With the development of triple combination antiretroviral therapy, routine HIV treatment eliminates nearly all actively infected cells. Nevertheless, the small reservoir of latently infected cells, which can remain dormant for long periods of time before becoming active and producing new virus particles, represents a crucial barrier to completely curing the disease. Identifying markers that identify latently infected cells or the biochemical factors that control latency activation could enable the effective use of a “shock and kill” strategy, where specific targeting or activation of latently infected cells eliminates the viral reservoir. Our recent work suggests that the global transcriptomic and epigenomic changes during hematopoietic differentiation affect viral latency and activation. Additionally, we recently found that global inhibition of histone deacetylase activity increases viral activation in these cells, further implicating epigenomic changes in activation. These results raise fundamental questions: What are the markers of latently infected cells? How do the transcriptomic and epigenomic state of a cell affect latency and activation? How does differentiation state relate to viral latency? Here, we leverage our experimental platform for identifying latently and actively infected cells, single cell transcriptome and epigenome sequencing, and our recently developed computational integration methods to investigate these questions. Our interdisciplinary team combines expertise in HIV basic science, HIV clinical treatment, and bioinformatics to develop an experimental and computational framework for integrated gene expression, chromatin accessibility, and lineage into a single picture of viral latency and activation. Specifically, this project will (1) use single-cell RNA-seq and single-cell ATAC-seq to map diversity of infected cells, (2) investigate the relationship between hematopoietic differentiation state and viral activation, (3) determine viral integration sites through single-cell RNA-seq, (4) computationally integrate single cell transcriptome and epigenome profiles, and (5) computationally infer cell lineage relationships among viral genomes and infected cells. To accomplish these goals, we will carry out the following aims: (1) Characterize lineage, transcriptomic and epigenomic diversity of single latently and actively infected primary cells. (2) Investigate latency and activation during in vitro differentiation. (3) Survey single cell diversity of re-activated and in vitro infected cells from cART-suppressed patients. Together, these aims will produce a comprehensive, integrated transcriptomic and epigenomic atlas of the HIV reservoir, identify DNA and RNA biomarkers of latency, and characterize clonal expansion patterns. Our work also develops a broadly applicable experimental and computational framework, laying a foundation for the discovery of novel insights into HIV latency and activation.
Antiretroviral medications suppress viral replication but do not eradicate cellular sources of integrated proviral genomes that are a major barrier to a cure. CD4+ hematopoietic stem and progenitor cells (HSPCs) have the capacity for life-long survival, self-renewal and the generation of daughter cells. They are also susceptible to HIV infection in vitro and in vivo. Combination antiretroviral therapy effectively suppresses viremia in HIV- infected people. However, residual plasma virus (RPV) can be detected with very sensitive assays. Recently published studies demonstrate that clusters of identical proviruses from HSPCs and their likely progeny often match RPV and are sometimes infectious. A higher proportion of these sequences matched RPV than proviral genomes from peripheral blood mononuclear cells that lacked evidence of clonal expansion. Furthermore, we provide examples of proviral genomes from progenitors that were latent in peripheral blood T cells while simultaneously contributing to RPV. The cellular source of RPV in these cases is not known but is unlikely to be peripheral blood T cells, which required latency reactivation for gene expression. We have developed a model based on these data. In this model, we propose that heterogeneous differentiated progeny of infected progenitors can support either active or latent infection depending on progeny epigenetic and transcriptional programs. The overall objective of this application is to test this hypothesis by comprehensively characterizing intact HIV in peripheral blood and tissue reservoirs. A secondary objective is to determine whether infected HSPCs are required for clonal provirus and RPV and to identify any alternative proliferative sources of non- HSPC generated clonal genomes. To accomplish this, we will: 1. Analyze intact near full length viral genomes to identify sources of clonally amplified proviral genomes in peripheral blood and to determine their relationship to proliferative sources such as HSPCs; 2. use viral outgrowth assays to confirm relationships amongst sources of infectious virus; and 3. determine the active and latently infected tissue sources of infectious virus and their relationships to proliferative sources such as HSPCs. Results from these aims will comprehensively identify sources of functional virus and RPV across multiple disparate tissue sites. They will determine the extent to which multipotent and/or restricted progenitors or other proliferative sources serve as the source for clonally expanded HIV proviral genomes present in the peripheral blood and tissue sites. These studies will provide important new information that has the potential to change the way we think about the source of functionally relevant HIV reservoirs.
Molecular mechanisms underlying HIV related intestinal epithelial barrier dysfunction https://reporter.nih.gov/search/PtMvALnGS0G6n4qCYcBJEA/project-details/10454418
The goal of proposed experiments in this application is to determine how HIV infection disrupts intestinal barrier function. It is now appreciated that microbial translocation across an impaired epithelial barrier leads to circulating LPS, persistent immune activation and chronic inflammation in people living with HIV (PLWH). These HIV associated effects are important contributors to premature development of neurocognitive disorders, cardiovascular disease, metabolic syndrome and bone abnormalities even in PLWH on optimal combination antiretroviral therapy (cART). Untreated infection is characterized by the production of proinflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNFα). Following therapy, cytokine levels decline but chronic inflammation continues. Prevention of inflammation-induced comorbidities requires the development of more specific therapeutics targeting the underlying cause. However, a gap exists in our understanding of the underlying molecular pathways involved. This proposal will capitalize on an established collaboration between investigators with expertise in HIV biology and intestinal barrier function/pathobiology. We have generated strong preliminary data that provides a framework for understanding the underlying link between disrupted intestinal epithelial barrier function and HIV infection. While the overall chronic inflammatory manifestations of HIV infection are likely to be multi-factorial, our exciting results support an overarching hypothesis that lamina propria HIV-1 infected primary human CD4+T lymphocytes that closely interact with intestinal epithelial initiate a process leading to enhanced production of pro-inflammatory cytokines that negatively impact epithelial homeostasis resulting in a leaky intestinal barrier. Given these important new insights, funding is requested to support a major collaborative effort between established investigators in the areas of HIV biology and intestinal inflammation/barrier disruption to determine the mechanism(s) through which primary human intestinal epithelial cells (IECs) and HIV-infected primary T cells synergize to cause intestinal pathobiology. Specifically, we will determine the HIV-dependent mechanisms that alter T-cell function and disrupt the intestinal barrier. In addition, we will identify the pathways altered in IECs exposed to HIV infected T-cells that lead to barrier dysfunction. Findings generated from these studies will allow a better understanding of the mechanisms underlying HIV related enteropathy that is known to be a major source of morbidity and mortality in HIV-infected individuals and will lead to development of new strategies to improve the health of HIV infected people. 1
Current combined antiretroviral therapies (cART) suppress viral levels in the blood but do not eradicate reservoirs of cells harboring integrated copies of HIV proviral genomes. These cells persist in part because the provirus maintains a latent state that evades the immune response and viral cytopathic effect. Approaches to clear reservoirs by reactivating latent cells have provided evidence that latency can be reversed in vivo, however reversal of latency alone has not been sufficient to reduce latent reservoirs. Efforts are now in place to couple latency reactivation with strategies to eradicate the infected cells – such as by design and activation of more efficacious anti-HIV cytotoxic T lymphocytes (CTLs). Another key player is Nef, an accessory protein encoded by HIV, which is a primary focus of our proposed research. Because Nef inhibits the activity of anti- HIV CTLs, a potent inhibitor of this protein would help achieve HIV eradication. One of the main functions of Nef is the down-modulation of major histocompatibility complex class I encoded proteins (MHC-I), masking infection from the host immune system and allowing HIV infected cells to persist. Combination therapy with latency antagonists plus Nef inhibitors could act synergistically to clear HIV reservoirs. To date, no Nef inhibitor has achieved potent restoration of MHC-I in the presence of Nef. We developed a high-throughput assay to identify inhibitors of Nef-mediated MHC-I downregulation, and a screen of natural product extracts (NPEs) yielded 10 hits with Nef inhibitory activity. We identified a number of related compounds, as the active component in several of these extracts. The pure natural products potently restore surface expression of MHC- I in the presence of Nef without inhibiting its other activities. We tested a number of structurally related compounds within this natural product family and identified two that possess pM to nM potencies in human primary cells. Based on this strong preliminary data, we believe that further enhancing the Nef inhibitory activity of these molecules through analog development will yield a safe anti-Nef drug. Therefore, we plan to (A) optimize these inhibitors by further separating and characterizing the anti-Nef effect from off-target activities to identify a lead drug candidate for development and (B) determine the mechanism by which the inhibitor disrupts Nef-mediated MHC-I downmodulation so that optimization can be conducted more intelligently. These goals will be achieved through the following specific aims: (1) Conduct lead compound structural optimization to improve pharmaceutical properties. (2) Perform a detailed functional analysis of all promising analogs to identify ideal lead compounds and (3) Determine the mechanism by which the natural product-derived inhibitor disrupts Nef-mediated MHC-I downmodulation including target identification and biochemical studies. From this work, we expect to generate a new class of compounds that are potent Nef inhibitors with high pharmaceutical potential. The addition of Nef inhibitory compounds to current cART cocktails is expected to enhance immune clearance of viral reservoirs, leading to the long-elusive HIV cure.