Targeting noncoding RNAs to reactivate or eliminate latent HIV reservoirs.

PURPOSE OF REVIEW: Expression of noncoding RNAs (ncRNAs) is more tissue and cell type-specific than expression of protein-coding genes. Understanding the mechanisms of action of ncRNAs and their roles in HIV replication and latency may inform targets for the latent HIV reservoir reactivation or elimination with high specificity to CD4 + T cells latently infected with HIV. RECENT FINDINGS: While the number of studies in the field of ncRNAs and HIV is limited, evidence points to complex interactions between different ncRNAs, protein-coding RNAs, and proteins. Latency-reversing agents modulate the expression of ncRNAs, with some effects being inhibitory for HIV reactivation. An important limitation of basic research on the ncRNA mechanisms of action is the reliance on cell lines. Because of cell type specificity, it is uncertain whether the ncRNAs function similarly in primary cells. SUMMARY: Comprehensive functional screens to uncover all ncRNAs that regulate HIV expression and the detailed exploration of their mechanisms of action in relevant cell types are needed to identify promising targets for HIV reservoir clearance. Classes of ncRNAs as a whole rather than individual ncRNAs might represent an attractive target for reservoir elimination. Compound screens for latency reversal should factor in the complexity of their effects on ncRNAs.

Single-cell RNA sequencing reveals common and unique gene expression profiles in primary CD4+ T cells latently infected with HIV under different conditions.

Background: The latent HIV reservoir represents the major barrier to a cure. One curative strategy is targeting diseased cells for elimination based on biomarkers that uniquely define these cells. Single-cell RNA sequencing (scRNA-seq) has enabled the identification of gene expression profiles associated with disease at the single-cell level. Because HIV provirus in many cells during latency is not entirely silent, it became possible to determine gene expression patterns in a subset of cells latently infected with HIV. Objective: The primary objective of this study was the identification of the gene expression profiles of single latently infected CD4+ T cells using scRNA-seq. Different conditions of latency establishment were considered. The identified profiles were then explored to prioritize the identified genes for future experimental validation. Methods: To facilitate gene prioritization, three approaches were used. First, we characterized and compared the gene expression profiles of HIV latency established in different environments: in cells that encountered an activation stimulus and then returned to quiescence, and in resting cells that were infected directly via cell-to-cell viral transmission from autologous activated, productively infected cells. Second, we characterized and compared the gene expression profiles of HIV latency established with viruses of different tropisms, using an isogenic pair of CXCR4- and CCR5-tropic viruses. Lastly, we used proviral expression patterns in cells from people with HIV to more accurately define the latently infected cells in vitro. Results: Our analyses demonstrated that a subset of genes is expressed differentially between latently infected and uninfected cells consistently under most conditions tested, including cells from people with HIV. Our second important observation was the presence of latency signatures, associated with variable conditions when latency was established, including cellular exposure and responsiveness to a T cell receptor stimulus and the tropism of the infecting virus. Conclusion: Common signatures, specifically genes that encode proteins localized to the cell surface, should be prioritized for further testing at the protein level as biomarkers for the ability to enrich or target latently infected cells. Cell- and tropism-dependent biomarkers may need to be considered in developing targeting strategies to ensure that all the different reservoir subsets are eliminated.

Beyond the Syndemic of Opioid Use Disorders and HIV: The Impact of Opioids on Viral Reservoirs.

People with HIV are more likely to have opioid use disorder and to be prescribed opioids for chronic pain than the general population; however, the effects of opioids on the immune system and HIV persistence have not been fully elucidated. Opioids may affect HIV reservoirs during their establishment, maintenance, and reactivation by enhancing HIV infectivity and replication due to upregulation of co-receptors and impairment of innate antiviral responses. Opioids may also modulate immune cell functioning and microbial translocation and can reverse viral latency. In this review, we summarize the current findings for and against the modulating effects of opioids on HIV cellular and anatomical reservoirs, highlighting the current limitations that affect in vitro, ex vivo, and in vivo studies in the field. We propose further research targets and potential strategies to approach this topic.

HIV Infection Elicits Differential Transcriptomic Remodeling in CD4+ T Cells with Variable Proliferative Responses to the T Cell Receptor Stimulus.

Identification of a cellular biomarker of latent HIV infection will facilitate the latent reservoir detection, quantification, and targeting for elimination. Unfortunately, the latency biomarkers reported in the literature define only a fraction of the entire reservoir. The latent HIV reservoir may be established in dividing cells that subsequently return to quiescence and in resting cells. The strength of the T cell receptor (TCR) signaling at the time of infection affects characteristics of the established reservoir, such as the ability to reactivate with latency reversing agents. To better understand the cellular environments before latency establishment, we characterized transcriptomic remodeling induced by the initial HIV infection in cells with differential proliferative responses to the TCR stimulus. Cell proliferation was monitored using the viable dye carboxyfluorescein diacetate succinimidyl ester. Cells that divided many times, a few times, or remained non-dividing were subjected to single-cell RNA sequencing. A subset of identified transcriptional changes induced by HIV infection was independent of the number of cell divisions; however, responses unique to different cell subsets were also detected. Some of these early gene expression changes were consistent with reported markers of latently infected cells. We pose that the latency biomarkers may depend on the cellular proliferative state at the time of infection.

An Evaluation on the Role of Non-Coding RNA in HIV Transcription and Latency: A Review.

The existence of latent cellular reservoirs is recognized as the major barrier to an HIV cure. Reactivating and eliminating "shock and kill" or permanently silencing "block and lock" the latent HIV reservoir, as well as gene editing, remain promising approaches, but so far have proven to be only partially successful. Moreover, using latency reversing agents or "block and lock" drugs pose additional considerations, including the ability to cause cellular toxicity, a potential lack of specificity for HIV, or low potency when each agent is used alone. RNA molecules, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are becoming increasingly recognized as important regulators of gene expression. RNA-based approaches for combatting HIV latency represent a promising strategy since both miRNAs and lncRNAs are more cell-type and tissue specific than protein coding genes. Thus, a higher specificity of targeting the latent HIV reservoir with less overall cellular toxicity can likely be achieved. In this review, we summarize current knowledge about HIV gene expression regulation by miRNAs and lncRNAs encoded in the human genome, as well as regulatory molecules encoded in the HIV genome. We discuss both the transcriptional and post-transcriptional regulation of HIV gene expression to align with the current definition of latency, and describe RNA molecules that either promote HIV latency or have anti-latency properties. Finally, we provide perspectives on using each class of RNAs as potential targets for combatting HIV latency, and describe the complexity of the interactions between different RNA molecules, their protein targets, and HIV.

A Camptothetin Analog, Topotecan, Promotes HIV Latency via Interference with HIV Transcription and RNA Splicing.

Low level HIV transcription during modern antiretroviral therapy (ART) in persons with HIV is linked to residual inflammation and associated diseases, like cardiovascular disease and cancer. The "block and lock" approach to hold HIV in a state of deep latency may help decrease residual inflammation in a person with HIV on ART and thus improve health. A camptothecin analog topotecan (TPT) was previously implicated as an inhibitor of active HIV replication. Using an in vitro primary T cell model of HIV latency, we demonstrated that (i) TPT reduces HIV transcriptional activity in latently infected cells; (ii) downregulation of HIV RNA by TPT cannot be reversed by latency reversing agents; (iii) several primary and secondary mechanism of action of TPT may be involved in control of HIV replication; (iv) regulation of HIV RNA by TPT is dependent on splicing complexity; (v) increase in proportion of unspliced HIV transcripts was facilitated by intron retention and upregulation of splicing factors, specifically SRSF6, by TPT. Although high TPT dosing (10 µM) was needed to achieve the observed effects, viability of primary CD4+ T cells was not greatly affected. Because toxicity can be observed with TPT in persons with cancer, TPT is unlikely to be used as an anti-HIV agent in clinic, but our study provides proof that camptothetin has "block and lock" activity. Other camptothetin analogs, which are less toxic than TPT, should be designed and tested as HIV "block and lock" agents. IMPORTANCE HIV survives in a state of very low activity, called latency, for long periods in persons with HIV on antiretroviral therapy. This low activity of HIV is linked to residual inflammation and associated diseases, such as heart disease and cancer. New strategies are being explored to further silence the HIV provirus and suppress residual inflammation. This study provides strong evidence that the camptothetin analog, Topotecan, can reduce residual activity of HIV in an experimental model of HIV latency. While Topotecan itself is likely not suitable for use in the clinic due to its toxicity, other camptothetin analogs should be designed and investigated as "block and lock" agents.

Monocytic-Myeloid Derived Suppressor Cells Suppress T-Cell Responses in Recovered SARS CoV2-Infected Individuals.

Coronavirus disease 2019 (COVID-19) caused by SARS Coronavirus 2 (CoV2) is associated with massive immune activation and hyperinflammatory response. Acute and severe CoV2 infection is characterized by the expansion of myeloid derived suppressor cells (MDSC) because of cytokine storm, these MDSC suppress T cell functions. However, the presence of MDSC and its effect on CoV2 antigen specific T cell responses in individuals long after first detection of CoV2 and recovery from infection has not been studied. We and others have previously shown that CD11b+CD33+CD14+HLA-DR-/lo monocytic MDSC (M-MDSC) are present in individuals with clinical recovery from viral infection. In this study, we compared the frequency, functional and transcriptional signatures of M-MDSC isolated from CoV2 infected individuals after 5-months of the first detection of the virus (CoV2+) and who were not infected with CoV2 (CoV2-). Compared to CoV2- individuals, M-MDSC were present in CoV2+ individuals at a higher frequency, the level of M-MDSC correlated with the quantity of IL-6 in the plasma. Compared to CoV2-, increased frequency of PD1+, CD57+ and CX3CR1+ T effector memory (TEM) cell subsets was also present in CoV2+ individuals, but these did not correlate with M-MDSC levels. Furthermore, depleting M-MDSC from peripheral blood mononuclear cells (PBMC) increased T cell cytokine production when cultured with the peptide pools of immune dominant spike glycoprotein (S), membrane (M), and nucleocapsid (N) antigens of CoV2. M-MDSC suppressed CoV2 S- antigen-specific T cell in ROS, Arginase, and TGFß dependent manner. Our gene expression, RNA-seq and pathway analysis studies further confirm that M-MDSC isolated from CoV2+ individuals are enriched in pathways that regulate both innate and adaptive immune responses, but the genes regulating these functions (HLA-DQA1, HLA-DQB1, HLA-B, NLRP3, IL1ß, CXCL2, CXCL1) remained downregulated in M-MDSC isolated from CoV2+ individuals. These results demonstrate that M-MDSC suppresses recall responses to CoV2 antigens long after recovery from infection. Our findings suggest M-MDSC as novel regulators of CoV2 specific T cell responses, and should be considered as target to augment responses to vaccine.

HIV-1 latency is established preferentially in minimally activated and non-dividing cells during productive infection of primary CD4 T cells.

Latently infected CD4 T cells form a stable reservoir of HIV that leads to life-long viral persistence; the mechanisms involved in establishment of this latency are not well understood. Three scenarios have been proposed: 1) an activated, proliferating cell becomes infected and reverts back to a resting state; 2) an activated cell becomes infected during its return to resting; or 3) infection is established directly in a resting cell. The aim of this study was, therefore, to investigate the relationship between T cell activation and proliferation and the establishment of HIV latency. Isolated primary CD4 cells were infected at different time points before or after TCR-induced stimulation. Cell proliferation within acutely infected cultures was tracked using CFSE viable dye over 14 days; and cell subsets that underwent varying degrees of proliferation were isolated at end of culture by flow cytometric sorting. Recovered cell subpopulations were analyzed for the amount of integrated HIV DNA, and the ability to produce virus, upon a second round of cell stimulation. We show that cell cultures exposed to virus, prior to stimulus addition, contained the highest levels of integrated and replication-competent provirus after returning to quiescence; whereas, cells infected during the height of cell proliferation retained the least. Cells that did not divide or exhibited limited division, following virus exposure and stimulation contained greater amounts of integrated and inducible HIV than did cells that had divided many times. Based on these results, co-culture experiments were conducted to demonstrate that latent infection could be established directly in non-dividing cells via cell-to-cell transmission from autologous productively infected cells. Together, the findings from our studies implicate the likely importance of direct infection of sub-optimally activated T cells in establishment of latently infected reservoirs in vivo, especially in CD4 lymphocytes that surround productive viral foci within immune tissue microenvironments.

Integrated proteomics and transcriptomics analyses identify novel cell surface markers of HIV latency.

Elimination of the latent HIV cell reservoir may be possible, if the molecular identity of latently infected cells were fully elucidated. We conducted comprehensive molecular profiling, at the protein and RNA levels, of primary T cells latently infected with HIV in vitro. Isobaric labelling quantitative proteomics and RNA sequencing identified 1453 proteins and 618 genes, altered in latently infected cells compared to mock-infected controls (p < 0.05). Biomarker selection was based on results from integrated data analysis. Relative enrichment for latently infected cells was monitored using flow cytometric sorting and the HIV integrant assay. Antibodies against selected proteins, encoded by CEACAM1 and PLXNB2, enabled enrichment of latently infected cells from cell mixtures by 3-10 fold (5.8 average, p < 0.001), comparable to levels obtained with biomarkers reported previously. Individual biomarkers are likely linked to subsets of latently infected cells, and an extended antibody panel will be required to inclusively target the latent HIV reservoir.

Micro RNA Targets in HIV Latency: Insights into Novel Layers of Latency Control.

Despite the considerable progress that has been made in identifying cellular factors and pathways that contribute to establishment and maintenance of the latent HIV reservoir, it remains the major obstacle to eradicating this virus. Most recently, noncoding genes have been implicated in regulation of HIV expression. In this study, small RNA sequencing was used to profile expression of microRNAs (miRNAs) in a primary CD4+ T cell in vitro model of HIV latency. Previously, we have shown that protein-coding genes dysregulated in this model were enriched for the p53 signaling pathway, which was confirmed experimentally. We further found a link between p53 signaling and dysregulated long noncoding RNAs. In this study, we hypothesized that miRNAs may provide an additional level of regulation of the p53 signaling pathway during HIV latency. Twenty-six miRNAs were identified to be dysregulated in our latency model. A subset of these miRNAs was validated by real-time quantitative polymerase chain reaction. Predicted messenger RNA (mRNA) targets and cellular pathways enriched for mRNA targets were identified using several analytical methods. Our analyses showed that many protein-coding genes and pathways targeted by dysregulated miRNAs have relevance to regulation of HIV expression or establishment of HIV latency. The p53 signaling pathway was found among pathways that were targeted by dysregulated miRNAs at a greater level than expected by chance. This study provides a mechanistic insight into regulation of the p53 pathway through miRNAs that may contribute to the establishment of latency.

Single-Cell Profiling Reveals Divergent, Globally Patterned Immune Responses in Murine Skin Inflammation.

Inflammatory response heterogeneity has impeded high-resolution dissection of diverse immune cell populations during activation. We characterize mouse cutaneous immune cells by single-cell RNA sequencing, after inducing inflammation using imiquimod and oxazolone dermatitis models. We identify 13 CD45+ subpopulations, which broadly represent most functionally characterized immune cell types. Oxazolone pervasively upregulates Jak2/Stat3 expression across T cells and antigen-presenting cells (APCs). Oxazolone also induces Il4/Il13 expression in newly infiltrating basophils, and Il4ra and Ccl24, most prominently in APCs. In contrast, imiquimod broadly upregulates Il17/Il22 and Ccl4/Ccl5. A comparative analysis of single-cell inflammatory transcriptional responses reveals that APC response to oxazolone is tightly restricted by cell identity, whereas imiquimod enforces shared programs on multiple APC populations in parallel. These global molecular patterns not only contrast immune responses on a systems level but also suggest that the mechanisms of new sources of inflammation can eventually be deduced by comparison to known signatures.

Use of GapmeRs for gene expression knockdowns in human primary resting CD4+ T cells.

Human primary resting CD4+ T cells are difficult to transfect while preserving viability. The present study evaluated gymnotic delivery and RNase H1-dependent gene expression knockdown mediated by antisense oligonucleotides, called GapmeRs. Exposure of primary resting CD4+ T cells to GapmeRs did not cause cell activation or affect cell viability. Gene expression knockdowns were stable at least up to 48 h after removal of GapmeRs from culture. Exposure to GapmeRs resulted in comparable levels of degradation along the entire transcript, which could be important when studying function of regulatory long non-coding RNAs. Efficiency of transcript degradation was not solely dependent on the dose of GapmeR, RNA target and its localization. When using GapmeRs, some optimization is required, and all targets have to be individually tested; however, using GapmeRs is advantageous in experiments where preservation of the resting state of the human primary CD4+ T cells and targeting nuclear RNAs are desired. In certain cases, combining GapmeR with siRNA for the same target may improve knockdown efficiency.

Long non-coding RNAs and latent HIV - A search for novel targets for latency reversal.

The latent cellular reservoir of HIV is recognized as the major barrier to cure from HIV infection. Long non-coding RNAs (lncRNAs) are more tissue and cell type-specific than protein coding genes, and may represent targets of choice for HIV latency reversal. Using two in vitro primary T-cell models, we identified lncRNAs dysregulated in latency. PVT1 and RP11-347C18.3 were up-regulated in common between the two models, and RP11-539L10.2 was down-regulated. The major component of the latent HIV reservoir, memory CD4+ T-cells, had higher expression of these lncRNAs, compared to naïve T-cells. Guilt-by-association analysis demonstrated that lncRNAs dysregulated in latency were associated with several cellular pathways implicated in HIV latency establishment and maintenance: proteasome, spliceosome, p53 signaling, and mammalian target of rapamycin (MTOR). PVT1, RP11-347C18.3, and RP11-539L10.2 were down-regulated by latency reversing agents, suberoylanilide hydroxamic acid and Romidepsin, suggesting that modulation of lncRNAs is a possible secondary mechanism of action of these compounds. These results will facilitate prioritization of lncRNAs for evaluation as targets for HIV latency reversal. Importantly, our study provides insights into regulatory function of lncRNA during latent HIV infection.

Histone deacetylase inhibitors induce complex host responses that contribute to differential potencies of these compounds in HIV reactivation.

Histone deacetylase (HDAC) inhibitors (HDACis) have been widely tested in clinical trials for their ability to reverse HIV latency but have yielded only limited success. One HDACi, suberoylanilide hydroxamic acid (SAHA), exhibits off-target effects on host gene expression predicted to interfere with induction of HIV transcription. Romidepsin (RMD) has higher potency and specificity for class I HDACs implicated in maintaining HIV provirus in the latent state. More robust HIV reactivation has indeed been achieved with RMD use ex vivo than with SAHA; however, reduction of viral reservoir size has not been observed in clinical trials. Therefore, using RNA-Seq, we sought to compare the effects of SAHA and RMD on gene expression in primary CD4+ T cells. Among the genes whose expression was modulated by both HDACi agents, we identified genes previously implicated in HIV latency. Two genes, SMARCB1 and PARP1, whose modulation by SAHA and RMD is predicted to inhibit HIV reactivation, were evaluated in the major maturation subsets of CD4+ T cells and were consistently either up- or down-regulated by both HDACi compounds. Our results indicate that despite having different potencies and HDAC specificities, SAHA and RMD modulate an overlapping set of genes, implicated in HIV latency regulation. Some of these genes merit exploration as additional targets to improve the therapeutic outcomes of "shock and kill" strategies. The overall complexity of HDACi-induced responses among host genes with predicted stimulatory or inhibitory effects on HIV expression likely contributes to differential HDACi potencies and dictates the outcome of HIV reactivation.

Transcriptional Modulation of Human Endogenous Retroviruses in Primary CD4+ T Cells Following Vorinostat Treatment.

The greatest obstacle to a cure for HIV is the provirus that integrates into the genome of the infected cell and persists despite antiretroviral therapy. A "shock and kill" approach has been proposed as a strategy for an HIV cure whereby drugs and compounds referred to as latency-reversing agents (LRAs) are used to "shock" the silent provirus into active replication to permit "killing" by virus-induced pathology or immune recognition. The LRA most utilized to date in clinical trials has been the histone deacetylase (HDAC) inhibitor-vorinostat. Potentially, pathological off-target effects of vorinostat may result from the activation of human endogenous retroviruses (HERVs), which share common ancestry with exogenous retroviruses including HIV. To explore the effects of HDAC inhibition on HERV transcription, an unbiased pharmacogenomics approach (total RNA-Seq) was used to evaluate HERV expression following the exposure of primary CD4+ T cells to a high dose of vorinostat. Over 2,000 individual HERV elements were found to be significantly modulated by vorinostat, whereby elements belonging to the ERVL family (e.g., LTR16C and LTR33) were predominantly downregulated, in contrast to LTR12 elements of the HERV-9 family, which exhibited the greatest signal, with the upregulation of 140 distinct elements. The modulation of three different LTR12 elements by vorinostat was confirmed by droplet digital PCR along a dose-response curve. The monitoring of LTR12 expression during clinical trials with vorinostat may be indicated to assess the impact of this HERV on the human genome and host immunity.

Relative efficacy of T cell stimuli as inducers of productive HIV-1 replication in latently infected CD4 lymphocytes from patients on suppressive cART.

Quantification of cell-associated replication-competent HIV, in blood samples from patients with undetectable plasma viremia, requires specialized culture conditions that include exogenous pan T cell stimulation. Different research groups have used several stimuli for this purpose; however, the relative efficacies of these T cell stimuli to induce productive HIV replication from latently infected cells ex vivo have not been systematically evaluated. To this end, we compared four commonly used T cell stimuli: 1) irradiated allogeneic cells plus phytohaemagglutinin (PHA); 2) PHA alone; 3) phorbol myristate acetate plus Ionomycin; and 4) immobilized αCD3 plus αCD28 antibodies. End-point dilutions of patient CD4 T cells were performed, using virion RNA production to quantify HIV induction. Our results demonstrated that these activation approaches were not equivalent and that antibody cross-linking of CD3 and CD28 membrane receptors was the most effective means to activate HIV replication from a resting cell state, closely followed by stimulation with irradiated allogeneic cells plus PHA.

Transcriptomic Analysis Implicates the p53 Signaling Pathway in the Establishment of HIV-1 Latency in Central Memory CD4 T Cells in an In Vitro Model.

The search for an HIV-1 cure has been greatly hindered by the presence of a viral reservoir that persists despite antiretroviral therapy (ART). Studies of HIV-1 latency in vivo are also complicated by the low proportion of latently infected cells in HIV-1 infected individuals. A number of models of HIV-1 latency have been developed to examine the signaling pathways and viral determinants of latency and reactivation. A primary cell model of HIV-1 latency, which incorporates the generation of primary central memory CD4 T cells (TCM), full-length virus infection (HIVNL4-3) and ART to suppress virus replication, was used to investigate the establishment of HIV latency using RNA-Seq. Initially, an investigation of host and viral gene expression in the resting and activated states of this model indicated that the resting condition was reflective of a latent state. Then, a comparison of the host transcriptome between the uninfected and latently infected conditions of this model identified 826 differentially expressed genes, many of which were related to p53 signaling. Inhibition of the transcriptional activity of p53 by pifithrin-α during HIV-1 infection reduced the ability of HIV-1 to be reactivated from its latent state by an unknown mechanism. In conclusion, this model may be used to screen latency reversing agents utilized in shock and kill approaches to cure HIV, to search for cellular markers of latency, and to understand the mechanisms by which HIV-1 establishes latency.

Acute psychological stress induces short-term variable immune response.

In spite of advances in understanding the cross-talk between the peripheral immune system and the brain, the molecular mechanisms underlying the rapid adaptation of the immune system to an acute psychological stressor remain largely unknown. Conventional approaches to classify molecular factors mediating these responses have targeted relatively few biological measurements or explored cross-sectional study designs, and therefore have restricted characterization of stress-immune interactions. This exploratory study analyzed transcriptional profiles and flow cytometric data of peripheral blood leukocytes with physiological (endocrine, autonomic) measurements collected throughout the sequence of events leading up to, during, and after short-term exposure to physical danger in humans. Immediate immunomodulation to acute psychological stress was defined as a short-term selective up-regulation of natural killer (NK) cell-associated cytotoxic and IL-12 mediated signaling genes that correlated with increased cortisol, catecholamines and NK cells into the periphery. In parallel, we observed down-regulation of innate immune toll-like receptor genes and genes of the MyD88-dependent signaling pathway. Correcting gene expression for an influx of NK cells revealed a molecular signature specific to the adrenal cortex. Subsequently, focusing analyses on discrete groups of coordinately expressed genes (modules) throughout the time-series revealed immune stress responses in modules associated to immune/defense response, response to wounding, cytokine production, TCR signaling and NK cell cytotoxicity which differed between males and females. These results offer a spring-board for future research towards improved treatment of stress-related disease including the impact of stress on cardiovascular and autoimmune disorders, and identifies an immune mechanism by which vulnerabilities to these diseases may be gender-specific.

Mixed effects of suberoylanilide hydroxamic acid (SAHA) on the host transcriptome and proteome and their implications for HIV reactivation from latency.

Suberoylanilide hydroxamic acid (SAHA) has been assessed in clinical trials as part of a "shock and kill" strategy to cure HIV-infected patients. While it was effective at inducing expression of HIV RNA ("shock"), treatment with SAHA did not result in a reduction of reservoir size ("kill"). We therefore utilized a combined analysis of effects of SAHA on the host transcriptome and proteome to dissect its mechanisms of action that may explain its limited success in "shock and kill" strategies. CD4+ T cells from HIV seronegative donors were treated with 1µM SAHA or its solvent dimethyl sulfoxide (DMSO) for 24h. Protein expression and post-translational modifications were measured with iTRAQ proteomics using ultra high-precision two-dimensional liquid chromatography-tandem mass spectrometry. Gene expression was assessed by Illumina microarrays. Using limma package in the R computing environment, we identified 185 proteins, 18 phosphorylated forms, 4 acetylated forms and 2982 genes, whose expression was modulated by SAHA. A protein interaction network integrating these 4 data types identified the HIV transcriptional repressor HMGA1 to be upregulated by SAHA at the transcript, protein and acetylated protein levels. Further functional category assessment of proteins and genes modulated by SAHA identified gene ontology terms related to NFκB signaling, protein folding and autophagy, which are all relevant to HIV reactivation. In summary, SAHA modulated numerous host cell transcripts, proteins and post-translational modifications of proteins, which would be expected to have very mixed effects on the induction of HIV-specific transcription and protein function. Proteome profiling highlighted a number of potential counter-regulatory effects of SAHA with respect to viral induction, which transcriptome profiling alone would not have identified. These observations could lead to a more informed selection and design of other HDACi with a more refined targeting profile, and prioritization of latency reversing agents of other classes to be used in combination with SAHA to achieve more potent induction of HIV expression.

Dose-responsive gene expression in suberoylanilide hydroxamic acid-treated resting CD4+ T cells.

DESIGN: Persistent latently infected CD4 T cells represent a major obstacle to HIV eradication. Histone deacetylase inhibitors (HDACis) are a proposed activation therapy. However, off-target effects on gene expression in host immune cells are poorly understood. We hypothesized that HDACi-modulated genes would be best identified with a dose-response analysis. METHODS: Resting primary CD4 T cells were treated with 0.34, 1, 3, or 10 µmol/l of the HDACi, suberoylanilide hydroxamic acid (SAHA), for 24 h and subjected to microarray gene expression analysis. Genes with dose-correlated expression were filtered to identify a subset with consistent up or downregulation at each SAHA dose. Histone modifications were characterized in six SAHA dose-responsive genes by chromatin immunoprecipitation (ChIP-RT-qPCR). RESULTS: A large number of genes were shown to be upregulated (N = 657) or downregulated (N = 725) by SAHA in a dose-responsive manner (FDR-corrected P-value ≤ 0.5, fold change ≥|2|). Several genes (e.g. CINNAL1, DPEP2, H1F0, IRGM, PHF15, and SELL) are potential in-vivo biomarkers of SAHA activity. SAHA dose-responsive genes included transcription factors, HIV restriction factors, histone methyltransferases, and host proteins that interact with HIV. Pathway analysis suggested net downregulation of T-cell activation with increasing SAHA dose. Histone acetylation was not correlated with host gene expression, but plausible alternative mechanisms for SAHA-modulated gene expression were identified. CONCLUSION: Numerous genes in CD4 T cells are modulated by SAHA in a dose-responsive manner, including genes that may negatively influence HIV activation from latency. Our study suggests that SAHA influences gene expression through a confluence of several mechanisms, including histone modification, and altered expression and activity of transcription factors.