), antigen with the appreciably altered by the disruption of UL97 under conditions
use of monoclonal antibody 1205B, and cellular actin loading control with the where replication levels are significantly reduced.
use of goat polyclonal antiserum. Size markers (in kDa) are indicated on the
right. M, mock-infected. UL97 Role in the Late Phase of Viral Replication. The differences in
DNA synthesis or protein expression did not account for the
poor replication of the UL97 mutant. Consequently, we inves-
less viral DNA accumulation at late times compared with wild tigated the behavior of mutant virus during the late phase of viral
type (Fig. 1A shows data for RC⌬97.08). To determine whether replication by employing the reversible encapsidation inhibitor
a preelongation step was involved in this difference, the revers- BDCRB. BDCRB is known to inhibit cleavage of concatemeric
ible antiviral compound PFA was used to block the elongation viral DNA molecules as well as packaging of progeny DNA into
step of wild-type and mutant virus DNA synthesis. The input nucleocapsids (13, 14). Accumulation and cleavage of viral DNA
viral DNA present during 96 h of PFA block remained barely during BDCRB block release were evaluated by DNA blot
detectable by blot hybridization (Fig. 1B, D96 lanes). Between 24 hybridization by following the appearance of L-S junction and

MICROBIOLOGY
and 48 h after drug release, DNA accumulation resumed and terminal fragments after digestion with EcoRI. Hybridization
increased through 96 h (Fig. 1B, R24 through R96 lanes). with EcoRI W probe revealed both terminal and junction
Analysis of the hybridization signal showed that mutant virus fragments in cleaved monomeric progeny DNA but only junction
DNA accumulation was similar to wild type at 96 h, although it fragments in concatemeric DNA intermediates. During BDCRB
appeared to be elevated above wild type at earlier times after block (96 hpi), only noncleaved DNA intermediates accumu-
drug release. These levels were also influenced by a slightly lated, as reflected by the presence of junction but not terminal
higher level of input mutant virus DNA at 24 hpi as measured fragments in EcoRI digests (Fig. 3, D96 lanes). After drug
by QC-PCR (data not shown). Similar results were obtained with release, terminal fragments were detectable in mutant infected
mutant virus RC⌬97.19 (data not shown). This finding was cells within 24 h, indicating that cleavage proceeded soon after
consistent with a role for UL97-mediated protein phosphoryla- BDCRB was removed, and the amount of viral DNA continued
tion events in the preelongation phase of viral DNA synthesis. to increase, indicating ongoing DNA synthesis. We observed a
significant increase in the appearance of junction fragment in
Viral Protein Accumulation. Alphaherpesvirus UL97 homologs both viruses up to 72 h after reversal, with a continued increase
such as HSV-1 UL13 mediate phosphorylation of regulatory in wild type to 96 h after reversal. The ratio of terminal to
proteins that affect viral protein synthesis (16–21). To determine junction fragment bands was difficult to compare, although this
whether putative protein modifications or the reduced level of ratio appeared to be greater in the mutant DNA samples. The
DNA synthesis in UL97 mutants were accompanied by reduced band representing the terminal fragment was sharper in mutant
or delayed protein expression, we examined the levels of repre- virus DNA than in wild type because of less a sequence
sentative early and late viral proteins after infection with mutant heterogeneity. Parental wild-type AD169 virus strain is highly
RC⌬97.19 or wild-type virus (Fig. 2). No differences in the heterogeneous, which makes direct comparison difficult (34) and
accumulation of ␣ proteins IE1491aa and IE2579aa were detected contributes to the apparent difference in the ratio of junction and
at 24, 48, 72, and 96 hpi in cells infected with either virus. terminal fragments. Whereas the DNA accumulation兾cleavage
Importantly, IE2 p40 protein (␥IE2331aa), a ␥2 (true late) protein, of wild type and mutant were synchronized by the BCDRB-
was readily detected in mutant infected cells at 96 hpi. The ␤2 induced block, the yield of mutant virus remained reduced by
(early-late) protein ppUL44 (DNA polymerase processivity fac- three orders of magnitude after BDCRB release (Fig. 4A). Thus,
tor) and the ␥1 (early-late) protein pp65 both accumulated in the difference between mutant and wild-type virus yield after
mutant infected cells to levels slightly less than those in wild type BDCRB synchronization was similar to the difference observed
at late times (96 hpi); however, ppUL44 appeared to be more when drug was not included (Fig. 4B and ref. 28). The discrep-

Wolf et al. PNAS 兩 February 13, 2001 兩 vol. 98 兩 no. 4 兩 1897

 Fig. 5. Analysis of total and encapsidated viral DNA in AD169-, RC⌬97.08-,
and RC⌬97.19-infected cells. (A) DNA blot hybridization showing accumula-
tion of total and DNase-resistant viral DNA in cells infected with mutant
RC⌬97.08 (⌬97) and parental strain (moi of 1). Total infected cell DNA was
isolated before or after DNase digestion (⫹DNase) at the indicated times (hpi).
Fig. 4. Growth curves for RC⌬97.08 and AD169. (A) Growth curves during Viral DNA digestion and hybridization were carried out to identify junction
BDCRB block-release. HFs were infected (moi of 0.1), and supernatant progeny and terminal EcoRI fragments (arrows) as indicated for Fig. 3. M, mock-
virus were harvested at the indicated times after infection or after drug infected. (B) Quantitative analysis of total and DNase-resistant (⫹DNase) viral
release (R) and titered on HF monolayers. (B) Growth curves for RC⌬97.08 and DNA in AD169-, RC⌬97.08-, and RC⌬97.19- (⌬97) infected cells at the times
AD169. HFs were infected (moi of 1), and supernatant progeny virus was indicated. Quantitation was carried out with the use of blot hybridization for
harvested at the indicated times after infection and titered on HF monolayers. all samples, except for the DNase-resistant mutant DNA, and QC-PCR was used
■, RC⌬97.08; E, AD169. for all samples, yielding values similar to blots at higher DNA levels. The mean
DNA copy number values, derived from three independent experiments, are
displayed. Empty bars, total AD169 DNA; stippled bars, DNase-resistant AD169
ancy between DNA accumulation兾cleavage synchronization and DNA; black bars, total RC⌬97 strains DNA; hatched bars, DNase-resistant
virus yield suggested an important second role for UL97 in RC⌬97 strains DNA.
maturation.
Based on QC-PCR, the ratios of total DNA to encapsidated
UL97 Role in Viral DNA Encapsidation. To determine whether the
DNA accumulating in mutant RC⌬97.08-infected cells was being DNA in mutant RC⌬97.08- or RC⌬97.19-infected cells was
packaged, total infected cell DNA was harvested either directly 200:1–300:1 at 72 or 96 hpi, whereas the ratio in wild-type AD169
from cells (total DNA) or after a DNase I digestion step virus-infected cells was 5:1–10:1. These results indicate a severe
(DNase-resistant) to degrade nonencapsidated viral DNA. encapsidation defect in the mutant virus.
When mutant virus-infected cells were subjected to DNase
treatment at 72 or 96 hpi, junction and terminal fragments were UL97 Role in Capsid Maturation. The presence and distribution of
not detected (Fig. 5A; compare Lane 10 to Lane 7, and Lane 11 capsid assembly intermediates were evaluated by transmission
to Lane 8), indicating that viral DNA was not encapsidated at electron microscopy in thin sections of HFs infected with mutant
these times during mutant virus infection. In contrast, the RC⌬97.08 or wild-type virus (Fig. 6). Empty capsids accumulated
junction fragment was reduced, but the terminal fragment was in the nuclei of mutant virus-infected cells at late times after
less affected when wild-type virus-infected cells were evaluated, infection that were morphologically similar to A capsids (Fig. 6 A
consistent with expected packaging of mature virion DNA (Fig. and B). These capsids contained neither viral DNA nor internal
5A; compare Lane 5 to Lane 2, and Lane 6 to Lane 3). Aliquots core structures (Fig. 6B). The mean ratio of mutant RC⌬97.08 A:C
of the same DNA samples were subjected to QC-PCR to capsids was 9:1, based on counts of representative fields from
compare the total and DNase-resistant (encapsidated) viral several different sections. Similar results were obtained with mutant
DNA (Fig. 5B). Given the detection sensitivity of 1–10 DNA RC⌬97.19 (data not shown). In contrast, the A:C capsid ratio in
copies per well, quantitation was possible on samples containing wild-type virus infected cells was 1:3, because of a marked increase
DNA at levels below the detection limits of blot hybridization. in the numbers of C capsids as well as a significantly reduced

1898 兩 www.pnas.org Wolf et al.

 was used. These results showed transcomplementation of the DNA
packaging defect exhibited by the UL97 mutant virus in UL97-
expressing cells, confirming the role of UL97 in the efficient
formation of DNA-containing capsids.

Discussion
This study has defined a role for the UL97 kinase, a herpesvirus-
conserved protein kinase, in two distinct phases of the viral
replication cycle, DNA replication and capsid assembly. A
preelongation step in DNA synthesis and a late step in genome
encapsidation are both less efficient in the absence of this kinase.
UL97 did not appear to influence viral protein expression levels
in the manner reported for UL97 homologs HSV UL13 and
varicella-zoster virus ORF 47, which are known to phosphorylate
viral immediate early regulatory proteins and to exert an impact
on viral gene expression (16–23). Even though the activities of
this herpesvirus-conserved kinase are not as well established in
CMV as in other herpesviruses (26, 35), the impact of UL97 on
DNA synthesis and capsid assembly is best ascribed to its role as
a protein kinase. This phenotype, which has been readily dem-
onstrated in cultured HFs with the CMV UL97 mutant, is much
more striking than that observed with kinase mutants of HSV-1
and varicella-zoster virus, where a critical requirement for the
viral kinase, when recognized, has been tissue specific (16, 23).
The partial replication defect of the CMV mutant and the lack
of a phenotype of either HSV-1 or varicella-zoster virus kinase
mutants in cell culture suggests that viral kinase function is
complemented by host cell kinases as a result of cell cycle or
differentiation state. The kinases related to UL97 carried by all
mammalian herpesviruses may therefore be predicted to have
roles similar to those described here.
DNA accumulation was consistently reduced in the UL97
deletion mutant compared with wild type, which, together with
the ability of PFA block-release to reverse the synthesis defect,
suggested that UL97-dependent protein phosphorylation events
precede replication fork assembly. These events may be linked to
origin recognition or protein–protein interactions that precede
active replication. Such a role for this viral enzyme is supported
by a preliminary report that UL97 controls the phosphorylation

MICROBIOLOGY
state of the DNA polymerase processivity factor ppUL44.储
Fig. 6. Transmission electron micrographs of virus-infected cells at 72 hpi. (A
and B) HFs infected with mutant RC⌬97.08. (C and D) HFs infected with strain
Furthermore, treatment with 1263W94, which directly blocks
AD169. (E) U373-vec cells infected with mutant RC⌬97.08. (F) UL97–9 cells UL97 kinase activity, is associated with disruption of DNA
infected with mutant RC⌬97.08. Arrows in B and E point to empty A capsids. replication compartments.** Interestingly, 1263W94 has also
Arrows in D and F point to DNA-containing capsids. [Magnification: A and C, been shown to inhibit the synthesis of Epstein–Barr virus DNA
⫻3,800; B, D–F, ⫻35,000. Size bars: A and C, 5 ␮m; B, D–F, 1 ␮m.] and the phosphorylation of the Epstein–Barr virus DNA pro-
cessivity factor (36), suggesting that a gammaherpesvirus UL97
homolog retains a function similar to that of UL97. Regardless
number of A capsids. In addition, wild-type virus-infected cells of the replication functions that may be targeted by UL97-
contained all three expected nuclear-localized capsid forms (A, B, mediated phosphorylation, the overall effect on DNA synthesis
and C capsids) and had DNA-containing virions in the cytoplasm is modest, suggesting that host cell kinases compensate for any
(Fig. 6D). DNA-containing virions were not observed in the critical UL97 role at this replication step.
cytoplasm of mutant virus-infected cells. To determine whether the A more substantial role for UL97 was observed during virion
presence of UL97 would compensate for the aberrant accumulation maturation and was revealed by a release of a BDCRB block to
of A capsids, stably transfected U373 cells constitutively expressing synchronize mutant and wild-type virus DNA encapsidation at
functional UL97 were established (UL97–9 and UL97–21) and capsid assembly. This process remains relatively poorly under-
evaluated after infection with mutant and wild-type viruses. G418- stood in CMV replication but was the most dramatically affected
resistant U373 cells carrying an empty vector construct (U373-vec) in UL97 mutant-infected cells and correlated best with the
were prepared as a control. Nuclear accumulation of A capsids was severely reduced mutant virus growth properties. The striking
observed at late times (Fig. 6E, data shown for RC⌬97.08) after accumulation of A capsids, along with the absence of detectable
infection of U373-vec cells with either RC⌬97.08 or RC⌬97.19. packaged DNA, revealed a role for this kinase in genome
These A-type capsid forms that accumulated in the nuclear periph- encapsidation or capsid assembly. By analogy with the double-
ery similar to those observed in HFs. When UL97–9 or UL97–21 stranded DNA bacteriophages, herpesvirus capsid maturation
cells were infected with mutant virus, DNA was clearly incorpo-
rated into nuclear-localized capsid forms (Fig. 6F) at levels similar
to those seen with AD169 infection of U373-vec, UL97–9, or 储Krosky, P. M., Baek, M. C., Barrera, I., Harvey, R. J., Biron, K. K., Coen, D. M., & Sethna, P.
UL97–21 cells (data not shown); however, the maturation of CMV B., 24th International Herpesvirus Workshop, July 17–23, 1999, Boston, MA, abstr. 12.016.
was not as efficient in this cell type. Mature virions were only rarely **Harvey, R. J., Chamberlain, S., Davis, M., Koszalka, G. W., Smith, A. & Biron, K. K., 22nd
observed in the cytoplasm of U373 cells, even when wild-type virus International Herpesvirus Workshop, August 2– 8, 1997, San Diego, CA, abstr. 245.

Wolf et al. PNAS 兩 February 13, 2001 兩 vol. 98 兩 no. 4 兩 1899

may proceed in a stepwise fashion and requires the concerted The phenotype of the UL97 mutant is also similar to an HSV-1
activities of cleavage-packaging proteins that function to cleave alkaline nuclease mutant that exhibits a DNA packaging defect
concatemeric DNA into unit-length genomes as well as to insert resulting from failure to resolve DNA intermediates that arise
DNA into capsids (3, 4). Cleavage-packaging machinery controls during replication (45, 46). Thus we cannot exclude an additional
docking and insertion of DNA into capsids, cleavage of genome impact of UL97-mediated phosphorylation on processing of DNA
ends, and maintenance of encapsidated DNA within capsids intermediates to create packageable forms. At the current level of
through the final stages of virion morphogenesis (3, 4). In analysis, any viral protein involved in packaging may be the critical
HSV-1, at least seven gene products have been shown to provide target of UL97 kinase. A role for this kinase in cleavage–packaging
critical DNA packaging functions, and mutations in most of them is further supported by the chemical similarity of 1263W94, which
inhibits UL97 directly, to the benzimidazole analog BDCRB, which
have been shown to result in the concomitant accumulation of
targets cleavage–packaging proteins (12–14, 36).
uncleaved concatemeric DNA and scaffold-containing B capsids
The behavior of UL97 as a ganciclovir kinase (8, 9) at first glance
(4, 37–44). In contrast, the UL97 deletion mutant demonstrated seems to distinguish this enzyme from homologs encoded by
a unique phenotype associated with concomitant production of alphaherpesvirus; however, its role in viral replication is consistent
cleaved unpackaged DNA and accumulation of A capsids. Type with that of a protein kinase. Although the differences observed
A capsids are regarded as aberrant end products of the capsid here may reflect viral UL97 function as well as the impact of host
assembly process, resulting from either incomplete insertion of cell factors induced during CMV infection, it is notable that UL97
DNA into capsids or the premature release of DNA from capsids was previously shown to compensate for HSV-1 UL13 in restoring
(4, 5). The viral replication cycle demands that DNA be stably efficient growth while only partially restoring protein modification
inserted into capsids before the completion of virion maturation, and without conferring ganciclovir sensitivity (26, 35). Regardless
but that it be readily released from those same capsids during of the differences we and other investigators have observed in the
entry in a subsequent round of infection. It is quite possible that behavior of these kinase homologs, two common functions retained
virion-associated kinases and phosphatases provide the modifi- by these enzymes may be regulation of DNA replication and
cations that throw this switch, either alone or in conjunction with encapsidation. These data are consistent with a common origin as
cellular kinases and phosphatases soon after entry. Most virion well as some divergence of the function during coevolution of this
maturation models (5) predict that DNA cleavage and packaging gene within each respective biologically distinct herpesvirus. Fur-
are coupled events, suggesting that UL97 phosphorylation ther studies of this enzyme and its targets will provide insight into
events regulate the efficiency of capsid assembly after the the regulation of CMV DNA packaging and capsid assembly
insertion of DNA into capsids. The phenotype of the UL97 and possibly reveal connections between distinct phases of viral
replication.
deletion mutant appears similar in both the pattern of A capsid
predominance and the accumulation of cleaved, nonencapsi- We thank Nafisa Ghori for her excellent assistance with electron
dated viral DNA to those displayed by a mutant in the HSV gene microscopy and A. Louise McCormick for comments on the manuscript.
UL25 (44), which encodes a minor herpesvirus-conserved capsid This work was supported by U.S. Public Health Service Grant RO1
component that stabilizes the capsid during packaging. AI20211 (to E.S.M.).

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