Antiretroviral therapies (ARTs) abrogate HIV replication; however, infection persists as long-lived reservoirs of infected cells with integrated proviruses, which reseed replication if ART is interrupted. A central tenet of our current understanding of this persistence is that infected cells are shielded from immune recognition and elimination through a lack of antigen expression from proviruses. Efforts to cure HIV infection have therefore focused on reactivating latent proviruses to enable immune-mediated clearance, but these have yet to succeed in reducing viral reservoirs. Here, we revisited the question of whether HIV reservoirs are predominately immunologically silent from a new angle: by querying the dynamics of HIV-specific T cell responses over long-term ART for evidence of ongoing recognition of HIV-infected cells. In longitudinal assessments, we show that the rates of change in persisting HIV Nef-specific responses, but not responses to other HIV gene products, were associated with residual frequencies of infected cells. These Nef-specific responses were highly stable over time and disproportionately exhibited a cytotoxic, effector functional profile, indicative of recent in vivo recognition of HIV antigens. These results indicate substantial visibility of the HIV-infected cells to T cells on stable ART, presenting both opportunities and challenges for the development of therapeutic approaches to curing infection.
Abstract Antiretroviral therapies (ART) durably suppress HIV replication to undetectable levels – however, infection persists in the form of long-lived reservoirs of infected cells with integrated proviruses, that re-seed systemic replication if ART is interrupted. A central tenet of our current understanding of this persistence is that infected cells are shielded from immune recognition and elimination through a lack of antigen expression from proviruses. Efforts to cure HIV infection have therefore focused on reactivating latent proviruses to enable immune-mediated clearance, but these have yet to succeed in driving reductions in viral reservoirs. Here, we revisited the question of whether HIV reservoirs are predominately immunologically silent from a new angle, by querying the dynamics of HIV-specific T-cell responses over long-term ART for evidence of ongoing recognition of HIV-infected cells. We show that T-cell responses to autologous reservoir viruses persist over years, and that the maintenance of HIV-Nef-specific responses was uniquely associated with residual frequencies of infected cells. These responses disproportionately exhibited a cytotoxic, effector functional profile, indicative of recent in vivo recognition of HIV-infected cells. These results indicate substantial visibility of the HIV reservoir to T-cells on stable ART, presenting both opportunities and challenges for the development of therapeutic approaches to curing HIV infection.
Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Human immunodeficiency virus (HIV) infection is currently incurable, due to the persistence of latently infected cells. The ‘shock and kill’ approach to a cure proposes to eliminate this reservoir via transcriptional activation of latent proviruses, enabling direct or indirect killing of infected cells. Currently available latency-reversing agents (LRAs) have however proven ineffective. To understand why, we used a novel HIV reporter strain in primary CD4+ T cells and determined which latently infected cells are reactivatable by current candidate LRAs. Remarkably, none of these agents reactivated more than 5% of cells carrying a latent provirus. Sequencing analysis of reactivatable vs. non-reactivatable populations revealed that the integration sites were distinguishable in terms of chromatin functional states. Our findings challenge the feasibility of ‘shock and kill’, and suggest the need to explore other strategies to control the latent HIV reservoir. https://doi.org/10.7554/eLife.34655.001 Introduction Antiretroviral therapy (ART) has transformed HIV infection from a uniformly deadly disease into a chronic lifelong condition, saving millions of lives. However, ART interruption leads to rapid viral rebound within weeks due to the persistence of proviral latency in rare, long-lived resting CD4+ T cells and possibly in tissue macrophages (Honeycutt et al., 2017). HIV latency is defined as the presence of a transcriptionally silent but replication-competent proviral genome. Latency allows infected cells to evade both immune clearance mechanisms and currently available ART, which is based solely on the elimination of actively replicating virus. An extensively investigated approach to purging latent HIV is the ‘shock and kill’ strategy, which consists of forcing the reactivation of latent proviruses (‘shock’ phase) with the use of latency reversing agents (LRAs), while maintaining ART to prevent de novo infections. Subsequently, reactivation of HIV expression would expose such cells (shocked cells) to killing by viral cytopathic effects and immune clearance (‘kill’ phase). A variety of LRAs have been explored in vitro and ex vivo, with only a few candidates being advanced to testing in pilot human clinical trials. Use of histone deacetylase inhibitors (HDACi: vorinostat, panobinostat, romidepsin, and disulfiram) in clinical studies has shown increases in cell-associated HIV RNA production and/or plasma viremia after in vivo administration (Archin et al., 2012a; Elliott et al., 2015; Elliott et al., 2014; Rasmussen et al., 2014; Søgaard et al., 2015). However, none of these interventions alone has succeeded in significantly reducing the size of the latent HIV reservoir (Rasmussen and Lewin, 2016). Several obstacles can explain the failure of LRAs, as reviewed in (Margolis et al., 2016; Rasmussen et al., 2016). However, the biggest challenge to date is our inability to accurately quantify the size of the reservoir. The absolute quantification (number of cells) of the latent reservoir in vivo (and ex vivo) has thus far been technically impossible. The most sensitive, quickest, and easiest assays to measure the prevalence of HIV-infected cells are PCR-based, quantifying total or integrated HIV DNA or RNA transcripts. However these assays substantially overestimate the number of latently infected cells, due to the predominance of defective HIV DNA genomes in vivo (Bruner et al., 2016; Ho et al., 2013). The best currently available assay to measure the latent reservoir is the relatively cumbersome viral outgrowth assay (VOA), which is based on quantification of the number of resting CD4+ T cells that produce infectious virus after a single round of maximum in vitro T-cell activation. After several weeks of culture, viral outgrowth is assessed by an ELISA assay for HIV-1 p24 antigen or a PCR assay for HIV-1 RNA in the culture supernatant. Importantly, the number of latently infected cells detected in the VOA is 300-fold lower than the number of resting CD4+ T cells that harbor proviruses detectable by PCR. This reliance on a single round of T-cell activation likely incorrectly estimates the viral reservoir for two reasons. First, the discovery of intact non-induced proviruses indicates that the size of the latent reservoir may be much greater than previously thought: the authors estimate that the number may be at least 60 fold higher than estimates based on VOA (Ho et al., 2013; Sanyal et al., 2017). This work and that of others (Chen et al., 2017) highlight the heterogeneous nature of HIV latency and suggest that HIV reactivation is a stochastic process that only reactivates a small fraction of latent viruses at any given time (Dar et al., 2012; Ho et al., 2013; Singh et al., 2010; Weinberger et al., 2005). Second, the ability of defective proviruses to be transcribed and translated in vivo (Pollack et al., 2017): this study shows that, although defective proviruses cannot produce infectious particles, they express viral RNA and proteins, which can be detectable by any p24 antigen or PCR assays used for the reservoir-size quantification. Thus, current assays underestimate the actual number of latently infected cells, both in vivo and ex vivo, and the real size of HIV reservoir is still to be determined. Therefore, it has been difficult to judge the potential of LRAs in in vitro (latency primary models), ex-vivo (patients’ samples) and in vivo (clinical trial) experiments. HIV latency is a complex, multi-factorial process (reviewed in [Dahabieh et al., 2015]). Its establishment and maintenance depend on: (a) viral factors, such as integrase that specifically interacts with cellular proteins, including LEDGF, (b) trans-acting factors (e.g., transcription factors) and their regulation by the activation state of T cells and the environmental cues that these cells receive, and (c) cis-acting mechanisms, such as the local chromatin environment at the site of integration of the virus into the genome. Recent evidence has also highlighted the association of specific HIV-1 integration sites with clonal expansion of latently infected cells (reviewed in [Maldarelli, 2016]). The role of the site of HIV integration into the cellular genome in the establishment and maintenance of HIV latency has remained controversial. While early studies found that the HIV integration site does affect both the entry into latency (Chen et al., 2017; Jordan et al., 2003; Jordan et al., 2001), and the viral response to LRAs (Chen et al., 2017), other studies have failed to find a significant role of integration sites in regulating the fate of HIV infection (Dahabieh et al., 2014; Sherrill-Mix et al., 2013). In this study, we have used a new dual color reporter virus, HIVGKO, to investigate the reactivation potential of various LRAs in pure latent population. We find that latency is heterogeneous and that only a small fraction (<5%) of the latently infected cells is reactivated by LRAs. We also show that both genomic localization and chromatin context of the integration site affect the fate of HIV infection and the reversal of viral latency. Results A second-generation dual-fluorescence HIV-1 reporter (HIVGKO) to study latency Our laboratory reported the development of a dual-labeled virus (DuoFluoI) in which eGFP is under the control of the HIV-1 promoter in the 5′ LTR and mCherry is under the control of the cellular elongation factor one alpha promoter (EF1α) (Calvanese et al., 2013). However, we noted that the model was limited by a modest number of latently infected cells (<1%) generated regardless of viral input (Figure 1—figure supplement 1A–1C), as well as a high proportion of productively infected cells in which the constitutive promoter EF1α was not active (GFP+, mCherry-). To address these issues, which we suspected were due to recombination between the 20–30 bp regions of homology at the N- and C-termini of the adjacent fluorescent proteins (eGFP and mCherry) (Salamango et al., 2013), we generated a new version of dual-labeled virus (HIVGKO), containing a codon-switched eGFP (csGFP) and a distinct, unrelated fluorescent protein mKO2 under the control of EF1α (Figure 1A). First, titration of HIVGKO input revealed that productively and latently infected cells increased proportionately as the input virus increased (Figure 1B and Figure 1—figure supplement 1), unlike the original DuoFluoI (Figure 1—figure supplement 1). Second, comparison of primary CD4+ T cells infected with HIVGKO or the original DuoFluoI revealed an increase in double-positive (csGFP+ mKO2+) infected cells in HIVGKO infected cells (Figure 1C). A small proportion of csGFP+ mKO2- cells were still visible in HIVGKO infected cells. We generated a HIVGKO virus lacking the U3 promoter region of the 3′LTR (ΔU3-GKO), resulting in an integrated virus devoid of the 5’ HIV U3 region. This was associated with a suppression of HIV transcription and an inversion of the latency ratio (ratios latent/productive = 0.34 for HIVGKO-WT-LTR and 8.8 for HIVGKO-ΔU3-3’LTR - Figure 1D). Finally, to further characterize the constituent populations of infected cells, double-negative cells, latently and productively infected cells were sorted using FACS and analyzed for viral mRNA and protein content. (Figures 1E and F, Figure 1—source data 1). As expected, productively infected cells (csGFP+) expressed higher amounts of viral mRNA and viral proteins, but latently infected cells (csGFP- mKO2+) had very small amounts of viral mRNA and no detectable viral proteins. Figure 1 with 1 supplement see all Download asset Open asset Second generation of dual-fluorescence HIV-1 reporter, HIVGKO to quantify stable latency. (A) Schematic representation of first (top: HIVDuoFluoI) and second generation (bottom: HIVGKO) of dual-labeled HIV-1 reporters. (B) Representative experiment of HIVGKO virus titration in activated primary CD4+ T cells (4 days post-infection). Primary CD4+ T cells were activated with αCD3/CD28 beads + 20 U/mL IL-2 for 3 days before infection with different amounts of HIVGKO (input, ng/p24) and analyzed by flow cytometry 4 days post-infection. (C) Comparison of HIVDuoFluoI and HIVGKO infection profiles by flow cytometry in activated primary CD4+ T-cells (4 days post-infection). Cells were treated as in (B). (D) Comparison of GKO-WT-LTR and GKO-ΔU3 3'LTR infection profiles by flow cytometry in cells treated as in (B). (E, F) Primary CD4+ T cells were treated as in (B). At 4 days post-infection, double-negative, productively infected, and latently infected cells were sorted out, and (E) the total RNA isolated from each population was subjected to Taqman RT-qPCR analysis (Source Data - Figure 1). Unspliced (US), singly spliced (SS), and multiply spliced (MS) HIV-1 mRNAs were quantified relative to cellular GAPDH. (F) Western blot analysis of each population. https://doi.org/10.7554/eLife.34655.002 Figure 1—source data 1 Taqman RT-qPCR analysis of unspliced (US), singly spliced (SS), and multiply spliced (MS) HIV-1 mRNAs in the uninfected, double negative, latent and productive populations. The experiment is detailed in the main text and Figure 1 legend. https://doi.org/10.7554/eLife.34655.004 Download elife-34655-fig1-data1-v3.xlsx Based on all these findings, the second-generation of dual-fluorescence reporter, HIVGKO, is able to more accurately quantify latent infections in primary CD4+ T cells than HIVDuoFluoI, and thus allows for the identification and purification of a larger number of latently infected cells. Using flow cytometry, we can determine infection and HIV productivity of individual cells and simultaneously control for cell viability. Correlation between LRA efficacy in HIV-infected patient samples and activity in HIVGKO latently infected cells Next, we evaluated the reactivation of latent HIVGKO in primary CD4+ T cells by LRAs, and compared it with the ability of the same LRAs to reverse latency in CD4+ T cells isolated from HIV-infected individuals. We tested the following LRAs: (a) the histone deacetylase inhibitor (HDACi) panobinostat (Rasmussen et al., 2013), (b) the bromodomain-containing protein 4 (BRD4) inhibitor JQ1, which acts through positive transcription elongation factor (P-TEFb) (Banerjee et al., 2012; Boehm et al., 2013; Filippakopoulos et al., 2010; Li et al., 2013; Zhu et al., 2012), and (c) the PKC activator, bryostatin-1 (del Real et al., 2004; Mehla et al., 2010). Viral reactivation mediated by these LRAs was compared to treatment of CD4+ T cells with αCD3/CD28 (Spina et al., 2013). Several studies have shown synergetic effects when combining different LRAs (Darcis et al., 2015; Jiang et al., 2015; Laird et al., 2015; Martínez-Bonet et al., 2015), therefore we also tested bryostatin-1 in combination with either panobinostat or JQ1. Drugs were used at concentrations previously shown to be effective at reversing latency in other model systems (Archin et al., 2012a; Bullen et al., 2014; Laird et al., 2015; Spina et al., 2013). To measure reactivation by LRAs in patient samples, we treated 5 million purified resting CD4+ T cells from four HIV infected individuals on suppressive ART (participant characteristics in Table 1) with single LRAs, combinations thereof, or vehicle alone for 24 hr. LRAs efficacy was assessed using a PCR-based assay, by measuring levels of intracellular HIV-1 RNA using primers and a probe that detect the 3′ sequence common to all correctly terminated HIV-1 mRNAs (Bullen et al., 2014). Of the LRAs tested individually, none exhibited a statistically significant effect (n=4 - Figure 2A, Figure 2—source data 1). Importantly, T-cell activation positive control, αCD3/CD28 (24.4-fold, Figure 2A), showed expected fold induction value (10 to 100-fold increases of HIV RNA in PBMCs [Bullen et al., 2014; Darcis et al., 2015; Laird et al., 2015]). Combinations of the PKC agonist bryostatin-1 with JQ1 or with panobinostat (fold-increases of 126.2- and 320.8-fold, respectively, Figure 2A), were highly more effective than bryostatin-1, JQ1 or panobinostat alone (fold-increases of 6.8, 1.7- and 2.9-fold, respectively, Figure 3A), and even greater than T-cell activation with αCD3/CD28. This observation is consistent with previous reports (Darcis et al., 2015; Jiang et al., 2015; Laird et al., 2015; Martínez-Bonet et al., 2015). Figure 2 Download asset Open asset LRAs efficacy in patient samples is predicted by activity in HIVGKO latently infected cells. (A) Intracellular HIV-1 mRNA levels in rCD4s, obtained from infected individuals and treated ex vivo with a single LRA or a combination of two LRAs for 24 hr in presence of raltegravir, presented as fold induction relative to DMSO control. (n = 4, mean +SEM) (Figure 2—source data 1). (B) Intracellular HIV-1 mRNA levels in HIVGKO latently infected CD4+ T-cells, and treated with a single LRA or a combination of two LRAs for 6 hr in presence of raltegravir, presented as fold induction relative to DMSO control. (n = 3 (different donors), mean +SEM, paired t-test) (Figure 2—source data 1). (C) Correlation between intracellular HIV-1 mRNA levels quantified in either 6 hr stimulated HIVGKO latently infected CD4+ T-cells from different donors, or 24 hr stimulated rCD4s from HIV infected patients, with a single LRA or a combination of two LRAs in presence of raltegravir. https://doi.org/10.7554/eLife.34655.005 Figure 2—source data 1 Intracellular HIV-1 mRNA levels in rCD4s, obtained from infected individuals, or in HIVGKO latently infected CD4+ T-cells. The experiment is detailed in the main text and Figure 2 legend. https://doi.org/10.7554/eLife.34655.006 Download elife-34655-fig2-data1-v3.xlsx Figure 3 Download asset Open asset Few HIVGKO latently infected primary CD4+ T cells are reactivated. (A) Schematic of experimental procedure with primary CD4+ T cells. Briefly, CD4+ T cells were purified from blood of healthy donors and activated for 72 hr with αCD3/CD28 beads and 100 U/ml IL-2 before infection with HIVGKO. Five days post-infection, latently infected cells (csGFP- mKO2+) cells were sorted, put back in culture overnight and stimulated with different LRAs in presence of raltegravir for 24 hr before performing FACS analysis. (B) Percentage of GFP+ cells is shown after stimulation of latently infected CD4+ T-cells with LRAs (n = 4 (different donors), mean +SEM, paired t-test) (Figure 3—source data 1). (C) Histogram plot of percent live cells for each drug treatment (n = 3 (different donors), mean + SEM, paired t-test) (Figure 3—source data 1). p-value: *p<0.05, **p<0.01 relative to DMSO. https://doi.org/10.7554/eLife.34655.007 Figure 3—source data 1 Percentage of GFP+ cells is shown after stimulation of latently infected CD4+ T-cells with LRAs as well as percent live cells for each drug treatment. The experiment is detailed in the main text and Figure 3 legend. https://doi.org/10.7554/eLife.34655.008 Download elife-34655-fig3-data1-v3.xlsx Table 1 Characteristics of HIV-1-infected study participants ABC, abacavir; DRV, darunavir; FTC, emtricitabine; RPV, rilpivirine; RTV, ritonavir; TCV, tivicay; TDF, tenofovir; 3TC, lamivudine; VL, viral load. https://doi.org/10.7554/eLife.34655.009 Scope IDAgeSexEthnicityCD4 CountDuration of infection (years)ART regimenDuration of ART (years)Peak reporter VL (copies/ml−1) 159756MMixed46919RPV/TDF/FTC545734 214759MAsian59728RPV/TDF/FTC23374000 246162MWhite66432RPV/TCV1920000 316254MWhite73429RTV, DRV, ABC/TCV/3TC20171000 The same LRAs and combinations were next tested after infection of human CD4+T cells in vitro with HIVGKO. Measurement of intracellular HIV-1 mRNA in HIVGKO latently infected cells showed an expected fold induction of latency in response to αCD3/CD28 (11.3-fold, Figure 2B, Figure 2—source data 1). Second, JQ1, panobinostat, and bryostatin-1 alone all caused limited reactivation of latent HIV (fold-increases of 1.1-, 5.6- and 6.2-fold, respectively, Figure 2B), as observed in patients’ samples. Finally, we observed low synergy when combining bryostatin and JQ1 (8-fold increase), but high synergy between bryostatin and panobinostat (67.3-fold increase). These data together demonstrate that HIVGKO closely mimics in vitro what is observed in ex vivo patients’ samples (correlation rate r2 = 0.88, p=0.0056 - Figure 2C), and validate the robustness and reliability of the dual-florescence HIV reporter as a model to study HIV-1 latency. HIV-1 LRAs target a minority of latently infected primary CD4+ T cells Current assays have relied on PCR-based assays to measure HIV RNA, and to evaluate the efficacy of different LRAs (Figure 2A). The use of dual-fluorescent HIV reporters, however, provides a tool to quantify directly the fraction of cells that become reactivated. To quantify the absolute number of latently infected cells reactivated following LRA treatment, primary CD4+ T cells were infected with HIVGKO, and cultured for 5 days (in the presence of IL-2) before sorting the pure latent population (GFP-, mKO2+). Cells were allowed to rest overnight and were treated for 24 hr with the various LRAs (same drug concentrations as in Figure 2) (Figure 3A, Figure 3—source data 1). Culture of DMSO-treated latently infected primary CD4+ T cells produced little spontaneous reactivation (average of four experiments: 1.4% of GFP+ cells). Unexpectedly, we found that none of the individual LRAs or their combinations reactivated more than 5.5% of the latently infected cells: JQ1 (1.7%) panobinostat (3.7%), bryostatin-1 (3%) αCD3/CD28 (4.5%), bryostatin-1 and JQ1 (3.3%.). bryostatin-1 and panobinostat (5.5%) (Figure 3B). Small fractional rate of latency reactivation is not explained by low cellular response to activation signals These data highlight two important facts: a) cell-associated HIV RNA quantification does not reflect the absolute number of cells undergoing viral reactivation, and b) induced cell-associated HIV RNA, in response to all reversing agents, comes from a small fraction of reactivated latent cells. This was particularly surprising with αCD3/CD28 stimulation, as a currently accepted model for HIV latency is that the state of T cell activation dictates the transcriptional state of the provirus. Treatment of latently infected primary CD4+ T cells with αCD3/CD28 stimulated HIV production in less than 5% of the cells, while the other 95% remained latent, even though after 24 hr of treatment nearly all of the cells had upregulated the early T cell activation marker CD69 (Figure 4—figure supplement 1). To further rule out the possibility that non-reactivated latently infected cells (NRLIC) simply represented a lack of efficient response to T-cell activation signals, we analyzed T-cell activation markers within the different populations (i.e., within uninfected, non reactivated (NRLIC) and reactivated latently infected cells (RLIC); Figure 4, Figure 4—source data 1). Briefly, 72h-stimulated CD4+ T cells were infected with HIVGKO, and 4 days later, GFP- cells were sorted, and allowed to rest overnight before restimulation with αCD3/CD28. After another 24 hr, cells were stained for the early, intermediate, and late markers of T cell activation CD69, CD25 and HLA-DR respectively. The three different populations, double negative, RLIC and NRLIC, had similar profiles of activated T-cell subsets, as shown in Figure 4, and were mainly composed of strongly activated cells (CD69+/CD25+/HLA-DR+/-). We only observed a statistically significant increase of NRLIC compared with RLIC in the CD69+/CD25-/HLA-DR+ population, however this small increase in a relatively minor population is insufficient to explain the low reactivation rate of latently infected cells. Overall, comparison of both reactivated and non reactivated latent populations showed little difference in their activation state. Figure 4 with 1 supplement see all Download asset Open asset Low-level latency reactivation is not explained by low cellular responses to activation signals. T-cell activation patterns between double negative, reactivated (RLIC) and non-reactivated (NRLIC) latently infected cells. Briefly, CD4+ T-cells were purified from blood of four healthy donors and activated for 72 hr with αCD3/CD28 beads and 20 U/ml IL-2 before infection with HIVGKO. At 4 days post-infection, csGFP- were sorted, cultured overnight and stimulated with αCD3/CD28 in presence of raltegravir. At 24 hr post-treatment, cells were stained for CD25, CD69, and HLA-DR activation markers before performing FACS analysis. (n = 4, mean +SEM, paired t-test; *p<0.05; **p<0.01) (Figure 4—source data 1). https://doi.org/10.7554/eLife.34655.010 Figure 4—source data 1 CD25, CD69, and HLA-DR activation markers patterns between double negative, reactivated (RLIC) and non-reactivated (NRLIC) latently infected cells. The experiment is detailed in the main text and Figure 4 legend. https://doi.org/10.7554/eLife.34655.012 Download elife-34655-fig4-data1-v3.xlsx Integration sites, gene expression, transcription units and the fate of HIV infection The role of the site of HIV integration into the genome in latency remains a subject of debate (Chen et al., 2017; Dahabieh et al., 2014; Jordan et al., 2003; Jordan et al., 2001; Sherrill-Mix et al., 2013). To identify possible differences in integration sites between reactivated and non-reactivated HIV genomes, primary CD4+ T-cells were infected with HIVGKO. At 5 days post-infection, productively infected cells (GFP+, PIC) were sorted and frozen. The GFP negative population (consisting of a mixture of latent and uninfected) was isolated and treated with αCD3/CD28. 48 hr post-induction, both non reactivated (NRLIC) and reactivated (RLIC) populations were isolated. Nine libraries (three donors, three samples/donor: PIC, RLIC, NRLIC) were constructed from genomic DNA as described (Cohn et al., 2015) and analyzed by high-throughput sequencing to locate HIV proviruses within the human genome. A total of 1803 virus integration sites were determined: 960 integrations in PIC, 681 in NRLIC, and 162 in RLIC (Integration Sites Source data). To determine whether integration within genes differentially expressed during T-cell activation predicted infection reactivation fate, we compared our HIV integration dataset with a published dataset for gene expression in resting and activated (48 hr - αCD3/CD28) CD4+ T cells from healthy individuals (Ye et al., 2014). The analysis revealed that most of the αCD3/CD28-induced latent proviruses were not integrated in genes responsive to T-cell activation signals (Figure 5A and B, Figure 5—source data 1). Interestingly, PIC and RLIC integration events were associated with genes whose basal expression was significantly higher than genes targeted in NRLIC, both in activated and resting T cells (Figure 5C, Figure 5—source data 2). Figure 5 Download asset Open asset Relative expression of HIV-1 integration targeted genes for each population, before or after TCR activation. (A) Scatter chart showing primary CD4+ T-cell gene expression changes after 48 hr of stimulation with αCD3/CD28 beads. Integration sites displayed outside of the two solid gray lines were targeted genes whose expression is at least ± twofold differentially expressed after 48 hr stimulation. Plot points size can be different, the bigger the plot point is, the more integration events happened within the same gene. (B) Fraction of integration sites from the different populations PIC, RLIC or NRLIC, integrated within genes whose expression is at least ± twofold differentially expressed after 48 hr of αCD3/CD28 stimulation (**p<0.01; ***p<0.001; two-proportion z test) (Figure 5—source data 1). (C) Relative expression of genes targeted by HIV-1 integration in PIC, RLIC or NRLIC before TCR stimulation and after αCD3/CD28 stimulation (n = 3, mean +SEM, paired t-test). ***p<0.001; ****p<0.0001. (Figure 5—source data 2). https://doi.org/10.7554/eLife.34655.013 Figure 5—source data 1 Fraction of integration sites from the different populations PIC, RLIC or NRLIC, integrated within genes whose expression is at least ± twofold differentially expressed after 48 hr of αCD3/CD28 stimulation. The experiment is detailed in the main text and Figure 5 legend. https://doi.org/10.7554/eLife.34655.014 Download elife-34655-fig5-data1-v3.xlsx Figure 5—source data 2 Relative expression of genes targeted by HIV-1 integration in PIC, RLIC or NRLIC before TCR stimulation and after 48 hr αCD3/CD28 stimulation. The experiment is detailed in the main text and Figure 6 legend. https://doi.org/10.7554/eLife.34655.015 Download elife-34655-fig5-data2-v3.xlsx Next, we investigated whether different genomic regions were associated with productive, inducible or non-inducible latent HIV-1 infections. In agreement with previous studies (Cohn et al., 2015; Dahabieh et al., 2014; Maldarelli et al., 2014; Wagner et al., 2014), the majority of integration sites were found within genes in each population (Figure 6A, Figure 6—source data 1), although the proportion of genic integrations in NRLIC was significantly lower than in PIC and RLIC samples. Moreover, integration events in the PIC and RLIC populations were more frequent in transcribed regions (64% and 58%, respectively, [sum of low + medium + high transcribed regions] (Figure 6B), Figure 6—source data 1), while these regions were significantly less represented in the NRLIC (31%) (Figure 6B). As expected since introns represent a much larger proportion of genes, genic integration events were more frequent in the introns for each population (>65%, Figure 6C, Figure 6—source data 1). Finally, viral orientation of proviruses with respect to the transcriptional unit did not correlate with the fate of HIV infection (latent vs productive) or the reactivation or absence thereof of HIV latency (Figure 6D, Figure 6—source data 1). Figure 6 Download asset Open asset Insertion landscapes of HIV-1. (A) Proportion of mapped insertions that are in genic or intergenic regions. (Figure 6—source data 1). (B) Proportion of integration sites in transcribed regions with high (top 1/8), medium (top 1/4–1/8), low expression (top 1/2–1/4), trace (bottom 1/2) or silent (0) expression. (Figure 6—source data 1). (C) Proportion of unique genic integration sites located in introns, exons, UTR or promoters. (Figure 6—source data 1). (D) Transcriptional orientation of integrated HIV-1 relative to host gene. (Figure 6—source data 1). p-value: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 using two-proportion z test. https://doi.org/10.7554/eLife.34655.016 Figure 6—source data 1 Proportion of mapped insertions that are in genic or intergenic regions; of integration sites in transcribed regions with high, medium, low expression, trace or silent expression; of unique genic integration sites located in introns, exons, UTR or promoters; and transcriptional orientation of integrated HIV-1 relative to
HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.
The primary reservoir for HIV is within memory CD4+ T cells residing within tissues, yet the features that make some of these cells more susceptible than others to infection by HIV is not well understood. Recent studies demonstrated that CCR5-tropic HIV-1 efficiently enters tissue-derived memory CD4+ T cells expressing CD127, the alpha chain of the IL7 receptor, but rarely completes the replication cycle. We now demonstrate that the inability of HIV to replicate in these CD127-expressing cells is not due to post-entry restriction by SAMHD1. Rather, relative to other memory T cell subsets, these cells are highly prone to undergoing latent infection with HIV, as revealed by the high levels of integrated HIV DNA in these cells. Host gene expression profiling revealed that CD127-expressing memory CD4+ T cells are phenotypically distinct from other tissue memory CD4+ T cells, and are defined by a quiescent state with diminished NFκB, NFAT, and Ox40 signaling. However, latently-infected CD127+ cells harbored unspliced HIV transcripts and stimulation of these cells with anti-CD3/CD28 reversed latency. These findings identify a novel subset of memory CD4+ T cells found in tissue and not in blood that are preferentially targeted for latent infection by HIV, and may serve as an important reservoir to target for HIV eradication efforts.
Abstract Persistent HIV reservoirs in CD4⁺ T-cells pose a barrier to curing HIV infection. We identified overexpression of enhancer of zeste homolog 2 (EZH2) in HIV-infected CD4⁺ T- cells that survive cytotoxic T lymphocyte (CTL) exposure, suggesting a mechanism of CTL resistance. Inhibition of EZH2 with the FDA-approved drug tazemetostat increased surface expression of major histocompatibility complex class I (MHC-I) on CD4⁺ T-cells, counterbalancing HIV Nef–mediated MHC-I downregulation. This improved CTL-mediated elimination of HIV-infected cells and suppressed viral replication in vitro. In a participant-derived xenograft mouse model, tazemetostat elevated MHC-I and the pro-apoptotic protein BIM in CD4⁺ T-cells, facilitating CD8⁺ T-cell–mediated reductions of HIV reservoir seeding. Additionally, tazemetostat promoted sustained skewing of CD8⁺ T-cells toward less differentiated and exhausted phenotypes. Our findings reveal EZH2 overexpression as a novel mechanism of CTL resistance and support the clinical evaluation of tazemetostat to enhance clearance of HIV reservoirs and improve CD8+ T-cell function.
Human immunodeficiency virus (HIV-1) indefinitely persists, despite effective antiretroviral therapy (ART), within a small pool of latently infected cells. These cells often display markers of immunologic memory and harbor both replication-competent and -incompetent proviruses at approximately a 1:100 ratio. Although complete HIV eradication is a highly desirable goal, this likely represents a bridge too far for our current and foreseeable technologies. A more tractable goal involves engineering a sustained viral remission in the absence of ART––a “functional cure.” In this setting, HIV remains detectable during remission, but the size of the reservoir is small and the residual virus is effectively controlled by an engineered immune response or other intervention. Biological precedence for such an approach is found in the post-treatment controllers (PTCs), a rare group of HIV-infected individuals who, following ART withdrawal, do not experience viral rebound. PTCs are characterized by a small reservoir, greatly reduced inflammation, and the presence of a poorly understood immune response that limits viral rebound. Our goal is to devise a safe and effective means for replicating durable post-treatment control on a global scale. This requires devising methods to reduce the size of the reservoir and to control replication of this residual virus. In the following sections, we will review many of the approaches and tools that likely will be important for implementing such a “reduce and control” strategy and for achieving a PTC-like sustained HIV remission in the absence of ART.
An ability to activate latent HIV-1 expression could benefit many HIV cure strategies, but the first generation of latency reversing agents (LRAs) has proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i's), SB-216763 and tideglusib (the latter already in phase II clinical trials) that activate AKT/mTOR signaling were tested. These GSK-3i's reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered
