Infected Primary CD4 Research Articles

Integrated Single-cell Multiomic Analysis of HIV Latency Reversal Reveals Novel Regulators of Viral Reactivation.

Despite the success of antiretroviral therapy, human immunodeficiency virus (HIV) cannot be cured because of a reservoir of latently infected cells that evades therapy. To understand the mechanisms of HIV latency, we employed an integrated single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) approach to simultaneously profile the transcriptomic and epigenomic characteristics of ∼ 125,000 latently infected primary CD4+ T cells after reactivation using three different latency reversing agents. Differentially expressed genes and differentially accessible motifs were used to examine transcriptional pathways and transcription factor (TF) activities across the cell population. We identified cellular transcripts and TFs whose expression/activity was correlated with viral reactivation and demonstrated that a machine learning model trained on these data was 75%-79% accurate at predicting viral reactivation. Finally, we validated the role of two candidate HIV-regulating factors, FOXP1 and GATA3, in viral transcription. These data demonstrate the power of integrated multimodal single-cell analysis to uncover novel relationships between host cell factors and HIV latency.

A histone deacetylase network regulates epigenetic reprogramming and viral silencing in HIV-infected cells

A long-lived latent reservoir of HIV-1-infected CD4 Tcells persists with antiretroviral therapy and prevents cure. We report that the emergence of latently infected primary CD4 Tcells requires the activity of histone deacetylase enzymes HDAC1/2 and HDAC3. Data from targeted HDAC molecules, an HDAC3-directed PROTAC, and CRISPR-Cas9 knockout experiments converge on a model where either HDAC1/2 or HDAC3 targeting can prevent latency, whereas all three enzymes must be targeted to achieve latency reversal. Furthermore, HDACi treatment targets features of memory Tcells that are linked to proviral latency and persistence. Latency prevention is associated with increased H3K9ac at the proviral LTR promoter region and decreased H3K9me3, suggesting that this epigenetic switch is a key proviral silencing mechanism that depends on HDAC activity. These findings support further mechanistic work on latency initiation and eventual clinical studies of HDAC inhibitors to interfere with latency initiation.

Sex and Age Impact CD4+ T Cell Susceptibility to HIV In Vitro through Cell Activation Dynamics.

Cellular composition and the responsiveness of the immune system evolve upon aging and are influenced by biological sex. CD4+ T cells from women living with HIV exhibit a decreased viral replication ex vivo compared to men's. We, thus, hypothesized that these findings could be recapitulated in vitro and infected primary CD4+ T cells with HIV-based vectors pseudotyped with VSV-G or HIV envelopes. We used cells isolated from twenty donors to interrogate the effect of sex and age on permissiveness over a six-day activation kinetics. Our data identified an increased permissiveness to HIV between 24 and 72 h post-stimulation. Sex- and age-based analyses at these time points showed an increased susceptibility to HIV of the cells isolated from males and from donors over 50 years of age, respectively. A parallel assessment of surface markers' expression revealed higher frequencies of activation marker CD69 and of immune checkpoint inhibitors (PD-1 and CTLA-4) in the cells from highly permissive donors. Furthermore, positive correlations were identified between the expression kinetics of CD69, PD-1 and CTLA-4 and HIV expression kinetics. The cell population heterogeneity was assessed using a single-cell RNA-Seq analysis and no cell subtype enrichment was identified according to sex. Finally, transcriptomic analyses further highlighted the role of activation in those differences with enriched activation and cell cycle gene sets in male and older female cells. Altogether, this study brought further evidence about the individual features affecting HIV replication at the cellular level and should be considered in latency reactivation studies for an HIV cure.

Crotonylation sensitizes IAPi-induced disruption of latent HIV by enhancing p100 cleavage into p52

SummaryThe eradication of HIV infection is difficult to achieve because of stable viral reservoirs. Here, we show that crotonylation enhances AZD5582-induced noncanonical NF-κB (ncNF-κB) signaling, further augmenting HIV latency reversal in Jurkat and U1 cell line models of latency, HIV latently infected primary CD4+ T cells and resting CD4+ T cells isolated from people living with HIV. Crotonylation upregulated the levels of the active p52 subunit of NF-κB following AZD5582. Biochemical analyses suggest that the ubiquitin E3 ligase TRIM27 is involved in enhanced p100 cleavage to p52. When TRIM27 was depleted, AZD5582-induced HIV latency reversal was reduced. TRIM27 small interfering RNA (siRNA) knockdown reduced both p100 and p52 levels without inhibiting p100 transcription, indicating that TRIM27 not only acts on p100 cleavage but also may impact p100/p52 stability. These observations reveal the complexity of HIV transcriptional machinery, particularly of NF-κB.

A Two-Color Haploid Genetic Screen Identifies Novel Host Factors Involved in HIV-1 Latency.

ABSTRACTTo identify novel host factors as putative targets to reverse HIV-1 latency, we performed an insertional mutagenesis genetic screen in a latent HIV-1 infected pseudohaploid KBM7 cell line (Hap-Lat). Following mutagenesis, insertions were mapped to the genome, and bioinformatic analysis resulted in the identification of 69 candidate host genes involved in maintaining HIV-1 latency. A select set of candidate genes was functionally validated using short hairpin RNA (shRNA)-mediated depletion in latent HIV-1 infected J-Lat A2 and 11.1 T cell lines. We confirmed ADK, CHD9, CMSS1, EVI2B, EXOSC8, FAM19A, GRIK5, IRF2BP2, NF1, and USP15 as novel host factors involved in the maintenance of HIV-1 latency. Chromatin immunoprecipitation assays indicated that CHD9, a chromodomain helicase DNA-binding protein, maintains HIV-1 latency via direct association with the HIV-1 5′ long terminal repeat (LTR), and its depletion results in increased histone acetylation at the HIV-1 promoter, concomitant with HIV-1 latency reversal. FDA-approved inhibitors 5-iodotubercidin, trametinib, and topiramate, targeting ADK, NF1, and GRIK5, respectively, were characterized for their latency reversal potential. While 5-iodotubercidin exhibited significant cytotoxicity in both J-Lat and primary CD4+ T cells, trametinib reversed latency in J-Lat cells but not in latent HIV-1 infected primary CD4+ T cells. Importantly, topiramate reversed latency in cell line models, in latently infected primary CD4+ T cells, and crucially in CD4+ T cells from three people living with HIV-1 (PLWH) under suppressive antiretroviral therapy, without inducing T cell activation or significant toxicity. Thus, using an adaptation of a haploid forward genetic screen, we identified novel and druggable host factors contributing to HIV-1 latency.

HIV-1 Envelope Glycoproteins Proteolytic Cleavage Protects Infected Cells from ADCC Mediated by Plasma from Infected Individuals.

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible but controls its transition from the unbound “closed” conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature “opening” of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the “closed” conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.

Influence of GlycoGag on the Incorporation of Host Membrane Proteins Into the Envelope of the Moloney Murine Leukemia Virus

Analysis of viral particle heterogeneity produced from infected cells has been limited by the inefficiency of traditional analytical methods to characterize large populations of viruses at an individual particle level. Flow virometry (FVM) is an emerging technique based on flow cytometry principles that enables a high throughput, multiparametric, and phenotypic characterization of viruses at a single particle resolution. Here, we performed FVM to analyze surface markers found on Murine Leukemia Virus (MLV) and glycosylated Gag-deficient (glycoGag) MLV. The glycoGag viral accessory protein has several roles in the MLV viral infection cycle including directing retroviral assembly and particle release at lipid rafts. Based on previous studies, we hypothesize that glycoGag modulates host protein incorporation into the viral envelope during viral assembly and budding. Here, by using FVM, we reveal that glycoGag is associated with an increased incorporation of the host-derived tetraspanins CD81 and CD63 along with the lipid raft marker and immune antigen Thy1.2 during the assembly and release of viral particles from infected NIH 3T3, EL4, and primary CD4+ T cells. Moreover, we show differences in the uptake of host proteins by viruses that are released from the two cell lines and primary T lymphocytes. Additionally, at the individual viral particle level, we observed a degree of expression heterogeneity of host-derived antigens within the viral population. Finally, certain cellular antigens can show either enrichment or exclusion from the viral envelope depending on whether glycoGag is expressed by the virus. This suggests that glycoGag is involved in a mechanism of selective host protein incorporation into the viral envelope.

Crotonylation Sensitizes IAPI-Induced Disruption of Latent HIV by Modulating ncNF-κB Signaling at the Step of p100 Cleavage Into p52

The eradication of infection with human immunodeficiency virus type 1 (HIV) is difficult to achieve, largely due to stable viral reservoirs. While many strategies have been tested, a clinical meaningful success has yet to be achieved, indicating that our understanding of HIV latency is incomplete and strategies to disrupt latency are not yet ideal. In this report, we show that crotonylation, a recently discovered epigenetic modification involved in HIV transcription from latency, also acts to enhance AZD5582-induced noncanonical NF-κB (ncNF-κB) signaling, further augmenting HIV latency reversal via this pathway. This was tested in Jurkat and U1 cell line models of latency, HIV latently infected primary CD4+ T cells and resting CD4+ T cells isolated from ART-suppressed people living with HIV ex vivo . Surprisingly, crotonylation upregulates the levels of the active p52 subunit of the NF-κB transcription factor induced by AZD5582 in Jurkat models of HIV latency. Quantitative mass spectrometry screening and biochemical analysis suggest that the ubiquitination E3 ligase TRIM27 may be involved in p100 cleavage into p52 activated by AZD5582, part of the heterodimer to activate NF-κB for the transcription of HIV. When TRIM27 is depleted, transcription of HIV and AZD5582-induced latency reversal are reduced. Interestingly, both p100 and p52 levels are reduced by TRIM27 siRNA knockdown or by pharmacologic inhibition of TRIM27 deubiquitinase partner protein USP7 during the induction of ncNF-κB signaling. These observations reveal the complexity of HIV transcriptional machinery, particularly of NF-κB factors known to be essential for HIV latency disruption.

Enhanced Ability of Plant-Derived PGT121 Glycovariants To Eliminate HIV-1-Infected Cells.

The activity of broadly neutralizing antibodies (bNAbs) targeting HIV-1 depends on pleiotropic functions, including viral neutralization and the elimination of HIV-1-infected cells. Several in vivo studies have suggested that passive administration of bNAbs represents a valuable strategy for the prevention or treatment of HIV-1. In addition, different strategies are currently being tested to scale up the production of bNAbs to obtain the large quantities of antibodies required for clinical trials. Production of antibodies in plants permits low-cost and large-scale production of valuable therapeutics; furthermore, pertinent to this work, it also includes an advanced glycoengineering platform. In this study, we used Nicotiana benthamiana to produce different Fc-glycovariants of a potent bNAb, PGT121, with near-homogeneous profiles and evaluated their antiviral activities. Structural analyses identified a close similarity in overall structure and glycosylation patterns of Fc regions for these plant-derived Abs and mammalian cell-derived Abs. When tested for Fc-effector activities, afucosylated PGT121 showed significantly enhanced FcγRIIIa interaction and antibody dependent cellular cytotoxicity (ADCC) against primary HIV-1-infected cells, both in vitro and ex vivo. However, the overall galactosylation profiles of plant PGT121 did not affect ADCC activities against infected primary CD4+ T cells. Our results suggest that the abrogation of the Fc N-linked glycan fucosylation of PGT121 is a worthwhile strategy to boost its Fc-effector functionality. IMPORTANCE PGT121 is a highly potent bNAb and its antiviral activities for HIV-1 prevention and therapy are currently being evaluated in clinical trials. The importance of its Fc-effector functions in clearing HIV-1-infected cells is also under investigation. Our results highlight enhanced Fc-effector activities of afucosylated PGT121 MAbs that could be important in a therapeutic context to accelerate infected cell clearance and slow disease progression. Future studies to evaluate the potential of plant-produced afucosylated PGT121 in controlling HIV-1 replication in vivo are warranted.

CBF-1 Promotes the Establishment and Maintenance of HIV Latency by Recruiting Polycomb Repressive Complexes, PRC1 and PRC2, at HIV LTR.

The C-promoter binding factor-1 (CBF-1) is a potent and specific inhibitor of the human immunodeficiency virus (HIV)-1 LTR promoter. Here, we demonstrate that the knockdown of endogenous CBF-1 in latently infected primary CD4+ T cells, using specific small hairpin RNAs (shRNA), resulted in the reactivation of latent HIV proviruses. Chromatin immunoprecipitation (ChIP) assays using latently infected primary T cells and Jurkat T-cell lines demonstrated that CBF-1 induces the establishment and maintenance of HIV latency by recruiting polycomb group (PcG/PRC) corepressor complexes or polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Knockdown of CBF-1 resulted in the dissociation of PRCs corepressor complexes enhancing the recruitment of RNA polymerase II (RNAP II) at HIV LTR. Knockdown of certain components of PRC1 and PRC2 also led to the reactivation of latent proviruses. Similarly, the treatment of latently infected primary CD4+ T cells with the PRC2/EZH2 inhibitor, 3-deazaneplanocin A (DZNep), led to their reactivation.

Proteasomal Inhibition Potentiates Latent HIV Reactivation.

Despite the success of antiretroviral therapy (ART), ART fails to eradicate the virus and HIV cure has remained beyond the reach of current treatments. ART targets replicating virally infected but not latently infected cells, which have limited expression of factors important for proliferation and cellular activity, including positive transcription elongation factor b (P-TEFb) and nuclear factor κB (NF-κB). Levels of the cyclin T1 (CycT1) subunit of P-TEFb are low to absent in resting T cells, and treatment with proteasome inhibitors (PIs) increases CycT1 protein levels to those of proliferating T cells. In this study, the clinically approved PI bortezomib reactivated latent HIV in latently infected primary CD4+ T cells. Bortezomib not only increased levels of CycT1 but also activated NF-κB. Strikingly, as opposed to most currently researched latency reversing agents (LRAs), bortezomib did not require a second LRA to potently reactivate latent HIV. Effects of bortezomib on resting T cells and reactivation of HIV suggest a possible direction for future attempts to diminish the viral reservoir in HIV+ individuals.

Dynamic nanopore long-read sequencing analysis of HIV-1 splicing events during the early steps of infection

BackgroundAlternative splicing is a key step in Human Immunodeficiency Virus type 1 (HIV-1) replication that is tightly regulated both temporally and spatially. More than 50 different transcripts can be generated from a single HIV-1 unspliced pre-messenger RNA (pre-mRNA) and a balanced proportion of unspliced and spliced transcripts is critical for the production of infectious virions. Understanding the mechanisms involved in the regulation of viral RNA is therefore of potential therapeutic interest. However, monitoring the regulation of alternative splicing events at a transcriptome-wide level during cell infection is challenging. Here we used the long-read cDNA sequencing developed by Oxford Nanopore Technologies (ONT) to explore in a quantitative manner the complexity of the HIV-1 transcriptome regulation in infected primary CD4+ T cells.ResultsONT reads mapping to the viral genome proved sufficiently long to span all possible splice junctions, even distant ones, and to be assigned to a total of 150 exon combinations. Fifty-three viral RNA isoforms, including 14 new ones were further considered for quantification. Relative levels of viral RNAs determined by ONT sequencing showed a high degree of reproducibility, compared favourably to those produced in previous reports and highly correlated with quantitative PCR (qPCR) data. To get further insights into alternative splicing regulation, we then compiled quantifications of splice site (SS) usage and transcript levels to build “splice trees”, a quantitative representation of the cascade of events leading to the different viral isoforms. This approach allowed visualizing the complete rewiring of SS usages upon perturbation of SS D2 and its impact on viral isoform levels. Furthermore, we produced the first dynamic picture of the cascade of events occurring between 12 and 24 h of viral infection. In particular, our data highlighted the importance of non-coding exons in viral RNA transcriptome regulation.ConclusionONT sequencing is a convenient and reliable strategy that enabled us to grasp the dynamic of the early splicing events modulating the viral RNA landscape in HIV-1 infected cells.

Phosphoproteomic landscaping of HIV-1 in vitro identified phosphorylated epitopes capable of eliciting CD8+ cytotoxic T cell (CTL) responses in patient samples ex vivo

Abstract Significant progress has been made in recent years in our abilities to simultaneously detect proteome and posttranslational modifications (PTMs), including phosphorylation responsible for regulating protein functions. We have an incomplete understanding of the role of phosphorylation in host-pathogen interaction. The objective of this study was to fine-map phosphorylation of HIV-1 proteins and subsequently study the consequences of these PTMs. Using mass-spectrometry, we identified over 30 unique phosphorylated sites on HIV-1 proteins detected in infected primary CD4+ T cells and in cell-free virus, with the majority being present on the viral proteins Gag, Pol, and Rev proteins. Interestingly, many of these phosphorylated sites were within previously identified optimal CTL epitopes. While CTL responses to phospho-neoepitopes is subject to recent research in cancer immunology, we lack any understanding of how phosphorylation of virus affects antiviral immune responses. We stimulated PBMCs from HIV-1 infected patients ex vivo in the presence of HIV-1 epitopes with or without phosphorylation, followed by assessment of IFN-γ, TNF-α, MIP1β, and CD107a expressions by flowcytometry. Two Gag-derived phospho-epitopes were able to elicit immune responses in CTLs, potentially indicating the genesis and functional responses to such modified epitopes in vivo in HIV-1 infected patients. Moreover, CTL clones of HLA B*5701 background responded with significantly more cytotoxic marker without any effector cytokine expression, suggesting that phospho-epitopes can modulate the quality of CTL responses. Taken together, our studies shed light on a new way of evaluating host-pathogen interaction with potential for vaccine research.

DCas9 Targeted Chromatin and Histone Enrichment for Mass Spectrometry (Catchet-MS) Identifies IKZF1 as a Novel Drug-Able Target for HIV-1 Latency Reversal

A major pharmacological strategy for HIV cure aims to reverse latency in infected cells to promote reservoir eradication. While the unbiased identification of putative molecular targets physically associated with the latent HIV-1 promoter would be crucial for the development of latency reversal drugs, due to technical limitations, this has not been possible. Here we use dCas9 targeted chromatin and histone enrichment strategy coupled to mass spectrometry (Catchet-MS) to isolate and identify the locus-bound protein complexes on the latent and activated HIV-1 promoter. Catchet-MS identified both known and novel host factors distinctly associated with the latent HIV-1 5’LTR. Among these, IKZF1 is a novel transcriptional repressor of HIV-1 required for latency. We find the drug iberdomide, which targets IKZF1 for degradation, to be a clinically advanced novel LRA that reverses latency in latent ex vivo HIV-1 infected primary CD4+ T cells and in cells isolated from virally suppressed HIV-1 infected participants.

HIV-1 Vpu Downregulates Tim-3 from the Surface of Infected CD4+ T Cells.

Along with other immune checkpoints, T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is expressed on exhausted CD4+ and CD8+ T cells and is upregulated on the surface of these cells upon infection by human immunodeficiency virus type 1 (HIV-1). Recent reports have suggested an antiviral role for Tim-3. However, the molecular determinants of HIV-1 which modulate cell surface Tim-3 levels have yet to be determined. Here, we demonstrate that HIV-1 Vpu downregulates Tim-3 from the surface of infected primary CD4+ T cells, thus attenuating HIV-1-induced upregulation of Tim-3. We also provide evidence that the transmembrane domain of Vpu is required for Tim-3 downregulation. Using immunofluorescence microscopy, we determined that Vpu is in close proximity to Tim-3 and alters its subcellular localization by directing it to Rab 5-positive (Rab 5+) vesicles and targeting it for sequestration within the trans- Golgi network (TGN). Intriguingly, Tim-3 knockdown and Tim-3 blockade increased HIV-1 replication in primary CD4+ T cells, thereby suggesting that Tim-3 expression might represent a natural immune mechanism limiting viral spread.IMPORTANCE HIV infection modulates the surface expression of Tim-3, but the molecular determinants remain poorly understood. Here, we show that HIV-1 Vpu downregulates Tim-3 from the surface of infected primary CD4+ T cells through its transmembrane domain and alters its subcellular localization. Tim-3 blockade increases HIV-1 replication, suggesting a potential negative role of this protein in viral spread that is counteracted by Vpu.

Induction of Transient Virus Replication Facilitates Antigen-Independent Isolation of SIV-Specific Monoclonal Antibodies

Structural characterization of the HIV-1 Envelope (Env) glycoprotein has facilitated the development of Env probes to isolate HIV-specific monoclonal antibodies (mAbs). However, preclinical studies have largely evaluated these virus-specific mAbs against chimeric viruses, which do not naturally infect non-human primates, in contrast to the unconstrained simian immunodeficiency virus (SIV)mac239 clone. Given the paucity of native-like reagents for the isolation of SIV-specific B cells, we examined a method to isolate SIVmac239-specific mAbs without using Env probes. We first activated virus-specific B cells by inducing viral replication after the infusion of a CD8β-depleting mAb or withdrawal of antiretroviral therapy in SIVmac239-infected rhesus macaques. Following the rise in viremia, we observed 2- to 4-fold increases in the number of SIVmac239 Env-reactive plasmablasts in circulation. We then sorted these activated B cells and obtained 206 paired Ab sequences. After expressing 122 mAbs, we identified 14 Env-specific mAbs. While these Env-specific mAbs bound to both the SIVmac239 SOSIP.664 trimer and to infected primary rhesus CD4+ T cells, five also neutralized SIVmac316. Unfortunately, none of these mAbs neutralized SIVmac239. Our data show that this method can be used to isolate virus-specific mAbs without antigenic probes by inducing bursts of contemporary replicating viruses in vivo.

Limitations of dual-fluorescent HIV reporter viruses in a model of pre-activation latency.

IntroductionHIV latency can be established in vitro following direct infection of a resting CD4+ T cell (pre‐activation latency) or infection of an activated CD4+ T cell which then returns to a resting state (post‐activation latency). We modified a previously published dual‐fluorescent reporter virus seeking to track the establishment and reactivation of pre‐activation latency in primary CD4+ T cells.MethodsA previously published dual‐fluorescent reporter virus was modified so that expression of enhanced green fluorescent protein (GFP) was under control of the elongation factor 1 alpha (EF1α) promoter to detect latent infection, and E2 crimson (E2CRM) was under control of the nef promoter to detect productive infection. NL4.3 that expressed GFP in place of nef was used as a positive control. We infected the Jurkat T‐cell line and primary CD4+ T cells that were either unstimulated or stimulated with either the chemokine CCL19 or phytohaemagglutinin (PHA)/IL‐2 and quantified the expression of both fluorescent proteins by flow cytometry. The study was carried out over a period of two years from September 2016 to October 2018.Results and DiscussionExpression of both fluorophores was detected following infection of the Jurkat T‐cell line while only low levels of the latent reporter were observed following infection of primary CD4+ T cells. In unstimulated and CCL19‐treated CD4+ T cells, expression of the GFP latent reporter, increased after further activation of the cells with PHA/phorbol 12‐myristate 13‐acetate (PMA).ConclusionsOur findings demonstrate that the EF1α promoter has poor constitutive expression in resting CD4+ T cells. Therefore, dual‐fluorescent reporter viruses with the EF1α promoter may underestimate the frequency of latent infection in resting CD4+ T cells.

PP 1.35 - An unbiased platform to identify regulators of HIV latency in infected primary human CD4 T Cells

PP 1.35 - An unbiased platform to identify regulators of HIV latency in infected primary human CD4 T Cells

Cell Size-Based Decision-Making of a Viral Gene Circuit

SUMMARYLatently infected T cells able to reinitiate viral propagation throughout the body remain a major barrier to curing HIV. Distinguishing between latently infected cells and uninfected cells will advance efforts for viral eradication. HIV decision-making between latency and active replication is stochastic, and drug cocktails that increase bursts of viral gene expression enhance reactivation from latency. Here, we show that a larger host-cell size provides a natural cellular mechanism for enhancing burst size of viral expression and is necessary to destabilize the latent state and bias viral decision-making. Latently infected Jurkat and primary CD4+ T cells reactivate exclusively in larger activated cells, while smaller cells remain silent. In addition, reactivation is cell-cycle dependent and can be modulated with cell-cycle-arresting compounds. Cell size and cell-cycle dependent decision-making of viral circuits may guide stochastic design strategies and applications in synthetic biology and may provide important determinants to advance diagnostics and therapies.

Histone H2A monoubiquitylation and p38-MAPKs regulate immediate-early gene-like reactivation of latent retrovirus HTLV-1.

It is not understood how the human T cell leukemia virus human T-lymphotropic virus-1 (HTLV-1), a retrovirus, regulates the in vivo balance between transcriptional latency and reactivation. The HTLV-1 proviral plus-strand is typically transcriptionally silent in freshly isolated peripheral blood mononuclear cells from infected individuals, but after short-term ex vivo culture, there is a strong, spontaneous burst of proviral plus-strand transcription. Here, we demonstrate that proviral reactivation in freshly isolated, naturally infected primary CD4+ T cells has 3 key attributes characteristic of an immediate-early gene. Plus-strand transcription is p38-MAPK dependent and is not inhibited by protein synthesis inhibitors. Ubiquitylation of histone H2A (H2AK119ub1), a signature of polycomb repressive complex-1 (PRC1), is enriched at the latent HTLV-1 provirus, and immediate-early proviral reactivation is associated with rapid deubiquitylation of H2A at the provirus. Inhibition of deubiquitylation by the deubiquitinase (DUB) inhibitor PR619 reverses H2AK119ub1 depletion and strongly inhibits plus-strand transcription. We conclude that the HTLV-1 proviral plus-strand is regulated with characteristics of a cellular immediate-early gene, with a PRC1-dependent bivalent promoter sensitive to p38-MAPK signaling. Finally, we compare the epigenetic signatures of p38-MAPK inhibition, DUB inhibition, and glucose deprivation at the HTLV-1 provirus, and we show that these pathways act as independent checkpoints regulating proviral reactivation from latency.