HIV-1 Reactivation Research Articles

Reanalysis of Gene Expression Profiles of CD4+ T Cells Treated with HIV-1 Latency Reversal Agents.

The human immunodeficiency virus (HIV-1) causes a progressive depletion of CD4+ T cells, hampering immune function. Current experimental strategies to fight the virus focus on the reactivation of latent HIV-1 in the viral reservoir to make the virus detectable by the immune system, by searching for latency reversal agents (LRAs). We hypothesize that if common molecular pathways elicited by the presence of LRAs are known, perhaps new, more efficient, “shock-and-kill” strategies can be found. Thus, the objective of the present study is to re-evaluate RNA-Seq assays to find differentially expressed genes (DEGs) during latency reversal via transcriptome analysis. We selected six studies (45 samples altogether: 16 negative controls and 29 LRA-treated CD4+ T cells) and 11 LRA strategies through a systematic search in Gene Expression Omnibus (GEO) and PubMed databases. The raw reads were trimmed, counted, and normalized. Next, we detected consistent DEGs in these independent experiments. AZD5582, romidepsin, and suberanilohydroxamic acid (SAHA) were the LRAs that modulated most genes. We detected 948 DEGs shared by those three LRAs. Gene ontology analysis and cross-referencing with other sources of the literature showed enrichment of cell activation, differentiation and signaling, especially mitogen-activated protein kinase (MAPK) and Rho-GTPases pathways.

PEBP1 suppresses HIV transcription and induces latency by inactivating MAPK/NF-κB signaling.

The latent HIV‐1 reservoir is a major barrier to viral eradication. However, our understanding of how HIV‐1 establishes latency is incomplete. Here, by performing a genome‐wide CRISPR‐Cas9 knockout library screen, we identify phosphatidylethanolamine‐binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein (RKIP), as a novel gene inducing HIV latency. Depletion of PEBP1 leads to the reactivation of HIV‐1 in multiple models of latency. Mechanistically, PEBP1 de‐phosphorylates Raf1/ERK/IκB and IKK/IκB signaling pathways to sequestrate NF‐κB in the cytoplasm, which transcriptionally inactivates HIV‐1 to induce latency. Importantly, the induction of PEBP1 expression by the green tea compound epigallocatechin‐3‐gallate (EGCG) prevents latency reversal by inhibiting nuclear translocation of NF‐κB, thereby suppressing HIV‐1 transcription in primary CD4+ T cells isolated from patients receiving antiretroviral therapy (ART). These results suggest a critical role for PEBP1 in the regulation of upstream NF‐κB signaling pathways governing HIV transcription. Targeting of this pathway could be an option to control HIV reservoirs in patients in the future.

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.

Filgotinib suppresses HIV-1-driven gene transcription by inhibiting HIV-1 splicing and T cell activation.

Despite effective antiretroviral therapy, HIV-1-infected cells continue to produce viral antigens and induce chronic immune exhaustion. We propose to identify HIV-1-suppressing agents that can inhibit HIV-1 reactivation and reduce HIV-1-induced immune activation. Using a newly developed dual-reporter system and a high-throughput drug screen, we identified FDA-approved drugs that can suppress HIV-1 reactivation in both cell line models and CD4+ T cells from virally suppressed HIV-1-infected individuals. We identified 11 cellular pathways required for HIV-1 reactivation as druggable targets. Using differential expression analysis, gene set enrichment analysis, and exon-intron landscape analysis, we examined the impact of drug treatment on the cellular environment at a genome-wide level. We identified what we believe to be a new function of a JAK inhibitor, filgotinib, that suppresses HIV-1 splicing. First, filgotinib preferentially suppresses spliced HIV-1 RNA transcription. Second, filgotinib suppresses HIV-1-driven aberrant cancer-related gene expression at the integration site. Third, we found that filgotinib suppresses HIV-1 transcription by inhibiting T cell activation and by modulating RNA splicing. Finally, we found that filgotinib treatment reduces the proliferation of HIV-1-infected cells. Overall, the combination of a drug screen and transcriptome analysis provides systematic understanding of cellular targets required for HIV-1 reactivation and drug candidates that may reduce HIV-1-related immune activation.

NF-κB-Interacting Long Noncoding RNA Regulates HIV-1 Replication and Latency by Repressing NF-κB Signaling.

NF-κB-interacting long noncoding RNA (NKILA) was recently identified as a negative regulator of NF-κB signaling and plays an important role in the development of various cancers. It is well known that NF-κB-mediated activation of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-driven gene expression is required for HIV-1 transcription and reactivation of latency. However, whether NKILA plays essential roles in HIV-1 replication and latency is unclear. Here, by ectopic expression and silencing experiments, we demonstrate that NKILA potently inhibits HIV-1 replication in an NF-κB-dependent manner by suppressing HIV-1 LTR promoter activity. Moreover, NKILA showed broad-spectrum inhibition on the replication of HIV-1 clones with different coreceptor tropisms as well as on LTR activity of various HIV-1 clinical subtypes. Chromatin immunoprecipitation (ChIP) assays revealed that NKILA expression abolishes the recruitment of p65 to the duplicated κB binding sites in the HIV-1 LTR. NKILA mutants disrupting NF-κB inhibition also lost the ability to inhibit HIV-1 replication. Notably, HIV-1 infection or reactivation significantly downregulated NKILA expression in T cells in order to facilitate viral replication. Downregulated NKILA was mainly due to reduced acetylation of histone K27 on the promoter of NKILA by HIV-1 infection, which blocks NKILA expression. Knockdown of NKILA promoted the reactivation of latent HIV-1 upon phorbol myristate acetate (PMA) stimulation, while ectopic NKILA suppressed the reactivation in a well-established clinical model of withdrawal of azidothymidine (AZT) in vitro These findings improve our understanding of the functional suppression of HIV-1 replication and latency by NKILA through NF-κB signaling.IMPORTANCE The NF-κB pathway plays key roles in HIV-1 replication and reactivation of HIV-1 latency. A regulator inhibiting NF-κB activation may be a promising therapeutic strategy against HIV-1. Recently, NF-κB-interacting long noncoding RNA (NKILA) was identified to suppress the development of different human cancers by inhibiting IκB kinase (IKK)-induced IκB phosphorylation and NF-κB pathway activation, whereas the relationship between NKILA and HIV-1 replication is still unknown. Here, our results show that NKILA inhibits HIV-1 replication and reactivation by suppressing HIV-1 long terminal repeat (LTR)-driven transcription initiation. Moreover, NKILA inhibited the replication of HIV-1 clones with different coreceptor tropisms. This project may reveal a target for the development of novel anti-HIV drugs.

Inhibition of Polo-like kinase 1 (PLK1) facilitates the elimination of HIV-1 viral reservoirs in CD4+ T cells ex vivo.

Although combination antiretroviral therapy is effective in controlling HIV-1 infection, latent HIV-1 proviruses cannot be eliminated. HIV-1 reactivation induced by the mere use of latency-reversing agents is insufficient to render death of reservoir cells, indicating that certain intrinsic survival mechanisms exist. We report that Polo-like kinase 1 (PLK1) plays a critical role in survival of CD4+ T cells that undergo HIV-1 reactivation from latency or de novo infection. PLK1 is elevated in both scenarios, which requires HIV-1 Nef. HIV-1 enhances PLK1 SUMOylation, causing its nuclear translocation and protein stabilization. Inhibition or knockdown of PLK1 markedly facilitates death of HIV-1-infected CD4+ T cells. Furthermore, PLK1 inhibitors strikingly reduce the size of HIV-1 latent reservoirs in primary CD4+ T cells. Our findings demonstrate that HIV-1 infection hijacks PLK1 to prevent cell death induced by viral cytopathic effects, and that PLK1 is a promising target for chemical "killing" of HIV-1 reservoir cells.

A targeted reactivation of latent HIV-1 using an activator vector in patient samples from acute infection.

BackgroundDuring combined anti-retroviral treatment, a latent HIV reservoir persists within resting memory CD4 T cells that initiates viral recrudescence upon treatment interruption. Strategies for HIV-1 cure have largely focused on latency reversing agents (LRAs) capable of reactivating and eliminating this viral reservoir. Previously investigated LRAs have largely failed to achieve a robust latency reversal sufficient for reduction of latent HIV pool or the potential of virus-free remission in the absence of treatment.MethodsWe utilize a polyvalent virus-like particle (VLP) formulation called Activator Vector (ACT-VEC) to ‘shock’ provirus into transcriptional activity. Ex vivo co-culture experiments were used to evaluate the efficacy of ACT-VEC in relation to other LRAs in individuals diagnosed and treated during the acute stage of infection. IFN-γ ELISpot, qRT-PCR and Illumina MiSeq were used to evaluate antigenicity, latency reversal, and diversity of induced virus respectively.FindingsUsing samples from HIV+ patients diagnosed and treated at acute/early infection, we demonstrate that ACT-VEC can reverse latency in HIV infected CD4 T cells to a greater extent than other major recall antigens as stimuli or even mitogens such as PMA/Iono. Furthermore, ACT-VEC activates more latent HIV-1 than clinically tested HDAC inhibitors or protein kinase C agonists.InterpretationTaken together, these results show that ACT-VEC can induce HIV reactivation from latently infected CD4 T cells collected from participants on first line combined antiretroviral therapy for at least two years after being diagnosed and treated at acute/early stage of infection. These findings could provide guidance to possible targeted cure strategies and treatments.FundingNIH and CIHR

In Situ Detection of Endogenous HIV Activation by Dynamic Nuclear Polarization NMR and Flow Cytometry.

We demonstrate for the first time in-cell dynamic nuclear polarization (DNP) in conjunction with flow cytometry sorting to address the cellular heterogeneity of in-cell samples. Utilizing a green fluorescent protein (GFP) reporter of HIV reactivation, we correlate increased 15N resonance intensity with cytokine-driven HIV reactivation in a human cell line model of HIV latency. As few as 10% GFP+ cells could be detected by DNP nuclear magnetic resonance (NMR). The inclusion of flow cytometric sorting of GFP+ cells prior to analysis by DNP-NMR further boosted signal detection through increased cellular homogeneity with respect to GFP expression. As few as 3.6 million 15N-labeled GFP+ cells could be readily detected with DNP-NMR. Importantly, cell sorting allowed for the comparison of cytokine-treated GFP+ and GFP− cells in a batch-consistent way. This provides an avenue for normalizing NMR spectral contributions from background cellular processes following treatment with cellular modulators. We also demonstrate the remarkable stability of AMUPol (a nitroxide biradical) in Jurkat T cells and achieved in-cell enhancements of 46 with 10 mM AMUPol, providing an excellent model system for further in-cell DNP-NMR studies. This represents an important contribution to improving in-cell methods for the study of endogenously expressed proteins by DNP-NMR.

Extracellular Vesicle Activation of Latent HIV-1 Is Driven by EV-Associated c-Src and Cellular SRC-1 via the PI3K/AKT/mTOR Pathway.

HIV-1 is a global health crisis that has infected more than 37 million people. Latent reservoirs throughout the body are a major hurdle when it comes to eradicating the virus. In our previous study, we found that exosomes, a type of extracellular vesicle (EV), from uninfected cells activate the transcription of HIV-1 in latent infected cells, regardless of combination antiretroviral therapy (cART). In this study, we investigated the specific mechanism behind the EV activation of latent HIV-1. We found that phosphorylated c-Src is present in EVs of various cell lines and has the ability to activate downstream proteins such as EGFR, initiating a signal cascade. EGFR is then able to activate the PI3K/AKT/mTOR pathway, resulting in the activation of STAT3 and SRC-1, culminating in the reversal of HIV-1 latency. This was verified by examining levels of HIV-1 TAR, genomic RNA and HIV-1 Gag p24 protein in cell lines and primary cells. We found that EVs containing c-Src rescued HIV-1 despite the presence of inhibitors, validating the importance of EV-associated c-Src in latent HIV-1 activation. Lastly, we discovered an increased recruitment of p300 and NF-κB in the nucleus of EV-treated infected cells. Collectively, our data suggest that EV-associated c-Src is able to activate latent HIV-1 via the PI3K/AKT/mTOR pathway and SRC-1/p300-driven chromatin remodeling. These findings could aid in designing new strategies to prevent the reactivation of latent HIV-1 in patients under cART.

Cellular Gene Modulation of HIV-Infected CD4 T Cells in Response to Serial Treatment with the Histone Deacetylase Inhibitor Vorinostat.

Histone deacetylase inhibitors (HDACi) are the most widely studied HIV latency-reversing agents (LRAs). The HDACi suberoylanilide hydroxamic acid (vorinostat [VOR]) has been employed in several clinical HIV latency reversal studies, as well as in vitro models of HIV latency, and has been shown to effectively induce HIV RNA and protein expression. Despite these findings, response to HDACi can vary, particularly with intermittent dosing, and information is lacking on the relationship between the host transcriptional response and HIV latency reversal. Here, we report on global gene expression responses to VOR and examine the longevity of the transcriptional response in various cellular models. We found that many genes are modulated at 6 h post-VOR treatment in HCT116, Jurkat, and primary resting CD4 T cells, yet return to baseline levels after an 18-h VOR-free period. With repeat exposure to VOR in resting CD4 T cells, we found similar and consistent transcriptional changes at 6 h following each serial treatment. In addition, serial exposure in HIV-infected suppressed donor CD4 T cells showed consistent transcriptional changes after each exposure to VOR. We identified five host genes that were strongly and consistently modulated following histone deacetylase (HDAC) inhibition; three (H1F0, IRGM, and WIPI49) were upregulated, and two (PHF15 and PRDM10) were downregulated. These genes demonstrated consistent modulation in peripheral blood mononuclear cell (PBMC) samples from HIV-positive (HIV+) participants who received either single or multiple doses of 400 mg of VOR. Interestingly, the host transcriptional response did not predict induction of cell-associated HIV RNA, suggesting that other cellular factors play key roles in HIV latency reversal in vivo despite robust HDACi pharmacological activity.IMPORTANCE Histone deacetylase inhibitors are widely studied HIV latency-reversing agents (LRAs). VOR, an HDACi, induces histone acetylation and chromatin remodeling and modulates host and HIV gene expression. However, the relationship between these events is poorly defined, and clinical studies suggest diminished HIV reactivation in resting CD4 T cells with daily exposure to VOR. Our study provides evidence that VOR induces a consistent level of host cell gene transcription following intermittent exposure. In addition, in response to VOR exposure a gene signature that was conserved across single and serial exposures both in vitro and in vivo was identified, indicating that VOR can consistently and reproducibly modulate transcriptional host responses. However, as the HIV response to HDACi declines over time, other factors modulate viral reactivation in vivo despite robust HDAC activity. The identified host gene VOR biomarkers can be used for monitoring the pharmacodynamic activity of HDAC inhibitors.

FOXO1 promotes HIV latency by suppressing ER stress in T cells.

Quiescence is a hallmark of CD4+ T cells latently infected with HIV-1. While reversing this quiescence is an effective approach to reactivate latent HIV from T cells in culture, it can cause deleterious cytokine dysregulation in patients. As a key regulator of T-cell quiescence, FOXO1 promotes latency and suppresses productive HIV infection. We report that in resting T cells, FOXO1 inhibition impaired autophagy and induced ER stress, thereby activating two associated transcription factors: activating transcription factor 4 (ATF4) and nuclear factor of activated T cells (NFAT). Both factors associate with HIV chromatin and were for HIV reactivation. Indeed, inhibition of PKR-like endoplasmic reticulum kinase (PERK), an ER stress sensor that can mediate the induction of ATF4, and calcineurin, a calcium-dependent regulator of NFAT, synergistically suppressed HIV reactivation induced by FOXO1 inhibition. Thus, our studies uncover a link between FOXO1, ER stress, and HIV infection that could be therapeutically exploited to selectively reverse T-cell quiescence and reduce the size of the latent viral reservoir.

Epigenetic Suppression of HIV in Myeloid Cells by the BRD4-Selective Small Molecule Modulator ZL0580.

Brain-resident microglia and myeloid cells (perivascular macrophages) are important HIV reservoirs in vivo, especially in the central nervous system (CNS). Despite antiretroviral therapy (ART), low-level persistent HIV replication in these reservoirs remains detectable, which contributes to neuroinflammation and neurological disorders in HIV-infected patients. New approaches complementary to ART to repress residual HIV replication in CNS reservoirs are needed. Our group has recently identified a BRD4-selective small molecule modulator (ZL0580) that induces the epigenetic suppression of HIV. Here, we examined the effects of this compound on HIV in human myeloid cells. We found that ZL0580 induces potent and durable suppression of both induced and basal HIV transcription in microglial cells (HC69) and monocytic cell lines (U1 and OM10.1). Pretreatment of microglia with ZL0580 renders them more refractory to latent HIV reactivation, indicating an epigenetic reprogramming effect of ZL0580 on HIV long terminal repeat (LTR) in microglia. We also demonstrate that ZL0580 induces repressive effect on HIV in human primary monocyte-derived macrophages (MDMs) by promoting HIV suppression during ART treatment. Mechanistically, ZL0580 inhibits Tat transactivation in microglia by disrupting binding of Tat to CDK9, a process key to HIV transcription elongation. High-resolution micrococcal nuclease mapping showed that ZL0580 induces a repressive chromatin structure at the HIV LTR. Taken together, our data suggest that ZL0580 represents a potential approach that could be used in combination with ART to suppress residual HIV replication and/or latent HIV reactivation in CNS reservoirs, thereby reducing HIV-associated neuroinflammation.IMPORTANCE Brain-resident microglia and perivascular macrophages are important HIV reservoirs in the CNS. Persistent viral replication and latent HIV reactivation in the CNS, even under ART, are believed to occur, causing neuroinflammation and neurological disorders in HIV-infected patients. It is critical to identify new approaches that can control residual HIV replication and/or latent HIV reactivation in these reservoirs. We here report that the BRD4-selective small molecule modulator, ZL0580, induces potent and durable suppression of HIV in human microglial and monocytic cell lines. Using an in vitro HIV-infected, ART-treated MDM model, we show that ZL0580 also induces suppressive effect on HIV in human primary macrophages. The significance of our research is that it suggests a potential new approach that has utility in combination with ART to suppress residual HIV replication and/or HIV reactivation in CNS reservoirs, thereby reducing neuroinflammation and neurological disorders in HIV-infected individuals.

HSV-2 infects T follicular helper cells to promote HIV reactivation

Abstract HSV-2 is one of the most significant factors fueling the global HIV epidemic and coinfection is associated with higher HIV plasma viral loads and increased rates of HIV transmission and HSV-2 recurrences. The mechanisms underlying this syndemic likely reflect local and systemic effects. To explore potential systemic effects, we characterized PBMC from a cohort of HIV-1+ women who were or were not HSV-2 seropositive (HSV-2+) and identified phenotypic changes in CD4 T cells including increased frequencies of activated cells and cells expressing co-receptors for HIV entry in HIV+/HSV-2+ vs. HIV+/HSV-2 seronegative (HSV-2−) women. Surprisingly, IL-32, a proinflammatory cytokine, was lower in CD4 T cells from HSV-2+ women. Addition of IL-32γ to CD4 T cells from HIV+ women blocked HIV reactivation following stimulation with phytohemagglutinin, suggesting that HSV-2 induces phenotypic changes in CD4 T cells to promote HIV reactivation. Subsequent RNAseq analysis of CD4 T cells infected ex vivo with HSV-2 also showed lower levels of IL-32, as well as elevated bcl6, itch, and irf4 transcripts along with decreased ccr7, psgl1, and s1pr1, suggesting a transcriptional landscape consistent with T follicular helper (TFH) cells. Flow cytometry studies using CD4 T cells isolated from PBMC from HIV− or HIV+ donors, as well as from tonsils, showed an increased frequency of TFH cells in HSV-2 infected CD4 T cells, suggesting preferential infection of this subpopulation, which is known to harbor latent HIV. HSV-2 infection of CD4 T cells isolated from HIV+ donors or latently infected T cell lines triggered HIV reactivation. These findings demonstrate that HSV-2 directly infects CD4 T cell subpopulations including TFH cells to promote HIV reactivation.

Tri-functional T-cell engagers target immune checkpoint inhibitors PD-1 and TIGIT to enhance CD8 effector T-cell functions in chronically SHIV-infected rhesus macaques

Abstract Checkpoint inhibitors, such as PD-1 and TIGIT, are crucial dysregulators of CD8 T cell function during chronic HIV/SIV infections. Importantly, these checkpoint inhibitors are highly expressed on the surface of CD4 T cells that harbor latent HIV. We have previously demonstrated that an anti-HIV/anti-CD3 bispecific T-cell engager (BiTE) can be used to redirect follicular CD8 (fCD8) T cells to kill HIV infected cells in vitro. Here, we hypothesize that adding an extra specificity to target PD-1 or TIGIT to the BiTE will further enhance the functional activities of CD8 T cells and simultaneously lower the threshold for reactivation of HIV latently infected cells. By using HIV-infected cell lines and primary CD4 T cells isolated from chronically SHIV-infected lymph nodes (LN) as target cells in in vitro and ex vivo killing assays, we found that trifunctional anti-HIV/anti-CD3-anti-TIGIT or anti-HIV/anti-CD3-anti-PD1 increased (1) T-cell specific activation, (2) antigen-specific CD107a degranulation, levels of granzyme B, cytokine/chemokine release by CD8 T cells, and (3) the killing capability of functionally compromised CD8 T cells when compared to the BiTE. When administered at 100 mcg/kg subcutaneously to chronically SHIV-infected animals, we demonstrated that anti-HIV/anti-CD3-anti-TIGIT could safely be used to augment activation and expansion of CD8 effector T cells that further resulted in a better elimination of the SHIV-infected cells as shown by the reduction in cell-associated SHIV gag DNA and RNA. Together, our results indicate that trifunctional T cell engagers targeting checkpoint inhibitors may serve as novel immunotherapeutic strategies to eliminate infected cells in HIV infected individuals.

Maturation State of HIV Infected CD4 T Cells Impacts the Inducible Response of the Latent Viral Reservoir

Abstract A structured microenvironment is necessary for proper immune cell function. While it is rarely possible to use ex vivo experimental models of human tissue (e.g. tonsil), culture of multiple immune cell types together in close contact may benefit cell survival and function. To study HIV latency in primary CD4 T cells, we use an in vitro model, which produces an infected cell hierarchy closely resembling that found in vivo: HIV frequency in effector memory (TEM) > central memory (TCM) > naïve (TN) cells. To enable mechanistic investigations, we developed a method to track CD4 cell phenotype origins following long-term culture and T cell receptor (TCR) stimulation. TN, TCM and TEM subsets were isolated by sorting and stained with live-cell tracking dyes: carboxyfluorescein (CFSE), Yellow and Violet dye, respectively. The tagged subsets were pooled with unstained CD4 cells to help preserve a healthy microenvironment. This mixture was co-cultured with autologous productively infected, TCR-activated cells to facilitate cell-to-cell HIV transmission and establishment of latency. After 3 days, dye-labeled cells were removed by sorting and rested an additional 3 days to complete provirus integration. Latent HIV reactivation by TCR stimulation was measured with HIV RNA expression by PrimeFlow analysis. Residual dye staining provided inference of initial subset origin on a single cell basis. Using this design improved viability and survival for all maturation subsets, compared to isolating subsets prior to stimulation. Results showed that HIV provirus in TN cells was the most responsive to TCR activation. In addition, our study highlights a novel approach that can easily be adapted to other rare cell subsets in biologically relevant settings.

Mechanisms of Endogenous HIV-1 Reactivation by Endocervical Epithelial Cells.

Pharmacological HIV-1 reactivation to reverse latent infection has been extensively studied. However, HIV-1 reactivation also occurs naturally, as evidenced by occasional low-level viremia ("viral blips") during antiretroviral treatment (ART). Clarifying where blips originate from and how they happen could provide clues to stimulate latency reversal more effectively and safely or to prevent viral rebound following ART cessation. We studied HIV-1 reactivation in the female genital tract, a dynamic anatomical target for HIV-1 infection throughout all disease stages. We found that primary endocervical epithelial cells from several women reactivated HIV-1 from latently infected T cells. The endocervical cells' HIV-1 reactivation capacity further increased upon Toll-like receptor 3 stimulation with poly(I·C) double-stranded RNA or infection with herpes simplex virus 2 (HSV-2). Notably, acyclovir did not eliminate HSV-2-induced HIV-1 reactivation. While endocervical epithelial cells secreted large amounts of several cytokines and chemokines, especially tumor necrosis factor alpha (TNF-α), CCL3, CCL4, and CCL20, their HIV-1 reactivation capacity was almost completely blocked by TNF-α neutralization alone. Thus, immunosurveillance activities by columnar epithelial cells in the endocervix can cause endogenous HIV-1 reactivation, which may contribute to viral blips during ART or rebound following ART interruption.IMPORTANCE A reason that there is no universal cure for HIV-1 is that the virus can hide in the genome of infected cells in the form of latent proviral DNA. This hidden provirus is protected from antiviral drugs until it eventually reactivates to produce new virions. It is not well understood where in the body or how this reactivation occurs. We studied HIV-1 reactivation in the female genital tract, which is often the portal of HIV-1 entry and which remains a site of infection throughout the disease. We found that the columnar epithelial cells lining the endocervix, the lower part of the uterus, are particularly effective in reactivating HIV-1 from infected T cells. This activity was enhanced by certain microbial stimuli, including herpes simplex virus 2, and blocked by antibodies against the inflammatory cytokine TNF-α. Avoiding HIV-1 reactivation could be important for maintaining a functional HIV-1 cure when antiviral therapy is stopped.

HIV Reactivation in Latently Infected Cells With Virological Synapse-Like Cell Contact.

HIV reactivation from latency is induced by cytokines but also by cell contact with other cells. To better understand this, J1.1 cells, a latent HIV-1-infected Jurkat derivative, were cocultured with its parental Jurkat. J1.1 cells became p17MA-positive and produced a high level of HIV p24CA antigen, only when they were cocultured with stimulated Jurkat with cell-to-cell contact. In contrast, very little p24CA was produced when they were cocultured without cell contact. Similar results were obtained when latent ACH-2 and its parental A3.01 cells were cocultured. Confocal microscopy revealed that not only HIV-1 p17MA and gp120Env but also LFA-1, CD81, CD59, and TCR CD3 accumulated at the cell contact site, suggesting formation of the virological synapse-like structure. LFA-1–ICAM-1 interaction was involved in the cell-to-cell contact. When J1.1 was cocultured with TCR-deficient Jurkat, the p17MA-positive rate was significantly lower, although the cell-to-cell contact was not impaired. Quantitative proteomics identified 54 membrane molecules, one of which was MHC class I, that accumulated at the cell contact site. Reactivation from latency was also influenced by the presence of stromal cells. Our study indicated that latent HIV-1 in J1.1/ACH-2 cells was efficiently reactivated by cell-to-cell contact with stimulated parental cells, accompanying the virological synapse-like structure.

4-Deoxyphorbol inhibits HIV-1 infection in synergism with antiretroviral drugs and reactivates viral reservoirs through PKC/MEK activation synergizing with vorinostat

Latent HIV reservoirs are the main obstacle to eradicate HIV infection. One strategy proposes to eliminate these viral reservoirs by pharmacologically reactivating the latently infected T cells. We show here that a 4-deoxyphorbol ester derivative isolated from Euphorbia amygdaloides ssp. semiperfoliata, 4β-dPE A, reactivates HIV-1 from latency and could potentially contribute to decrease the viral reservoir. 4β-dPE A shows two effects in the HIV replication cycle, infection inhibition and HIV transactivation, similarly to other phorboids PKC agonists such PMA and prostratin and to other diterpene esters such SJ23B. Our data suggest 4β-dPE A is non-tumorigenic, unlike the related compound PMA. As the compounds are highly similar, the lack of tumorigenicity by 4β-dPE A could be due to the lack of a long side lipophilic chain that is present in PMA. 4β-dPE activates HIV transcription at nanomolar concentrations, lower than the concentration needed by other latency reversing agents (LRAs) such as prostratin and similar to bryostatin. PKCθ/MEK activation is required for the transcriptional activity, and thus, anti-latency activity of 4β-dPE A. However, CD4, CXCR4 and CCR5 receptors down-regulation effect seems to be independent of PCK/MEK, suggesting the existence of at least two different targets for 4β-dPE A. Furthermore, NF-κb transcription factor is involved in 4β-dPE HIV reactivation, as previously shown for other PKCs agonists. We also studied the effects of 4β-dPE A in combination with other LRAs. When 4β-dPE A was combined with another PKC agonists such as prostratin an antagonic effect was achieved, while, when combined with an HDAC inhibitor such as vorinostat, a strong synergistic effect was obtained. Interestingly, the latency reversing effect of the combination was synergistically diminishing the EC50 value but also increasing the efficacy showed by the drugs alone. In addition, combinations of 4β-dPE A with antiretroviral drugs as CCR5 antagonist, NRTIs, NNRTIs and PIs, showed a consistent synergistic effect, suggesting that the combination would not interefer with antiretroviral therapy (ART). Finally, 4β-dPE A induced latent HIV reactivation in CD4 + T cells of infected patients under ART at similar levels than the tumorigenic phorbol derivative PMA, showing a clear reactivation effect. In summary, we describe here the mechanism of action of a new potent deoxyphorbol derivative as a latency reversing agent candidate to decrease the size of HIV reservoirs.

Virtual memory CD8+ T cells restrain the viral reservoir in HIV-1-infected patients with antiretroviral therapy through derepressing KIR-mediated inhibition.

The viral reservoir is the major hurdle in developing and establishing an HIV cure. Understanding factors affecting the size and decay of this reservoir is crucial for the development of therapeutic strategies. Recent work highlighted that CD8+ T cells are involved in the control of viral replication in ART-treated HIV-1-infected individuals, but how CD8+ T cells sense and restrict the HIV reservoir are not fully understood. Here, we demonstrate that a population of unconventional CD45RA+, PanKIR+, and/or NKG2A+ virtual memory CD8+ T cells (TVM cells), which confer rapid and robust protective immunity against pathogens, plays an important role in restraining the HIV DNA reservoir in HIV-1-infected patients with effective ART. In patients undergoing ART, TVM cells negatively correlate with HIV DNA and positively correlate with circulating IFN-α2 and IL-15. Moreover, TVM cells constitutively express high levels of cytotoxic granule components, including granzyme B, perforin and granulysin, and demonstrate the capability to control HIV replication through both cytolytic and noncytolytic mechanisms. Furthermore, by using an ex vivo system, we showed that HIV reactivation is effectively suppressed by TVM cells through KIR-mediated recognition. This study suggests that TVM cells are a promising target to predict posttreatment virological control and to design immune-based interventions to reduce the reservoir size in ART-treated HIV-1-infected individuals.

Mycobacterium tuberculosis Reactivates HIV-1 via Exosome-Mediated Resetting of Cellular Redox Potential and Bioenergetics.

The synergy between Mycobacterium tuberculosis and human immunodeficiency virus-1 (HIV-1) interferes with therapy and facilitates the pathogenesis of both human pathogens. Fundamental mechanisms by which M. tuberculosis exacerbates HIV-1 infection are not clear. Here, we show that exosomes secreted by macrophages infected with M. tuberculosis, including drug-resistant clinical strains, reactivated HIV-1 by inducing oxidative stress. Mechanistically, M. tuberculosis-specific exosomes realigned mitochondrial and nonmitochondrial oxygen consumption rates (OCR) and modulated the expression of host genes mediating oxidative stress response, inflammation, and HIV-1 transactivation. Proteomics analyses revealed the enrichment of several host factors (e.g., HIF-1α, galectins, and Hsp90) known to promote HIV-1 reactivation in M. tuberculosis-specific exosomes. Treatment with a known antioxidant-N-acetyl cysteine (NAC)-or with inhibitors of host factors-galectins and Hsp90-attenuated HIV-1 reactivation by M. tuberculosis-specific exosomes. Our findings uncover new paradigms for understanding the redox and bioenergetics bases of HIV-M. tuberculosis coinfection, which will enable the design of effective therapeutic strategies.IMPORTANCE Globally, individuals coinfected with the AIDS virus (HIV-1) and with M. tuberculosis (causative agent of tuberculosis [TB]) pose major obstacles in the clinical management of both diseases. At the heart of this issue is the apparent synergy between the two human pathogens. On the one hand, mechanisms induced by HIV-1 for reactivation of TB in AIDS patients are well characterized. On the other hand, while clinical findings clearly identified TB as a risk factor for HIV-1 reactivation and associated mortality, basic mechanisms by which M. tuberculosis exacerbates HIV-1 replication and infection remain poorly characterized. The significance of our research is in identifying the role of fundamental mechanisms such as redox and energy metabolism in catalyzing HIV-M. tuberculosis synergy. The quantification of redox and respiratory parameters affected by M. tuberculosis in stimulating HIV-1 will greatly enhance our understanding of HIV-M. tuberculosis coinfection, leading to a wider impact on the biomedical research community and creating new translational opportunities.