viruses

Review
Experimental Systems for Measuring HIV Latency
and Reactivation
Koh Fujinaga 1, * and Daniele C. Cary 2
1 Division of Rheumatology, Department of Medicine, School of Medicine, University of California,
San Francisco, CA 94143-0703, USA
2 Department of Medicine, Microbiology, and Immunology, School of Medicine, University of California,
San Francisco, CA 94143-0703, USA; carydc@hotmail.com
* Correspondence: koh.fujinaga@ucsf.edu; Tel.: +1-415-502-1908




Received: 15 August 2020; Accepted: 5 November 2020; Published: 9 November 2020


Abstract: The final obstacle to achieving a cure to HIV/AIDS is the presence of latent HIV reservoirs
scattered throughout the body. Although antiretroviral therapy maintains plasma viral loads below
the levels of detection, upon cessation of therapy, the latent reservoir immediately produces infectious
progeny viruses. This results in elevated plasma viremia, which leads to clinical progression to AIDS.
Thus, if a HIV cure is ever to become a reality, it will be necessary to target and eliminate the latent
reservoir. To this end, tremendous effort has been dedicated to locate the viral reservoir, understand
the mechanisms contributing to latency, find optimal methods to reactivate HIV, and specifically kill
latently infected cells. Although we have not yet identified a therapeutic approach to completely
eliminate HIV from patients, these efforts have provided many technological breakthroughs in
understanding the underlying mechanisms that regulate HIV latency and reactivation in vitro. In this
review, we summarize and compare experimental systems which are frequently used to study HIV
latency. While none of these models are a perfect proxy for the complex systems at work in HIV+
patients, each aim to replicate HIV latency in vitro.

Keywords: HIV; latency; latency reversing agents; transcription; P-TEFb; silencing; transcription
interference; NFkB

1. Introduction
Antiretroviral therapy (ART) regimens keep the virus below the level of detection in HIV+
individuals. However, upon cessation of therapy, plasma viremia rapidly rebounds, leading to the
progression to AIDS [1]. Although ART is successful at suppressing HIV, it is unable to completely
eradicate the virus due to the presence of latent HIV reservoirs [2]. These reservoirs are long-lived viral
sanctuaries scattered throughout the body. In HIV+ individuals, HIV-infected cells can be detected not
only in the peripheral blood and lymph nodes, but also in the brain, lungs, kidneys, liver, adipose
tissues, gastrointestinal tract, genitourinary systems, and bone marrow. Lymphoid tissues, such as
spleen, thymus, and particularly gut-associated lymphoid tissues (GALT), are the most important
viral reservoirs, which remain relatively dormant, but account for the immediate rebounds in viremia
following cessation of ART [1,3]. One currently investigated approach to target the latent reservoir is
therapeutic reactivation of HIV, also known as “shock and kill” [4,5]. A latency reversing agent (LRA)
or combinations of LRAs would be used to reactivate and shock the virus out of hiding, leaving it
vulnerable to immune killing machinery or cytolysis by the virus [4–7]. However, sufficient levels of
HIV reactivation will be required for “shock and kill” therapies to significantly impact the reservoirs.
Therefore, to develop the most effective reactivation strategies, it is essential to understand the precise
cellular and molecular mechanisms by which HIV latency is established and controlled [8].

Viruses 2020, 12, 1279; doi:10.3390/v12111279 www.mdpi.com/journal/viruses

Viruses 2020, 12, 1279 2 of 22

There are no animal models which fully recapitulate HIV pathogenesis as observed in HIV+
individuals [9]. Many studies on HIV latency and reactivation instead rely on tissue culture systems
using immortalized human cell lines or primary cells derived from human blood or tissues [10,11].
These in vitro culture models utilize different combinations of growth factors, feeder cells, and cytokines
to drive HIV-infected, activated T cells into a quiescent state, with the goal of modeling the conditions
of latently infected T central or effector memory (Tcm or Tem) cells [12–14]. While all of these models
produce resting T cells with transcriptionally silent HIV, it is unclear whether any of these in vitro
latent HIV models phenocopy latent reservoirs in HIV+ individuals. In the following review, we
present several stable cell line and primary cell models used to study HIV latency in order to provide a
comprehensive comparison of the different available systems.

2. Mechanisms Regulating HIV Transcription and Latency

HIV transcription is initiated at the transcription start site (TSS) located in the HIV long terminal
repeat (LTR) region, which consists of a typical inducible promoter/enhancer sequences [15] (Figure 1).
Once integrated, the HIV LTR acts like a typical inducible cellular gene promoter, responding to
various cellular signaling cues [15]. HIV transcription is regulated by host transcription factors
(TFs) which control host gene expression. Following infection, activated cells can return to a resting
state. These quiescent cells have vanishing low levels of important TFs. This maintains viral latency
by keeping these cells in a transcriptionally silent state. Multiple cellular signaling pathways can
control HIV transcription positively: positive transcription elongation factor b (P-TEFb), the nuclear
factor kappa B (NFκB) pathway [16], the nuclear factor of activated T cells (NFAT) pathway [17],
the mitogen-activated protein kinase (MAPK) pathway [18], the toll-like receptor (TLR) pathway [19],
and the mammalian target of rapamycin (mTOR) pathway [20]); as well as negatively: via recruitment
of the polycomb repressive complex (PRC) [21–23], the Cullin 3 E3 ligase pathway [24], and the
zinc-finger protein 304 [25]. The proviral 50 -LTR is an inducible promoter containing a TATA-box and
upstream enhancer binding regions [26]. For example, a typical HIV LTR contains two (clade B) or
three (clade C) NFκB sites and two SP1 sites [26].
Immediately after initiation of transcription and promoter clearance, the host RNA polymerase
II (RNAPII) is stalled by the negative elongation factor (NELF) and DRB-sensitivity inducing factor
(DSIF), resulting in accumulation of short premature RNAs with a length of less than 100 nucleotides
that form a stem-loop structure called the transactivation response (TAR) element [27]. When there is
sufficient HIV Tat, it binds to TAR and the cyclin T1 (CycT1) subunit of P-TEFb. The cyclin-dependent
kinase 9 (CDK9) subunit of P-TEFb then hyperphosphorylates the C-terminal domain (CTD) of RNAPII
at the second serine residues (S2) of the heptapeptide repeats (YSPTSPS), NELF and the SPT5 subunit
of DSIF. NELF is then removed, and DSIF becomes a positive transcription factor, which accompanies
RNAPII as it progresses through the gene body [28–31]. Recruitment of P-TEFb is a main switch
between non-productive and productive transcription of HIV. Importantly, in quiescent CD4+ T cells,
such as memory T cells [32,33] and hematopoietc stem cells [34–36], which represent the bulk of the
latent reservoir, the expression of CycT1 is severely downregulated post-transcriptionally [37,38].
This downregulation of CycT1 causes a block of transcription elongation from the HIV LTR.
In the late stage of HIV transcription, another cellular CDK/cyclin complex (CDK11/CycL) is
recruited to RNAII elongation complex transcribing HIV [39,40]. CDK11 phosphorylates RNAPII’s
S2 and recruits the transcription export (TREX/THO) complex, which promotes the cleavage and
polyadenylation of HIV transcripts, stabilizing HIV mRNAs and enhancing their nuclear export and
translation of viral proteins [39]. Interestingly, CDK11/CycL expression is also severely downregulated
in quiescent CD4+ T cells [41]. Therefore, at least two major cellular CDK/cyclin complexes play key
roles in HIV latency [42] (Figure 1). Recent studies from Yukl and colleagues demonstrated that HIV
transcription is blocked at multiple steps including initiation, elongation, splicing, termination, and
polyadenylation [43].
Viruses 2020, 12, 1279 3 of 22
Viruses 2020, 12, x FOR PEER REVIEW 3 of 23

Figure 1. Two major cellular cyclin/Cdk complexes play key roles in HIV transcription. P-TEFb
Figure 1. Two major cellular cyclin/Cdk complexes play key roles in HIV transcription. P-TEFb
(CycT1:CDK9, green) is recruited to the transcription machinery on HIV LTR by various factors.
(CycT1:CDK9, green)
Epigenetic factor Brd4,is DNA-bound
recruited to the transcription
transactivator machinery
NFκB, on HIV
and viral LTR by various
Tat (associated factors.
with cellular
Epigenetic factor Brd4, DNA-bound transactivator NFκB, and viral Tat (associated with cellular
super elongation complex) bind to CycT1 and recruit P-TEFb to HIV provirus in an active chromatin super
elongation complex)
environment. bind phophorylates
P-TEFb then to CycT1 andRNAPII
recruit and
P-TEFb to HIV
negative provirus elongation
transcription in an active chromatin
factors NELF
environment. P-TEFb then phophorylates RNAPII and negative transcription elongation
and DSIF, which augments transcriptional elongation. CycL:Cdk11 complex (yellow), in turn, associated factors
NELFtranscription/export
with and DSIF, which augments
and THOtranscriptional elongation.isCycL:Cdk11
complex (TREX/THOC) recruited to complex (yellow), in turn,
RNAPII transcribing HIV
associated with transcription/export and THO complex (TREX/THOC)
genes. Cdk11 phosphorylates the CTD of RNAPII and promotes the assembly of cleavage is recruited to RNAPII
and
transcribing HIV(CPA)
polyadenylation genes. Cdk11
factors phosphorylates
at the thewhich
30 end of genes, CTD ensures
of RNAPII and expression
optimal promotes the assembly
of HIV mRNAs. of
cleavage and polyadenylation (CPA) factors at the 3′ end of genes, which ensures optimal expression
of HIV
HIV mRNAs. are integrated into host chromosomal DNA at random locations. There is a
proviruses
preference to integrate inside actively transcribing genes [44,45], which have less compact chromatin
In theThus,
structure. late stage of HIV transcription,
HIV integration site is a key another
factor tocellular CDK/cyclin
understanding how complex
viral latency(CDK11/CycL)
is established is
recruited
and to RNAIIWhen
maintained. elongation
HIV iscomplex
integrated transcribing HIV [39,40]. CDK11
into a transcriptionally activephosphorylates
gene, HIV transcriptionRNAPII’s
S2 and recruits
0 the transcription export (TREX/THO) complex, which
from its 5 -LTR is silenced by a mechanism called transcription interference (TI) (Figure 2). RNAPII promotes the cleavage and
polyadenylation
actively transcribing of HIV transcripts,
cellular stabilizing
genes reads through HIV
themRNAs
boundary and enhancing
between hosttheir
genenuclear
and HIV export and
provirus,
translation viral
preventing of viral proteinsinitiated
transcription [39]. at Interestingly,
the TSS in theCDK11/CycL
50 -LTR, producing expression
host–viralis hybrid
also severely
(HVH)
downregulated
RNA in quiescent
[46,47], although a certain CD4+
levelTofcells
HIV [41]. Therefore,
transcription at least
occurs at thetwo
TSSmajor
when cellular CDK/cyclin
HIV is integrated in
complexes
the play key as
same orientation roles
hostingenes
HIV [47].
latency Since[42]HIV (Figure
provirus1). Recent
can be studies
integrated from Yukl
in the andor
same colleagues
opposite
demonstrated
direction to thethat HIVgene,
cellular transcription
HVH can is blocked
contain at or
sense multiple
antisensesteps
(AS)including
HIV RNA. initiation,
These RNAs elongation,
are not
splicing, termination, and polyadenylation [43].
transcribed from HIV’s canonical TSS and therefore they do not produce HIV proteins. To reverse TI
HIV proviruses
and increase HIV mRNAs, are integrated into to
it is necessary hostblockchromosomal
the upstream DNA at random
cellular locations.
transcription There is a
and stimulated
preference to integrate inside actively transcribing
HIV transcription from its canonical TSS at the same time [46]. genes [44,45], which have less compact chromatin
structure. Thus, HIV integration site is a key factor to understanding how viral
It is also plausible that HIV transcription is regulated epigenetically via histone modifications, in a latency is established
manner similar toWhen
and maintained. HIV is
host genes integratedEarly
[11,48–52]. into seminal
a transcriptionally active gene,
studies by Verdin HIV transcription
and colleagues demonstrated from
its 5′-LTR is silenced by a mechanism called transcription interference (TI)
that a highly ordered nucleosome structure, called Nuc 1, is built into the region of HIV TSS. Chromatin (Figure 2). RNAPII actively
transcribing of
remodeling cellular
Nuc1 is genes reads with
associated through HIVthe boundary
latency between host
and reactivation [53].gene
While andtheHIV roleprovirus,
of DNA
preventing viral transcription initiated at the TSS in the 5′-LTR, producing
methylation on HIV latency is still debatable [54], histone modifications play critical roles in host–viral hybrid (HVH)
HIV
RNA [46,47], although a certain level of HIV transcription occurs
latency and reactivation [55]. In particular, repressive histone marks such as H3K9 methylation at the TSS when HIV is integrated
in the
and samemethylation
H3K27 orientation as canhost genes at
be found [47].
theSince HIV provirus
integrated HIV LTRcan be integrated
in latently infected incells.
the sameH3K27or
opposite direction to the cellular gene, HVH can contain sense
methylation is mediated by the PRC via direct recruitment of the EZH histone methyltransferaseor antisense (AS) HIV RNA. These
RNAs are
(HMT) not transcribed
subunit to HIV LTR from HIV’sAcanonical
[22,56]. selective TSS EZH2 and therefore
inhibitor they do not
exhibited producereactivation
synergistic HIV proteins. of
To reverse TI and increase HIV mRNAs, it is necessary to block the
HIV from latently infected cells when used in combination with other LRAs [56]. H3K9 methylation upstream cellular transcription
andthe
on stimulated
HIV LTR HIV occurs transcription
via variousfrom HMTs: its SUV39H,
canonical G9a, TSS at the same
and/or SETDB1time[57,58].
[46]. It has recently been
Viruses 2020, 12, 1279 4 of 22

suggested that crosstalk between H3K9 and H3K27 methylation might occur at the HIV LTR, driving
HIV into a deeper latency [59,60]. On the other hand, transcriptionally active histone marks such
as H3K4 and H3K27 acetylation can be found upon HIV reactivation [55]. In latently infected cells,
these lysine residues are deacetylated by various histone deacetylases (HDAC) recruited to the HIV
LTR [61,62]. HDAC inhibitors such as vorinostat were among the first compounds used in clinical
trials for HIV latency reversal and functional cure [63]. Additionally, a recently discovered histone
modification, crotonylation, may be involved in HIV latency [64]. Induction of histone crotonylation
by the crotonyl-CoA-producing enzyme acyl-CoA synthetase short-chain family member 2 (ACSS2)
reactivates HIV
Viruses 2020, 12, from
x FOR latently
PEER REVIEWinfected cells [65]. 4 of 23

Figure 2.2. Host–viral hybrid RNAs

Figure RNAs are are expressed
expressed by by TI
TI in
in latently
latentlyinfected
infectedcells.
cells. (A)
(A) When
When HIV HIV
integrates
integratesininthe
thesense
senseorientation
orientationwithin
withinaahost
hostcell
cellgene,
gene,the
thetranscribing
transcribingRNAPII
RNAPIIreads
readsthrough
throughthe the
boundary 0 -LTR
boundarybetween
betweenthethehost
hostgene
geneand thethe
and 5’LTR
5’LTRor the HIVHIV
or the proviral DNA,
proviral and and
DNA, terminates in thein5the
terminates 5′-
(past
LTR TAR
(pastnear
TARthe poly
near A poly
the site), A
resulting in the expression
site), resulting of host–viral
in the expression hybrid (HVH)
of host–viral RNAs.
hybrid (HVH)(B) RNAs.
When
HIV integrates
(B) When HIVinintegrates
the antisense orientation
in the within
antisense a host gene,
orientation withinRNAPII
a hostreads through
gene, RNAPII without
readsstopping
through
and HVH RNAs containing HIV antisense RNA are produced.
without stopping and HVH RNAs containing HIV antisense RNA are produced.

3. Viral Expression in Latently Infected Cells

It is also plausible that HIV transcription is regulated epigenetically via histone modifications,
in aAlthough
manner similar
latentlyto host genes
infected cells are[11,48–52].
not activelyEarly seminal and
replicating, studies
do notby express
Verdin detectable
and colleagues
viral
demonstrated
antigens, these that
cellsaproduce
highly ordered nucleosome
several different viralstructure,
RNAs and/or calledproteins
Nuc 1, is built into
[43,66]. the regionthe
In particular, of
HIV TSS.ofChromatin
majority HIV proviruses remodeling
detectedofinNuc1
HIV+ isindividuals
associated with HIV latency
are defective and reactivation
and unable to produce[53]. While
infectious
the rolebecause
viruses of DNAof methylation on HIVby
mutations caused latency is still
cellular debatable [54],
RNA-editing enzymeshistone
such modifications
as APOBEC play critical
3G [66–69].
roles bearing
Cells in HIV theselatency and reactivation
defective proviruses[55]. canIn particular,
still produce repressive
viral proteinshistone marks [66,67].
and RNAs such as Short
H3K9
methylation
viral and H3K27
transcripts, formedmethylation
through thecan be found
block at the integrated
at transcription HIV LTR
elongation, werein among
latentlytheinfected cells.
first viral
H3K27detected
RNAs methylation is mediated
in peripheral by the PRC cells
blood mononuclear via (PBMCs)
direct recruitment of the EZH
from asymptomatic histone
HIV-infected
methyltransferase
individuals [43,70–72].(HMT) Withsubunit to HIV LTR
the development [22,56].
of highly A selective
sensitive EZH2quantitative
and specific inhibitor exhibited
reverse
synergistic reactivation
transcription PCR (RT-qPCR) of HIV fromthroughput
and high latently infected cells when(HT-RNAseq),
RNA-sequencing used in combination
different with other
viral RNA
LRAs [56].
species, H3K9 multiply
including methylation on thespliced
and singly HIV LTR and occurs
unspliced via HIV
various
mRNAs, HMTs: SUV39H,
as well G9a, and/or
as nonproductive
SETDB1
HVH RNA [57,58]. It has
resulting recently
from been
TI, are ablesuggested that crosstalk
to be detected between
[43,46,47,73]. H3K9of
Analysis and H3K27
which methylation
viral RNAs are
might occur
produced at the
by latent HIV
cells, LTR,
either driving
while still atHIV
rest orinto
upon a reactivation,
deeper latency can be[59,60].
used to On the other
further hand,
understand
transcriptionally
HIV latency. The HIV activeLTRhistone marks
can also suchantisense
produce as H3K4 (AS) and H3K27 acetylation
transcripts, as is thecan
casebeoffound upon
most of HIV
cellular
reactivation
promoters [55]. InHowever,
[74–77]. latently infected
it is still cells,
unclear these lysinethe
whether residues areAS
levels of deacetylated
HIV transcriptsby various
correlatehistone
with
deacetylases
HIV (HDAC)
latency. These AS recruited to the HIV
HIV transcripts couldLTRencode[61,62]. HDAC
proteins, butinhibitors suchif as
it is unclear thesevorinostat
proteinswere
are
among theinfirst
expressed compounds
latently infected used in clinical
cells [78]. trials
The ratio offor HIV
sense to latency reversalproduced
AS transcripts and functional cure [63].
by a reactivated
Additionally,
cell can inform awhetherrecentlya strategy
discovered histone modification,
is successful crotonylation,
in truly reactivating the latent may be Some
virus. involved
HIVin HIV
RNAs
latency [64].
expressed Induction
in latently of histone
infected cells crotonylation
can be exported by the crotonyl-CoA-producing
as extracellular enzyme
vesicles, in either acyl-CoA
exosomes or
synthetase short-chain family member 2 (ACSS2) reactivates HIV from latently infected cells [65].

3. Viral Expression in Latently Infected Cells

Although latently infected cells are not actively replicating, and do not express detectable viral
antigens, these cells produce several different viral RNAs and/or proteins [43,66]. In particular, the
Viruses 2020, 12, 1279 5 of 22

virus-like particles. Exosomes containing HIV components such as Nef proteins or TAR RNA are
detected in HIV-infected individuals [79–86]. Exosomes in patient sera or cell supernatants can be
isolated and analyzed for the presence of Nef or TAR to further understand latency. The majority of
HIV-infected cells contain defective HIV proviruses, and a very low population of HIV-infected cells
retain the ability to produce infectious progeny viruses [87]. Defective HIV proviruses often contain a
large deletion in the LTR and/or coding regions [88–90]. It is becoming clear that some model systems
overestimate the reactivatable reservoir, by being unable to distinguish between defective and infective
clones [91].

4. LRAs and How They Work

To increase HIV transcription from latently infected cells, it is necessary to manipulate cellular
pathways that are involved in the establishment and maintenance of HIV latency. However, optimal
HIV reactivation can only be achieved by stimulating HIV transcription to produce enough Tat to boost
HIV transcription with the aid of the cellular P-TEFb [15,27,92,93]. Initial rounds of HIV transcription
must occur without Tat, which is a very inefficient process. Once sufficient Tat is produced, viral
transcription is amplified hundreds to thousands of times. Thus, this self-fueling feedback mechanism
regulated by Tat is the primary “on/off” molecular switch between latent and productive infection of
HIV [15,27,92,93].
Most LRAs aim to increase initial HIV transcription [19,94]. To date, there are several categories
of LRAs being tested preclinically and clinically: (1) P-TEFb releaser/activators [42], (2) protein kinase
c (PKC) agonist/canonical NFκB inducers [95], (3) non-canonical NFκB inducers, (4) Toll-like receptor
agonists [19], (5) epigenetic modulators [96], (6) mTOR inhibitors [20], (7) protease inhibitors (PIs),
(8) MAPK agonists [97], etc. Examples of LRAs are presented in Table 1. These LRAs were identified and
tested with various latency models including cell lines, primary cells derived from HIV-seronegative
individuals infected with HIV reporter virus ex vivo, or primary cells derived from HIV+ individuals.
However, since mechanisms of HIV latency vary depending on cell types or even cell clones, none of
these LRAs optimally reactivate HIV in all latency models.

Table 1. Examples of LRAs.

Category Compounds Mechanism of Action References

Prostratin, ingenol, Induce NFkB (canonical
PKC agonist euphorbia kansui, pathway) [98–106]
bryostatin-1 Increase P-TEFb proteins
Induce NFkB (non-canonical
SMAC mimetics AZD5582 [6,107,108]
pathway)
Panovinostat, Release P-TEFb from 7SK
HDAC inhibitors romidepsin, vorinostat snRNP [22,52,63,94,109–111]
(SAHA) Increase H3K27Ac
Release P-TEFb from 7SK
BET inhibitors JQ1, iBET snRNP [71,112–115]
Release Brd4 from H3K27Ac
Proteasome Induce NFkB
Bortezomib [116,117]
inhibitors Increase P-TEFb proteins
CPG 7909, Pam3CSK4,
TLR agonists Multiple pathways? [19,118–123]
MGN1703, GS-9620
Procyanidine, cacao
MAPK activator Induce the MAPK pathway [97,124]
extract
Reverse epigenetic silencing,
DMNT1 inhibitors 5-aza-20 -deoxycytidine Release P-TEFb from 7SK [48,125,126]
snRNP
3-deazaneplanocin A, Reverse polycomb-mediated
HKMT inhibitors [22,127]
EPZ-6438, UNC-0638 transcriptional suppression
Viruses 2020, 12, 1279 6 of 22

Cellular factors and cellular pathways mediating HIV latency and reactivation also vary among
cell types. It is still largely unclear exactly where HIV-infected cells reside and how HIV latency is
established and maintained in HIV+ individuals in vivo. Because of the complexities of viral latency
and insufficient knowledge about the viral latency in vivo, many compounds that reactivate HIV
transcription in cell line systems do not reverse latency in HIV+ individuals [128,129]. Therefore, it is
necessary to understand mechanisms of HIV latency in different models to identify effective LRAs that
can be used in the HIV anti-latency therapies (HALT) and to choose appropriate latency models to
screen new compounds. In the following sections, we will discuss the differences between different
systems that model HIV latency.

5. HIV Molecular Clones

Infectious molecular clones are an important tool of modern virology, which can be used to
produce infectious viral particles and enable genetic analysis of virally encoded genes [130–132].
For HIV, several infectious molecular clones were constructed immediately after the discovery of the
virus. Since HIV can infect non-dividing cells, molecular clones were further modified to develop
safe and efficient lentiviral delivery for non-HIV gene therapies including treatment for cancer and
genetic disorders such as Wiskott-Aldrich Syndrome and metachromatic leukodystrophy [133–137].
To prevent spreading infection, lentiviral vectors do not contain viral structural and regulatory genes
which are essential for viral replication, such as gag, pol, env, which can be supplemented by different
plasmids [138]. These lentiviruses do not produce progeny viruses after infection, hence they do not
replicate, resulting in a single round of infection. The envelope protein of Vesicular Stomatitis Virus
(VSV-G) is often packaged with the replication incompetent HIV in order to produce virus particles
which can enter into the host cells independent of the HIV envelope protein (Env) [138]. Lentiviral
vectors encoding reporter genes including cDNAs for fluorescent proteins (FPs) and luciferase (Luc)
have been constructed to study aspects of HIV infection in vitro. In particular, EGFP and mCherry
are useful tools to measure viral gene expression and viral gene integration independently [139–142].
The gene expressing the fluorescent protein can be placed after the HIV promoter to measure HIV
transcription, or from a separate promoter to measure expression of the gene in which HIV has been
integrated [139–142].

6. Choosing the Best Latency Models

The choice of appropriate models to study HIV latency and reactivation depends on the objectives
of the study. Since HIV only infects and causes AIDS in humans, there are no animal models that
precisely recapitulate the course of HIV infection. Among a few exceptions are primates infected with an
HIV-simian immunodeficiency virus (SIV) hybrid virus (SHIV) and severe combined immunodeficiency
(SCID) mice transplanted with human hematolymphoid tissues (SCID-hu mice) infected with HIV
(since animal models are beyond the scope of this review, please refer to other reviews for further
reading) [143–146].
For cell culture systems, a major question is whether to use primary cells or cell lines. Since HIV
infects CD4+ T cells and macrophages, primary CD4+ T cells and macrophages from PBMCs or
lymphoid tissues are the experimental systems closest to HIV infection in humans [14,147]. However,
isolating and culturing these cells from biopsy samples are generally cumbersome processes that
require experience and skill [148]. These cells can be kept ex vivo for only a certain length of time,
usually 2–4 weeks, although it is possible to prolong these cells by ectopically expressing anti-apoptotic
factors [149,150]. Moreover, the efficiency of transfection or electroporation of exogenous genes or
proteins into these cells are generally low. Sample-to-sample variations can be large and it might
require preparation of a large number of samples to obtain statistically reliable data.
On the other hand, immortalized cell lines are easy to maintain for a long time with higher
transfection efficiencies. Popular cell lines used in HIV researches are Jurkat, CEM, MOLT4, MT 4 for
T cells, and THP1 and U937 for monocyte/macrophages [11]. However, since cell lines are actively
Viruses 2020, 12, 1279 7 of 22

replicating, the cellular characteristics are different from hematopoietic cells in humans. Of particular
importance, the level of transcription machinery, namely P-TEFb, is severely downregulated in
primary resting CD4+ T cells and macrophages whereas these factors are highly expressed in cell
lines [15,41,42]. Other cellular transcription machinery components and/or transcription factors
are similarly downregulated in quiescent cells, resulting in blockages of multiple steps of HIV
transcription [43]. HIV transcription seems to be blocked at different steps between circulating cells
(blood) and lymphoid-associated cells (GALT, for example) based on RNA expression analysis [151,152].
On the other hand, because of the nature of active cell replication, global transcription is kept at
relatively high level in cell line models. Regulation of HIV latency occurs largely at the step of
transcription elongation [153], due to impaired recruitment of Tat/P-TEFb complex to HIV TAR via
transcription interference or mutations in Tat and/or TAR [153]. This may affect cellular responses to
LRAs in cell lines compared to primary cell systems. In the following section, we will further discuss
several HIV latency models.

7. Primary Cells (PBMCs) from HIV+ Individuals vs. Ex Vivo Infection Models
Primary CD4+ T cells and macrophages can be obtained from blood samples or tissue biopsies
from HIV+ or HIV-seronegative individuals. Cells from HIV+ individuals contain naturally occurring
latently infected cells. However, to obtain and use HIV+ primary cells requires special safety precautions
and facilities, as well as lengthy administrative process. The proportion of latently HIV-infected cells in
primary cells is estimated to be 1 in million or less. Therefore, it is very difficult to detect HIV-expressing
cells and measure HIV reactivation at the single-cell level. On the other hand, de novo infection
with HIV reporter virus of primary cells from HIV-seronegative individuals generally provides a
larger proportion of HIV latently infected cells and therefore is an appropriate model to study viral
latency and reactivation at the molecular/single cell level. In general, quiescent CD4+ T cells are
isolated from PBMCs or lymphoid tissues such as tonsils or the gut [147,148,154], and infected with
pseudotyped single-round HIV encoding FPs or other reporters. When a dual-color HIV reporter virus
such as Duo-Fluo [139,140] or RGH [141,142] is used, a bulk of HIV-infected cells are sorted by the
internal promoter-driven mCherry expression, then latently infected cells are negatively selected by
HIV-driven EGFP expression. These cells are further used to study the mechanisms by which HIV
latency is established and maintained, and to measure responses to different LRAs. Although lentiviral
transduction normally requires cell stimulation, direct infection of resting CD4+T can be achieved,
with less than 1% efficiency, by mixing Tcm or Tem with a high titer of HIV (m.o.i ~22–150) [155].
Recently, Greene and colleagues developed an efficient method to transduce a lentiviral vector into
quiescent cells by using a helper virus containing SIV’s Vpx (Vpx-VLP) [156].
There are several different well-established protocols of latent cell isolation and HIV infection
established by various laboratories. In particular, the following methods were designed to obtain
latently infected cells without T cell stimulation/re-entry to latency. Karn and colleagues demonstrated
that a high number of latently infected cells are obtained by co-culturing HIV-infected CD4+ T cells
and H80 feeder cells [157]. The method established by Bosque and Planelles uses TCR stimulation in
the presence of TGF-β and anti-IL4/anti-IL2 to differentiate CD4+ T cells into non-polarized subsets,
representing Tcm [158]. In the Siliciano method, cells are first set to a resting state in the presence
of anti-apoptotic factor Bcl2 to prolong the culture, then cells are infected with HIV and latently
infected cells are sorted [159]. Spina and colleagues found that HIV infection can be established by
directly infecting freshly isolated resting CD4+ T cells [14]. The method developed by Greene and
colleagues also uses direct HIV infection of resting CD4+ T cells with spinoculation in order to increase
the interaction between HIV and CD4+ T cells [160]. The model established in the Lewin lab uses
chemokines such as CCR7, CXCR3 or CCR6 to increase the efficiency of HIV infection in resting CD4+
T cells [161,162]. While no one model perfectly replicates latently infected cells, each of these model
systems attempts to recapitulate the in vivo conditions to create a cell system that will permit the
latently infected cells to be manipulated in culture. Planelles and colleagues compared the responses
Viruses 2020, 12, 1279 8 of 22

of different classes of LRAs known to reactivate HIV in latently infected cells on different primary
latent model cells, and demonstrated that none of the tested models accurately capture the response
characteristics of latently infected cells from HIV+ individuals [14]. Chomont and colleagues recently
demonstrated that LRAs such as HDACis and PKC agonists display variable activities in different
subsets of primary memory CD4+ T cells, suggesting that combination of several compounds might be
required for an optimal latency reversal in all memory subset [33].

8. Measuring HIV Transcription/Gene Expression in Primary Cells Derived

HIV-Positive Individuals
While HIV gene expression can be measured by various sensitive methods in ex vivo infection
models using pseudotyped reporter HIV, there are limited number of methods available for measuring
viral gene expression in latently infected cells derived from HIV+ individuals. The most sensitive assay
is to directly measure HIV mRNAs is using RT-qPCR. By using different sets of PCR primers, different
HIV mRNA species (unspliced, singly or doubly spliced, AS, HVH, etc) can be measured independently.
However, some HIV species (particularly, short premature/unproductive RNAs) may be already highly
expressed in latently infected cells. Therefore, primers specific for the gag genes, to detect unspliced
HIV RNA, are often used to measure viral reactivation [163]. Recently, a new PCR technology called
droplet digital PCR (ddPCR) was developed and applied to measure HIV RNAs [43,72]. In this system,
a sample is separated into tens of thousands of droplets of water/oil emulsion and PCR reactions are
performed in each droplet. This technology provides absolute quantification of PCR targets with higher
efficiency and precision than conventional qPCR. Recent studies employing this technology revealed
that various steps of HIV transcription including elongation, termination, and splicing are blocked
in latently infected cells [43,72]. Although qPCR is a powerful and sensitive technique to detect and
measure HIV RNAs, it does not differentiate signals between intact (productive) and defective RNAs,
and therefore it might overestimate the size of the reservoir [5,164].
A commonly used technology to specifically measure replication-competent HIV is the quantitative
viral outgrowth assay (QVOA). In this assay, stimulated PBMCs from HIV+ individuals are co-cultured
with CD4+ T cells purified from HIV-seronegative individuals. Replication-competent HIV produced
from HIV+ PBMCs are amplified via infecting HIV-seronegative CD4+ T cells so that viral replication
can be measured by p24 ELISA or reverse transcription (RT) assays [165–167]. Although QVOA has
been frequently used as a gold standard to measure viral reservoir bearing replication-competent HIV,
there are several limitations. The assay takes weeks to obtain results, and the experiments requires
a large volume blood from multiple donors. Therefore, it is time-consuming, labor-intensive and
expensive. In addition, since the HIV growth depends of the condition of PBMCs, the sample-to-sample
variation is often very large. More importantly, not all replication-competent viruses are stimulated
and spread in this assay, resulting in an underestimation of viral reservoirs [168]. To circumvent
the problem of low sensitivity, a more sensitive version of QVOA using primary cells or tissues of
humanized mice [169]

9. Cell Line Models for HIV Latency

Using cell lines to study HIV latency and reactivation has several advantages. First, these cells are
easy to maintain. HIV latently infection is easily established and single cell clones can be isolated with
relative ease. High levels of DNAs, RNAs, and proteins can be ectopically expressed by lipofection or
electroporation into cell lines. This facilitates genetic analysis of cellular factors and pathways involved
in HIV latency and reactivation. There are several well-characterized HIV latency models commonly
used to study HIV latency and test LRAs.

10. HIV Indicator Cells

The simplest models to study HIV transcription are highly permissive cell lines such as HeLa
and HEK 293 cells which stably carry HIV LTR-driven Luc and b-Galactosidase (β-Gal), as well as
Viruses 2020, 12, 1279 9 of 22

cellular HIV receptors CD4 and CXCR5a. MAGI [170] and TZM-bl cells [171] are examples of such
reporter cell lines. HIV can infect these cells and activates Luc genes after integration and expression
of viral Tat protein. Therefore, these cells are commonly used to determine infectivity as measured by
HIV titers. However, HIV LTR-driven transcription is not silenced in these cells and hence they are
not an appropriate model for HIV latency. Viral Tat can still amplify HIV LTR-driven reporter gene
expression by ~100 fold. Therefore, these cells are often used to study Tat-dependent transcription, but
are not appropriate to study latency.

11. HIV Latently Infected Cell Clones

CD4+ T cells or monocyte/macrophage-originated cell lines are more physiologically related to
primary cell models. There are already several different HIV latent cell clones carrying intact HIV
proviruses or reporter genes under the control of HIV. Before fluorescent reporter HIV technologies were
established, many HIV latent clones such as U1, A3.01, OM1.1 and J1.1. were established in the Folks
and colleagues using promonocytic U937 cells, T-lymphocytic CEM and, Jurkat cells, or promyeolocytic
HL60 cells chronically infected with intact HIV laboratory strains [172–176]. In these cells, HIV gene
expression is kept at low to undetectable levels but increased by various stimulations [173]. Similarly,
MOLT20-2 is isolated from lymphoblastic MOLT4 cells infected with HIV NL43 [177,178]. Early studies
on HIV latency primarily used these latency models. However, measurement of HIV gene expression
required labor -or cost-intensive assays such as Gag p24 ELISA, RT assay, RT-qPCR, or Western blotting.
In the following sections, we will describe some of commonly used HIV latently infected cell clones
(summarized in Table 2). All clones are available free to all researchers of non-profit organizations at
the NIH AIDS Reagent Program (https://www.aidsreagent.org).

Table 2. Examples of HIV latency clones.

HIV
Surrogate Integration Exon Mechanism of
Cells Host Cells Replication/ References
Marker Site /Intron Latency
VLP Production
Attenuated Tat
U1 U937 Yes/Yes multiple [175,179]
(H13L)
Point mutation in
ACH2 A3.01 Yes/Yes multiple [174,179]
TAR
J1.1 Jurkat Yes/Yes multiple ND [176]
OM10.2 OM Yes/Yes ND ND [172]
MOLT20-2 MOLT4 Yes/Yes ND ND [177,178]
JLAT8.4 Jurkat No/Yes EGFP FUBP/NEXN exon TI [46,180]
JLAT9.2 Jurkat No/Yes EGFP PPP5C intron TI [46,180]
JLAT10.6 Jurkat No/Yes EGFP SEC16A ND [180]
JLAT15.4 Jurkat No/Yes EGFP UBA2 TI [46,180]
JLAT-A2 Jurkat No/No EGFP KDM6A ND [180]
JLAT5A8 Jurkat No/Yes EGFP MAT2a intron ND [181,182]
J-Lat H2 Jurkat No/No EGFP SLC25A12 [180]
Attenuated Tat
2D10 Jurkat No/No d2EGFP * SEPX1 exon [183]
(H13L)
TI/NELF-mediated
CA5 Jurkat No/Yes EGFP RBM12 exon [184,185]
RNAPII pausing?
TI/NELF-mediated
BA2 Jurkat No/Yes EGFP PDZD8 intron [184,185]
RNAPII pausing?
TI/NELF-mediated
11B2 Jurkat No/Yes EGFP HELZ intron [184,185]
RNAPII pausing?
* shorter half-life.

12. U1 and ACH2

U1 and ACH2 cells were among the first HIV latency models, established in the Fauci lab [174,175]
by isolating U937 and A.3.01 cells chronically infected with replication-competent laboratory strains of
HIV. These cells contain intact HIV proviruses without surrogate markers such as GFP or Luc. Hence,
analysis of HIV gene expression from these cells requires the same methods as that of clinical samples
from HIV+ individuals. Nonetheless, these cells have been widely used to study HIV latency and test
Viruses 2020, 12, 1279 10 of 22

LRAs. In both cells, HIV expression is blocked at the transcription level [175,176,186]. While U1 cells
can be activated by ectopically expressed Tat proteins, ACH2 does not respond to Tat [186]. Genetic
analysis of cDNAs and proviral DNAs revealed that U1 cells express a mutant Tat protein (H13L)
which is impaired in interacting with P-TEFb [179,187]. ACH2 contains a point mutation in the TAR
region that diminishes the interaction between the Tat and the TAR [188]. Therefore, while U1 cells are
suitable to measure Tat-dependent and Tat-independent HIV transcription, ACH2 cells are particularly
useful to study Tat-independent transcription. Recent studies revealed that HIV proviruses in both U1
and ACH2 are integrated in many different sites, 28 and 100 sites per 100,000 cells, respectively. This is
in spite of the fact that both cells were originally isolated as single cell clones [189], indicating that the
replication-competent HIV proviruses in these cells replicate over multiple passages. Nonetheless,
the overall behavior of these cells as bulk does not seem to have changed, implying that proviral
integration sites are not a main determination factor of HIV latency and reactivation in these cells.

13. J-Lat Series

J-Lat cells were established in the Verdin lab by infecting Jurkat cells with a VSV-G-pseudotyped
replication-incompetent HIV-based lentivirus, where the HIV nef gene was replaced with an egfp
gene [180]. From a pool of HIV-infected cells, five clones were extensively characterized: 6.3, 8.4,
9.2, 10.6, and 15.4 [180]. In unstimulated condition, all clones express less than 1% GFP positive
cells. However, upon TNF-α stimulation, 27 to 96% GFP expression is induced [180]. Full-length
HIV-based lentiviral vector, containing the intact gag-pol gene and the env gene with premature stop
codon to create replication-incompetent virus, was used to make these cells Upon stimulation, J-Lat
cells produce HIV VLPs into culture supernatants. Therefore, GFP expression can be measured by flow
cytometry, as well as Gag p24 ELISA and the RT assay of culture supernatants, in order to analyze HIV
reactivation. Simultaneously, similar HIV latent cells were established with a simpler retroviral vector
containing only Tat-IRES-EGFP, Tat and EGFP are translated from the same mRNA RNA via an IRES
sequence, under the control of HIV LTR [180], and several clones were isolated and characterized: A72,
82, H2, A2, and A10. These clones do not express GFP in unstimulated conditions, but after TNF-α
stimulation, they highly express GFP. However, these clones do not produce VLPs since the lentiviral
vector used to establish them does not encode the gag-pol gene [180].
All J-Lat clones are activated by most LRAs tested [14,71,109]. However, only 20–80% of
LRA-stimulated cells express EGFP, even with optimal doses of LRAs [14], suggesting that a robust
stimulation protocol is required for a maximum reactivation of HIV in these cells. Peterlin and
colleagues demonstrated that potent TI is the cause of HIV latency in J-Lat 9.2, 8.4, and 15.4 cells,
resulting in exclusive expression of HVH RNA in the unstimulated condition [46]. Further RNA-seq
experiments confirmed this notion (Fujinaga, submitted). HIV provirus is integrated in highly expressed
genes in these cells: ppp5c, fubp, and uba2 for J-Lat 9.2, 8.4, and 15.4, respectively [46,189]. In J-Lat 8.4,
a full-length HIV provirus is integrated into the last exons of fubp in the same orientation and the nexn
gene in the opposite orientation. To reverse TI and achieve optimal HIV transcription, it is required
to block on-going RNAPII transcribing the upstream or downstream host gene and induce a potent
transcriptional initiation and elongation at the same time [46]. This could be fulfilled by blockage of
RNAPII on the host gene and initiation of HIV transcription from viral LTR by PKC agonists combined
with recruitment of active P-TEFb by P-TEFb releasers. Combinations of at least two LRAs stimulating
different pathways will be required for robust HIV reactivation from latency maintained by TI [46].

14. 2D10
2D10 cells were established in the Karn laboratory by cloning Jurkat cells infected with an
HIV-based lentiviral vector, pHR0 -d2EGFP, which encodes an attenuated Tat with the same point
mutation (H13L) as Tat in U1 cells [183]. Among more than 10 latent clones isolated and characterized,
2D10 cells have been most widely used to study HIV latency. 2D10 cells contain one copy of HIV
provirus integrated in an exon of Selenoprotein X in the reverse orientation [183]. A main mechanism
Viruses 2020, 12, 1279 11 of 22

of latency in 2D10 is attenuated Tat activity, which is less capable of releasing RNAPII paused at the
viral 5’ LTR by cellular NELF, although epigenetic silencing also occurs in these cells [30,183]. Although
less than 1% of 2D10 cells express detectable GFP, as a surrogate marker for HIV gene expression in
unstimulated culture condition, these cells respond to many LRAs robustly [97,98,183].
Since pHR0 -d2EGFP encodes an EGFP fused with the d2 peptide, which promotes rapid
degradation via a ubiquitin-dependent proteasome pathway, the EGFP proteins expressed by external
stimuli have a very short half-life in 2D10 cells: 3.6 hrs for d2EGFP vs. >24 h for wild-type EGFP.
This facilitates an efficient measurement of transcription rate. However, it also requires a special
precaution for using 2D10 cell to study HIV latency particularly when protease inhibitors (PIs),
including clinically approved bortezomib, are used as LRAs [116,117] since PIs can also stabilize
EGFP proteins.

15. Other HIV Latent Clones

Other Jurkat-based latent HIV clones similar to J-Lat cells have been established in various
laboratories. For example, Kutsch and colleagues isolated several HIV latent clones: A5, 3F12, 11B10,
and BA5 after establishing chronic infection of HIV LTR EGFP-containing lentiviral vector to Jurkat
cells. Characterization of these clones suggests that TI might be a main mechanism of viral latency [184]
although subsequent studies also indicated that RNAPII-pausing by NELF might be sufficient for
maintaining viral latency as suggested in 2D10 cells [185]. Similar to the J-Lat clones established in the
Verdin lab, 5A8 cells were established in the Greene lab by isolating Jurkat cells chronically infected
with the full-length/replication incompetent HIV EGFP virus. These cells exhibit characteristics similar
to J-Lat clones [14,181,182].

16. HIV Latency Model vs. “Actual” HIV Latent Reservoir in HIV+ Patients
How closely do these available HIV latent models mimic the actual latent reservoir in HIV-positive humans?
A simple answer for this question would be “probably not very much”. However, it is true that we
still do not know exactly where in the human body the latent reservoirs reside, whether there are
specific subsets of cells or molecular markers associated with latently infected cells, and how latency
is established and maintained in these cells. Therefore, it is probably safe to say each latent model
reflects at least some aspects of “real” HIV latency in vivo. Therefore, these HIV latent models are very
useful to study molecular mechanisms of viral latency and search best strategies to reverse latency
(“shock and kill”) or maintain latency (“block and lock”) [2,4] with the ultimate goal of achieving a
functional cure of AIDS. Knowing the precise mechanism of establishment and maintenance of HIV
latency in each model, choosing the right model(s), and/or using various types of models are keys to
obtain highly applicable results that can lead to the development of effective therapies.

17. Perspective/Closing Remarks

A functional cure of AIDS requires (1) activation of replication-competent HIV from all latently
infected cells plus complete elimination of these cells by cell killing mediated by host immune
activity [190], virus-induced cytotoxicity [191] or killing by the LRAs themselves [192] for the “shock
and kill” approach [7], or (2) complete and permanent silencing of latently infected cells bearing
replication-competent HIV even after cessation of cART for the “block and lock” approach [193,194].
At this point, we have not yet developed clinically successful strategies for either approaches and a
functional cure of AIDS seems far in the future. However, recent rapidly developing technologies in
biomedical sciences have advanced our knowledge of how HIV persists in patients. From what we
have learnt so far about the mechanisms of HIV latency, a “cocktail” of multiple compounds targeting
different pathways involved in the establishment and maintenance of HIV latency will be required
to optimally reactivate HIV from latently infected cells [99,195]. Taking advantage of a combination
of different latency models will facilitate this approach, and provide a well-rounded picture of the
mechanisms that regulate HIV latency.
Viruses 2020, 12, 1279 12 of 22

Funding: K.F. is supported by grants from the National Institute of Health (1R56AI136683 and R21AI127274).
Acknowledgments: The authors thank B. Matija Peterlin (UCSF) for his constructive comments.
Conflicts of Interest: The authors declare no conflict of interest.

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