HIV-1 Reactivation Research Articles

CD4+ CD151+ T cells may serve as preferential reservoir cells of latent HIV infection events.

Abstract We have demonstrated that the tetraspanin CD151 marks a hyperactivated, pro-inflammatory human CD4+ T cell population. Expansion of this population in HIV patients on ART may explain the associated risk of inflammation-related co-morbidities. However, CD151+ CD4 T cells can also proliferate independent of cognate antigen recognition, driven by just IL-2, a feature that is reminiscent of latently infected T cells which have been reported to maintain the latent reservoir size by homeostatic proliferation mechanisms. The objective of this study was to test our hypothesis that the CD4+CD151+ T cell population is more likely to host latent HIV infection events. First, we noted that all latently HIV-1 infected T cell clones we investigated had upregulated CD151 expression. Using samples from PLWH on ART (n=5) with undetectable viral loads and CD4 count >500 cells/mm3 we enriched CD4+CD151+ and CD4+CD151− T cell populations. Using a nested ALU/GAG qPCR assay to determine the level of proviral HIV-1 integration events, we found a 3.6-fold increase in integration events in the enriched CD4+CD151+ T cell population. Given the demonstrated correlation of proviral DNA frequencies and latent reservoir size, this would suggest that CD151+ T cells disproportionately harbor latent HIV infection, and that CD151+ T cells could be used as a surrogate population to study pertinent features of latently HIV-1 infected T cells. We here begin to address the conundrum of how T cells with a hyperactivated phenotype can host latent HIV-1 infection events and dissect the influence of CD151 expression on latency establishment and reactivation. Characterization of this T cell compartment may reveal new molecular targets to efficiently trigger HIV-1 reactivation. Supported by grants from NIH: NIH R01-AI122842 NIH R33-AI116188 NIH R33-AI133679

Loop-Mediated Isothermal Amplification of Trypanosoma cruzi DNA for Point-of-Care Follow-Up of Anti-Parasitic Treatment of Chagas Disease.

A loop-mediated isothermal amplification assay was evaluated as a surrogate marker of treatment failure in Chagas disease (CD). A convenience series of 18 acute or reactivated CD patients who received anti-parasitic treatment with benznidazole was selected—namely, nine orally infected patients: three people living with HIV and CD reactivation, five chronic CD recipients with reactivation after organ transplantation and one seronegative recipient of a kidney and liver transplant from a CD donor. Fifty-four archival samples (venous blood treated with EDTA or guanidinium hydrochloride-EDTA buffer and cerebrospinal fluid) were extracted using a Spin-column manual kit and tested by T. cruzi Loopamp kit (Tc-LAMP, index test) and standardized real-time PCR (qPCR, comparator test). Of them, 23 samples were also extracted using a novel repurposed 3D printer designed for point-of-care DNA extraction (PrintrLab). The agreement between methods was estimated by Cohen’s kappa index and Bland–Altman plot analysis. The T. cruzi Loopamp kit was as sensitive as qPCR for detecting parasite DNA in samples with parasite loads higher than 0.5 parasite equivalents/mL and infected with different discrete typing units. The agreement between qPCR and Tc-LAMP (Spin-column) or Tc-LAMP (PrintrLab) was excellent, with a mean difference of 0.02 [CI = −0.58–0.62] and −0.04 [CI = −0.45–0.37] and a Cohen’s kappa coefficient of 0.78 [CI = 0.60–0.96] and 0.90 [CI = 0.71 to 1.00], respectively. These findings encourage prospective field studies to validate the use of LAMP as a surrogate marker of treatment failure in CD.

Quantification of transcription noise's impact on cell fate commitment with digital resolutions.

Gene transcription is a random and noisy process. Tremendous efforts in single-cell studies have been mapping transcription noises to phenotypic variabilities between isogenic cells. However, the exact role of the noise in cell fate commitment remains largely descriptive or even controversial. For a specified cell fate, we define the jumping digit I of a critical gene as a statistical threshold that a single cell has approximately an equal chance to commit the fate as to have at least I transcripts of the gene. When the transcription is perturbed by a noise enhancer without changing the basal transcription level E0, we find a crossing digit k such that the noise catalyzes cell fate change when I > k while stabilizes the current state when I < k; k remains stable against enormous variations of kinetic rates. We further test the reactivation of latent HIV in 22 integration sites by noise enhancers paired with transcriptional activators. Strong synergistic actions are observed when the activators increase transcription burst frequency, whereas no synergism, but antagonism, is often observed if activators increase burst size. The synergistic efficiency can be predicted accurately by the ratio I/E0. When the noise enhancers double the noise, the activators double the burst frequency, and I/E0≥7, their combination is 10 times more effective than their additive effects across all 22 sites. The jumping digit I may provide a novel probe to explore the phenotypic consequences of transcription noise in cell functions. Code is freely available at http://cam.gzhu.edu.cn/info/1014/1223.htm. The data underlying this article are available in the article and in its online supplementary material. Supplementary data are available at Bioinformatics online.

Clearance of HIV-1 or SIV reservoirs by promotion of apoptosis and inhibition of autophagy: Targeting intracellular molecules in cure-directed strategies.

The reservoirs of the HIV display cellular properties resembling long-lived immune memory cells that could be exploited for viral clearance. Our interest in developing a cure for HIV stems from the studies of immunologic memory against infections. We and others have found that long-lived immune memory cells employ prosurvival autophagy and antiapoptotic mechanisms to protect their longevity. Here, we describe the rationale for the development of an approach to clear HIV-1 by selective elimination of host cells harboring replication-competent HIV (SECH). While reactivation of HIV-1 in the host cells with latency reversing agents (LRAs) induces viral gene expression leading to cell death, LRAs also simultaneously up-regulate prosurvival antiapoptotic molecules and autophagy. Mechanistically, transcription factors that promote HIV-1 LTR-directed gene expression, such as NF-κB, AP-1, and Hif-1α, can also enhance the expression of cellular genes essential for cell survival and metabolic regulation, including Bcl-xL, Mcl-1, and autophagy genes. In the SECH approach, we inhibit the prosurvival antiapoptotic molecules and autophagy induced by LRAs, thereby allowing maximum killing of host cells by the induced HIV-1 proteins. SECH treatments cleared HIV-1 infections in humanized mice in vivo and in HIV-1 patient PBMCs ex vivo. SECH also cleared infections by the SIV in rhesus macaque PBMCs ex vivo. Research efforts are underway to improve the efficacy and safety of SECH and to facilitate the development of SECH as a therapeutic approach for treating people with HIV.

β-catenin regulates HIV latency and modulates HIV reactivation.

Latency is the main obstacle towards an HIV cure, with cure strategies aiming to either elicit or prevent viral reactivation. While these strategies have shown promise, they have only succeeded in modulating latency in a fraction of the latent HIV reservoir, suggesting that the mechanisms controlling HIV latency are not completely understood, and that comprehensive latency modulation will require targeting of multiple latency maintenance pathways. We show here that the transcriptional co-activator and the central mediator of canonical Wnt signaling, β-catenin, inhibits HIV transcription in CD4+ T cells via TCF-4 LTR binding sites. Further, we show that inhibiting the β-catenin pathway reactivates HIV in a primary TCM cell model of HIV latency, primary cells from cART-controlled HIV donors, and in CD4+ latent cell lines. β-catenin inhibition or activation also enhanced or inhibited the activity of several classes of HIV latency reversing agents, respectively, in these models, with significant synergy of β-catenin and each LRA class tested. In sum, we identify β-catenin as a novel regulator of HIV latency in vitro and ex vivo, adding new therapeutic targets that may be combined for comprehensive HIV latency modulation in HIV cure efforts.

ADAP1 promotes latent HIV-1 reactivation by selectively tuning KRAS\u2013ERK\u2013AP-1 T cell signaling-transcriptional axis

Immune stimulation fuels cell signaling-transcriptional programs inducing biological responses to eliminate virus-infected cells. Yet, retroviruses that integrate into host cell chromatin, such as HIV-1, co-opt these programs to switch between latent and reactivated states; however, the regulatory mechanisms are still unfolding. Here, we implemented a functional screen leveraging HIV-1’s dependence on CD4+ T cell signaling-transcriptional programs and discovered ADAP1 is an undescribed modulator of HIV-1 proviral fate. Specifically, we report ADAP1 (ArfGAP with dual PH domain-containing protein 1), a previously thought neuronal-restricted factor, is an amplifier of select T cell signaling programs. Using complementary biochemical and cellular assays, we demonstrate ADAP1 inducibly interacts with the immune signalosome to directly stimulate KRAS GTPase activity thereby augmenting T cell signaling through targeted activation of the ERK–AP-1 axis. Single cell transcriptomics analysis revealed loss of ADAP1 function blunts gene programs upon T cell stimulation consequently dampening latent HIV-1 reactivation. Our combined experimental approach defines ADAP1 as an unexpected tuner of T cell programs facilitating HIV-1 latency escape.

Gingival tissue as a reservoir for human immunodeficiency virus type 1: Preliminary results of a cross-sectional observational study.

Despite combined antiretroviral therapy (cART), total cure of immunodeficiency virus type 1 (HIV-1) infection remains elusive. Chronic periodontitis (CP) is strongly associated with HIV-1 infection. This condition is characterized by an intense inflammatory infiltrate mainly constituted of immune cells which in turn may be a valuable source of HIV-1 reactivation. This study aimed to determine if gingival tissue could act as a reservoir for HIV-1. Twelve patients with HIV-1 and CP and 12 controls (no HIV-1-infection and no CP) were evaluated in a cross-sectional study. RNA viral load and interleukin (IL) levels were determined in blood plasma and saliva. Histological sections of gingival tissue were stained with fluorescent antibodies against p24 antigen and different cellular biomarkers. In six of the 12 patients, HIV RNA load was detected, despite cART; in three of them, expression of viral RNA was also detected in saliva. The levels of IL-2, IL-6, and IL-12 were higher in blood and saliva of patients with HIVand CP than controls. HIV-1 p24 antigen was detected by immunostaining in gingival biopsies of 10 of the 12 patients but in no controls. Immune markers for T cells and antigen-presenting cells were also identified in most patients and some controls. These preliminary data showing the detection of HIV-1 p24 antigen in the gingival biopsies of a significant part of patients with HIV-1 and CP under cART together with the presence of immune cells, plead for the existence of a HIV-1 reservoir in the gingival tissue of this population.

Pharmacological suppression of glycogen synthase kinase-3 reactivates HIV-1 from latency via activating Wnt/β-catenin/TCF1 axis in CD4+ T cells

ABSTRACT HIV-1 latency posts a major obstacle for HIV-1 eradication. Currently, no desirable latency reversing agents (LRAs) have been implicated in the “Shock and Kill” strategy to mobilize the latently infected cells to be susceptible for clearance by immune responses. Identification of key cellular pathways that modulate HIV-1 latency helps to develop efficient LRAs. In this study, we demonstrate that the Wnt downstream β-catenin/TCF1 pathway is a crucial modulator for HIV-1 latency. The pharmacological activation of the β-catenin/TCF1 pathway with glycogen synthase kinase-3 (GSK3) inhibitors promoted transcription of HIV-1 proviral DNA and reactivated latency in CD4+ T cells; the GSK3 kinase inhibitor 6-bromoindirubin-3′-oxime (6-BIO)-induced HIV-1 reactivation was subsequently confirmed in resting CD4+ T cells from cART-suppressed patients and SIV-infected rhesus macaques. These findings advance our understanding of the mechanisms responsible for viral latency, and provide the potent LRA that can be further used in conjunction of immunotherapies to eradicate viral reservoirs.

EK-16A liposomes enhance HIV replication in ACH2 or J-Lat 10.6 cell engrafted NSG mice.

Background: Numbers of HIV latency reversal agents (LRAs) have been tested in clinical trials, but with limited effect. EK-16A is an ingenol derivative that isolated from Euphorbia kansui. Our prior studies have suggested that it could reactivate latent HIV and meanwhile inhibit HIV infection in vitro. Here, we further advanced the research in vivo.Methods: In vitro, the activity of EK-16A liposomes was measured in HIV latently infected cells. In serum pharmacology test, BALB/c mice were orally administered with EK-16A liposomes, serum was separated and co-cultured with cells, HIV reactivation was measured. In vivo, NSG mice were transplanted with human cells for 3 weeks and then administered with EK-16A liposomes for 3 days. In ACH2 cell engrafted NSG mice, P24 in plasma and cell-associated HIV RNA in tissues was measured. In J-Lat 10.6 cell engrafted NSG mice, GFP expression of J-Lat 10.6 cells in diverse tissues was measured. Hematoxylin and eosin (HE) staining was carried out for histopathological examination in both mice.Results: EK-16A liposomes can reactivate latent HIV in ACH2 and J-Lat 10.6 cells. Serum pharmacological test showed that EK-16A retained activity after oral administration. Importantly, in ACH2 cell engrafted NSG mice, EK-16A liposomes increased the secretion of P24 in plasma and the expression of cell-associated HIV RNA in tissues. In J-Lat 10.6 cell engrafted NSG mice, EK-16A liposomes increased the GFP expression of J-Lat 10.6 cells in diverse tissues, including the bone marrow, spleen, liver, lung and peripheral blood. Furthermore, there was no obvious histopathological change associated with the use of EK-16A liposomes in both mice.Conclusions: Our results confirmed the enhancing HIV replication activity and preliminary security of EK-16A in human cell engrafted NSG mice, laying the foundation for research in clinical trials.

An In Vitro System to Model the Establishment and Reactivation of HIV-1 Latency in Primary Human CD4+ T Cells.

HIV-1 establishes latency primarily by infecting activated CD4+ T cells that later return to quiescence as memory cells. Latency allows HIV-1 to evade immune responses and to persist during antiretroviral therapy, which represents an important problem in clinical practice. Here we describe both the original and a simplified version of HIV-1 latency models that mimics this process using replication competent viruses. Our model allows generation of large numbers of latently infected CD4+ T cell to dissect molecular mechanisms of HIV latency and reactivation.

Examining the Impact of Galectin-9 on Latent HIV Transcription.

The β-galactoside-binding protein Galectin-9 (Gal-9) functions as a double-edged sword during HIV infection. On the one hand, Gal-9 can reactivate HIV latently infected cells, the main barrier to achieving HIV eradication, making them visible to immune clearance. On the other hand, Gal-9 induces latent HIV transcription by activating T cell Receptor (TCR) signaling pathways. These signaling pathways induce undesirable pro-inflammatory responses. While these unwanted responses can be mitigated by rapamycin without impacting Gal-9-mediated latent HIV reactivation, this effect raises the concern that Gal-9 may play a role in the chronic immune activation/inflammation that persists in people living with HIV despite antiretroviral therapy. Together, these data highlight the need to understand the positive and negative impacts of galectin interactions on immunological functions during HIV infection. In this chapter, we describe methods that can be used to investigate the effects of galectins, in particular Gal-9, on latent HIV transcription in vitro and ex vivo.

Fighting HIV-1 Persistence: At the Crossroads of "Shoc-K and B-Lock".

Despite the success of highly active antiretroviral therapy (HAART), integrated HIV-1 proviral DNA cannot be eradicated from an infected individual. HAART is not able to eliminate latently infected cells that remain invisible to the immune system. Viral sanctuaries in specific tissues and immune-privileged sites may cause residual viral replication that contributes to HIV-1 persistence. The “Shock or Kick, and Kill” approach uses latency reversing agents (LRAs) in the presence of HAART, followed by cell-killing due to viral cytopathic effects and immune-mediated clearance. Different LRAs may be required for the in vivo reactivation of HIV-1 in different CD4+ T cell reservoirs, leading to the activation of cellular transcription factors acting on the integrated proviral HIV-1 LTR. An important requirement for LRA drugs is the reactivation of viral transcription and replication without causing a generalized immune activation. Toll-like receptors, RIG-I like receptors, and STING agonists have emerged recently as a new class of LRAs that augment selective apoptosis in reactivated T lymphocytes. The challenge is to extend in vitro observations to HIV-1 positive patients. Further studies are also needed to overcome the mechanisms that protect latently infected cells from reactivation and/or elimination by the immune system. The Block and Lock alternative strategy aims at using latency promoting/inducing agents (LPAs/LIAs) to block the ability of latent proviruses to reactivate transcription in order to achieve a long term lock down of potential residual virus replication. The Shock and Kill and the Block and Lock approaches may not be only alternative to each other, but, if combined together (one after the other), or given all at once [namely “Shoc-K(kill) and B(block)-Lock”], they may represent a better approach to a functional cure.

Histone deacetylase inhibition reduces deleterious cytokine release induced by ingenol stimulation

Latency reversing agents (LRAs), such as protein kinase C (PKC) agonists, constitute a promising strategy for exposing and eliminating the HIV-1 latent reservoir. PKC agonists activate NF-κB and induce deleterious pro-inflammatory cytokine production. Adjuvant pharmacological agents, such as ruxolitinib, a JAK inhibitor, have previously been combined with LRAs to reduce deleterious pro-inflammatory cytokine secretion without inhibiting HIV-1 reactivation in vitro. Histone deacetylase inhibitors (HDACi) are known to dampen pro-inflammatory cytokine secretion in the context of other diseases and synergize with LRAs to reactivate latent HIV-1. This study investigates whether a panel of epigenetic modifiers, including HDACi, could dampen PKC-induced pro-inflammatory cytokine secretion during latency reversal. We screened an epigenetic modifier library for compounds that reduced intracellular IL-6 production induced by the PKC agonist Ingenol-3,20-dibenzoate. We further tested the most promising epigenetic inhibitor class, HDACi, for their ability to reduce pro-inflammatory cytokines and reactivate latent HIV-1 ex vivo. We identified nine epigenetic modulators that reduced PKC-induced intracellular IL-6. In cells from aviremic individuals living with HIV-1, the HDAC1-3 inhibitor, suberohydroxamic acid (SBHA), reduced secretion of pro-inflammatory cytokines TNF-α, IL-5, IL-2r, and IL-17 but did not significantly reactivate latent HIV-1 when combined with Ingenol-3,20-dibenzoate. Combining SBHA and Ingenol-3,20-dibenzoate reduces deleterious cytokine production during latency reversal but does not induce significant viral reactivation in aviremic donor PBMCs. The ability of SBHA to reduce PKC-induced pro-inflammatory cytokines when combined with Ingenol-3,20-dibenzoate suggests SBHA can be used to reduced PKC induced pro-inflammatory cytokines but not to achieve latency reversal in the context of HIV-1.

Design and implementation of a cohort study of persons living with HIV infection who are initiating medication treatment for opioid use disorder to evaluate HIV-1 persistence

BackgroundOpioid use disorder (OUD) negatively impacts the HIV continuum of care for persons living with HIV (PLH). Medication treatment for OUD (MOUD) may have differential biological effects in individuals with HIV and OUD. To understand the role of MOUD – opioid agonist methadone, partial agonist buprenorphine and antagonist naltrexone – in HIV-1 persistence and reactivation, we will use molecular virology approaches to carry out the first prospective, longitudinal studies of adults living with HIV with OUD initiating MOUD. One of the major challenges to studying the impact of MOUD on HIV persistence is the low retention rate of study participants and the requirement of large-volume blood sampling to study the HIV proviral landscape and expression profiles. MethodsA prospective cohort study is underway to study the HIV-1 expression, proviral landscape, and clonal expansion dynamics using limited blood sampling from persons with DSM-5 diagnosed OUD who are living with HIV infection and initiating treatment with methadone, buprenorphine, or extended-release naltrexone. ResultsWe describe the recruitment, laboratory, and statistical methods of this study as well as the protocol details of this on-going study. Out of the 510 screened for enrollment into the study, 35 (7%) were eligible and 27 were enrolled thus far. Retention through month 3 has been high at 95%. ConclusionsThis on-going study is evaluating the impact of MOUD on HIV persistence at the molecular virology level using limited blood sampling via a prospective, longitudinal study of people living with HIV DSM-5 OUD initiating treatment with MOUD.

Inhibition of the H3K27 demethylase UTX enhances the epigenetic silencing of HIV proviruses and induces HIV-1 DNA hypermethylation but fails to permanently block HIV reactivation.

One strategy for a functional cure of HIV-1 is "block and lock", which seeks to permanently suppress the rebound of quiescent HIV-1 by epigenetic silencing. For the bivalent promoter in the HIV LTR, both histone 3 lysine 27 tri-methylation (H3K27me3) and DNA methylation are associated with viral suppression, while H3K4 tri-methylation (H3K4me3) is correlated with viral expression. However, H3K27me3 is readily reversed upon activation of T-cells through the T-cell receptor. In an attempt to suppress latent HIV-1 in a stable fashion, we knocked down the expression or inhibited the activity of UTX/KDM6A, the major H3K27 demethylase, and investigated its impact on latent HIV-1 reactivation in T cells. Inhibition of UTX dramatically enhanced H3K27me3 levels at the HIV LTR and was associated with increased DNA methylation. In latently infected cells from patients, GSK-J4, which is a potent dual inhibitor of the H3K27me3/me2-demethylases JMJD3/KDM6B and UTX/KDM6A, effectively suppressed the reactivation of latent HIV-1 and also induced DNA methylation at specific sites in the 5'LTR of latent HIV-1 by the enhanced recruitment of DNMT3A to HIV-1. Nonetheless, suppression of HIV-1 through epigenetic silencing required the continued treatment with GSK-J4 and was rapidly reversed after removal of the drug. DNA methylation was also rapidly lost after removal of drug, suggesting active and rapid DNA-demethylation of the HIV LTR. Thus, induction of epigenetic silencing by histone and DNA methylation appears to be insufficient to permanently silence HIV-1 proviral transcription.

MAT2A-Mediated S-Adenosylmethionine Level in CD4+ T Cells Regulates HIV-1 Latent Infection.

Antiretroviral drugs effectively halt HIV-1 replication and disease progression, however, due to the presence of a stable viral latent reservoir, the infection cannot be cured by antiretroviral drugs alone. Elucidating the molecular mechanisms underlying HIV-1 latent infection remains a critical hurdle that precludes the development of novel therapeutic strategies aiming for a potential functional cure. Cellular metabolism has been reported to affect HIV-1 replication in CD4+ T cells, but it remains largely unclear whether it is involved in the regulation of HIV-1 latency. Here, we performed a sub-pooled CRISPR library knockout screen targeting 1773 metabolic-related genes in a cell model of HIV-1 latent infection and found that Methionine Adenosyltransferase 2A (MAT2A) contributes to HIV-1 latency. MAT2A knockout enhanced the reactivation of latent HIV-1 while MAT2A overexpression did the opposite. Mechanistically, MAT2A modulates HIV-1 latency through S-Adenosylmethionine (SAM)-mediated one-carbon flux. MAT2A knockout resulted in a significant downregulation of DNA and histone methylation at the HIV-1 5’-LTR. Importantly, we found that the plasma level of SAM is positively correlated with HIV-1 DNA in PBMCs from ART-treated infected individuals, suggesting SAM could serve as a potential biomarker for the latent viral reservoir. Overall, this study reveals an important role of MAT2A-mediated one-carbon metabolism in regulating HIV-1 latency and provides a promising target for the development of new strategies for a functional cure of HIV-1.

Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways.

The switch between HIV latency and productive transcription is regulated by an auto-feedback mechanism initiated by the viral trans-activator Tat, which functions to recruit the host transcription elongation factor P-TEFb to proviral HIV. A heterodimeric complex of CDK9 and one of three cyclin T subunits, P-TEFb is expressed at vanishingly low levels in resting memory CD4+ T cells and cellular mechanisms controlling its availability are central to regulation of the emergence of HIV from latency. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4+ T cells, we characterized specific T-cell receptor (TCR) signaling pathways that regulate the generation of transcriptionally active P-TEFb, defined as the coordinate expression of cyclin T1 and phospho-Ser175 CDK9. Protein kinase C (PKC) agonists, such as ingenol and prostratin, stimulated active P-TEFb expression and reactivated latent HIV with minimal cytotoxicity, even in the absence of intracellular calcium mobilization with an ionophore. Unexpectedly, inhibition-based experiments demonstrated that PKC agonists and TCR-mobilized diacylglycerol signal through MAP kinases ERK1/2 rather than through PKC to effect the reactivation of both P-TEFb and latent HIV. Single-cell and bulk RNA-seq analyses revealed that of the four known isoforms of the Ras guanine nucleotide exchange factor RasGRP, RasGRP1 is by far the predominantly expressed diacylglycerol-dependent isoform in CD4+ T cells. RasGRP1 should therefore mediate the activation of ERK1/2 via Ras-Raf signaling upon TCR co-stimulation or PKC agonist challenge. Combined inhibition of the PI3K-mTORC2-AKT-mTORC1 pathway and the ERK1/2 activator MEK prior to TCR co-stimulation abrogated active P-TEFb expression and substantially suppressed latent HIV reactivation. Therefore, contrary to prevailing models, the coordinate reactivation of P-TEFb and latent HIV in primary T cells following either TCR co-stimulation or PKC agonist challenge is independent of PKC but rather involves two complementary signaling arms of the TCR cascade, namely, RasGRP1-Ras-Raf-MEK-ERK1/2 and PI3K-mTORC2-AKT-mTORC1.

The Effect of JAK1/2 Inhibitors on HIV Reservoir Using Primary Lymphoid Cell Model of HIV Latency.

HIV eradication is hindered by the existence of latent HIV reservoirs in CD4+ T cells. Therapeutic strategies targeting latent cells are required to achieve a functional cure, however the study of latently infected cells from HIV infected persons is extremely challenging due to the lack of biomarkers that uniquely characterize them. In this study, the dual reporter virus HIVGKO was used to investigate latency establishment and maintenance in lymphoid-derived CD4+ T cells. Single cell technologies to evaluate protein expression, host gene expression, and HIV transcript expression were integrated to identify and analyze latently infected cells. FDA-approved, JAK1/2 inhibitors were tested in this system as a potential therapeutic strategy to target the latent reservoir. Latent and productively infected tonsillar CD4+ T cells displayed similar activation profiles as measured by expression of CD69, CD25, and HLADR, however latent cells showed higher CXCR5 expression 3 days post-infection. Single cell analysis revealed a small set of genes, including HIST1-related genes and the inflammatory cytokine, IL32, that were upregulated in latent compared to uninfected and productively infected cells suggesting a role for these molecular pathways in persistent HIV infection. In vitro treatment of HIV-infected CD4+ T cells with physiological concentrations of JAK1/2 inhibitors, ruxolitinib and baricitinib, used in clinical settings to target inflammation, reduced latent and productive infection events when added 24 hr after infection and blocked HIV reactivation from latent cells. Our methods using an established model of HIV latency and lymphoid-derived cells shed light on the biology of latency in a crucial anatomical site for HIV persistence and provides key insights about repurposing baricitinib or ruxolitinib to target the HIV reservoir.

Effect of Xanthium Strumarium on HIV-1 5'-LTR Transcriptional Activity and Viral Reactivation in Latently Infected Cells.

Chinese herbal medicines (CHMs) are widely used in Asian countries. They show multiple pharmacological activities, including antiviral activities. The 5′-long terminal repeat (LTR) region of HIV-1, required for viral transcription, is a potential drug target for HIV-1 reactivation and intrinsic cell death induction of infected or latently infected cells. Modulation of HIV-1 reactivation requires interactions between host cell proteins and viral 5′-LTR elements. By evaluation of two CHMs- Xanthium strumarium and Pueraria montana, we found that 1) X. strumarium reactivated HIV-1 latently infected cells in J-Lat 8.4, J-Lat 9.2, U1, and ACH-2 cells in vitro; 2) 27 nuclear regulatory proteins were associated with HIV-1 5′-LTR using deoxyribonucleic acid affinity pull-down and LC-MS/MS analyses; and 3) among them, silencing of XRCC6 reactivated HIV-1 5′-LTR transcriptional activity. We found that X. strumarium inhibits the 5′-LTR associated XRCC6 nuclear regulatory proteins, increases its viral 5′-LTR promoter transcriptional activity, and reactivates HIV-1 latently infected cells in vitro. These findings may contribute to understanding the 5′-LTR activity and the host cell nuclear regulatory protein machinery for reactivating HIV-1 and for future investigations to eradicate and cure HIV-1 infection.

Monitoring reactivation of latent HIV by label-free gradient light interference microscopy

SummaryHuman immunodeficiency virus (HIV) can infect cells and take a quiescent and nonexpressive state called latency. In this study, we report insights provided by label-free, gradient light interference microscopy (GLIM) about the changes in dry mass, diameter, and dry mass density associated with infected cells that occur upon reactivation. We discovered that the mean cell dry mass and mean diameter of latently infected cells treated with reactivating drug, TNF-α, are higher for latent cells that reactivate than those of the cells that did not reactivate. Cells with mean dry mass and diameter less than approximately 10 pg and 8 μm, respectively, remain exclusively in the latent state. Also, cells with mean dry mass greater than approximately 28-30 pg and mean diameter greater than 11–12 μm have a higher probability of reactivating. This study is significant as it presents a new label-free approach to quantify latent reactivation of a virus in single cells.