Probes for HIV-1

Subtype C HIV-1 is responsible for the largest proportion of people living with HIV-1 infection. However, there is limited information about the roles of the brain and its cell types as a potential sanctuary for this subtype and how the sanctuary may be affected by the administration of anti-retroviral therapy (ART). To address this issue, we collected postmortem brain tissues from ART treated HIV-1 infected Zambian individuals who experienced complete viral suppression and those who did not. Tissues from various brain compartments were collected from each individual as frozen and formalin-fixed paraffin embedded brain specimens, for detection and quantification of HIV-1 genomes and identification of the infected cell type. Genomic DNA and RNA were extracted from frozen brain tissues. The extracted DNA and RNA were then subjected to droplet digital PCR for HIV-1 quantification. RNA/DNAscope in situ hybridization (ISH) for HIV-1 was performed on formalin-fixed paraffin embedded brain tissues in conjugation with immunohistochemistry to identify the infected cell types. Droplet digital PCR revealed that HIV-1 gag DNA and RNA were detectable in half of the cases studied regardless of ART success or failure. The presence of HIV-1 lacked specific tissue compartmentalization since detection was random among various brain tissues. When combined with immunohistochemistry, RNA/DNAscope ISH demonstrated co-localization of HIV-1 DNA with CD68 expressing cells indicative of microglia or peripheral macrophage. Our study showed that brain is a potential sanctuary for subtype C HIV-1, as HIV-1 can be detected in the brain of infected individuals irrespective of ART treatment outcome and no compartmentalization of HIV-1 to specific brain compartments was evident.

Identifying the source and dynamics of persistent HIV-1 at single-cell resolution during cART is crucial for the design of strategies to eliminate the latent HIV-1 reservoir. An assay to measure latent HIV-1 that can distinguish inducible from defective proviruses with high precision is essential to evaluate the efficacy of HIV-1 cure efforts but is presently lacking. The primary aim of this study was therefore to identify transcription and translation competent latently infected cells through detection of biomolecules that are dependent on transcriptional activation of the provirus. We investigated the applicability of two commercially available assays; PrimeFlowTM RNA Assay (RNAflow) and RNAscope™ ISH (RNAscope) for evaluation of the efficacy of latency reversal agents (LRAs) to reactivate the HIV-1 latent reservoir. The J-Lat cell model (clones 6.3, 9.3, and 10.6) and four LRAs was used to evaluate the sensitivity, specificity, and lower detection limit of the RNAflow and RNAscope assays for the detection and description of the translation-competent HIV-1 reservoir. We also checked for HIV-1 subtype specificity of the RNAscope assay using patient-derived subtype A1, B, C, and CRF01_AE recombinant plasmids following transfection in 293T cells and the applicability of the method in patient-derived peripheral blood mononuclear cells (PBMCs). The lower detection limit of RNAflow was 575 HIV-1 infected cells/million and 45 cells/million for RNAscope. The RNAscope probes, designed for HIV-1B, also detected other subtypes (A1, B, C, and CRF01_AE). RNAscope was applicable for the detection of HIV-1 in patient-derived PBMCs following LRA activation. In conclusion, our study showed that RNAscope can be used to quantify the number of directly observed individual cells expressing HIV-1 mRNA following LRA activation. Therefore, it can be a useful tool for characterization of translation-competent HIV-1 in latently infected cell at single-cell resolution in the fields of HIV-1 pathogenesis and viral persistence.

CD4+ T cells are the primary HIV-1 target cell, with the vast majority of these cells residing within lymphoid tissue compartments throughout the body. Predictably, HIV-1 infection, replication, localization, reservoir establishment and persistence, as well as associated host immune and inflammatory responses and disease pathology principally take place within the tissues of the immune system. By virture of the fact that the virus-host struggle is played out within lymphoid and additional tissues compartments in HIV-1 infected individuals it is critical to understand HIV-1 infection and disease within these relevant tissue sites; however, there are obvious limitations to studying these dynamic processes in humans. Nonhuman primate (NHP) research has provided a vital bridge between basic and preclinical research and clinical studies, with experimental SIV infection of NHP models offering unique opportunities to understand key processes of HIV-1 infection and disease that are either not practically feasible or ethical in HIV-1 infected humans. In this review we will discuss current approaches to studying the tissue based immunopathogenesis of AIDS virus infection in NHPs, including both analyses of tissues obtained at biopsy or necropsy and complementary non-invasive imaging approaches that may have practical utility in monitoring HIV-1 disease in the clinical setting.

Human immunodeficiency virus type one (HIV-1) tissue compartments are established soon after viral infection. However, the timing in which virus gains a permanent foothold in tissue and the cellular factors that control early viral-immune events are incompletely understood. These are critical events in studies of HIV-1 pathogenesis and in the development of viral reservoirs after antiretroviral therapy. Moreover, factors affecting the permanence of viral-tissue interactions underlie barriers designed to eliminate HIV-1 infection. To this end we investigated the temporal and spatial viral and host factors during HIV-1 seeding of tissue compartments. Two humanized NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ mouse models were employed. In the first, immune deficient mice were reconstituted with human CD34+ cord blood hematopoietic stem cells (HSC) (hu-HSC) and in the second mice were transplanted with adult mature human peripheral lymphocytes (hu-PBL). Both, in measure, reflect relationships between immune activation and viral infection as seen in an infected human host. Following humanization both mice models were infected with HIV-1ADA at 104 50% tissue culture infective doses. Viral nucleic acids and protein and immune cell profiles were assayed in brain, lung, spleen, liver, kidney, lymph nodes, bone marrow, and gut from 3 to 42 days. Peripheral CD4+ T cell loss began at 3 days together with detection of HIV-1 RNA in both mouse models after initiation of HIV-1 infection. HIV-1 was observed in all tested tissues at days 3 and 14 in hu- PBL and HSC mice, respectively. Immune impairment was most prominent in hu-PBL mice. T cell maturation and inflammation factors were linked directly to viral tissue seeding in both mouse models. We conclude that early viral tissue compartmentalization provides a roadmap for investigations into HIV-1 elimination.

The present study demonstrates HIV-1 Tat-mediated epigenetic downregulation of microglial miR-124 and its association with microglial activation. Exposure of mouse primary microglia isolated from newborn pups of either sex to HIV-1 Tat resulted in decreased expression of primary miR-124-1, primary miR-124-2 as well as the mature miR-124. In parallel, HIV-1 Tat exposure to mouse primary microglial cellsresulted in increased expression of DNA methylation enzymes, such as DNMT1, DNMT3A, and DNMT3B that were also accompanied by increased global DNA methylation. Bisulfite-converted genomic DNA sequencing in the HIV-1 Tat exposed mouse primary microglial cellsfurther confirmed increased DNA methylation of the primary miR-124-1 and primary miR-124-2 promoters. Bioinformatic analyses identified MECP2 as a novel 3'-UTR target of miR-124. This was further validated in mouse primary microglial cells wherein HIV-1 Tat-mediated downregulation of miR-124 resulted in increased expression of MECP2, leading in turn to further repression of miR-124 via the feedback loop. In addition to MECP2, miR-124 also modulated the levels of STAT3 through its binding to the 3'-UTR, leading to microglial activation. Luciferase assays and Ago2 immunoprecipitation determined the direct binding between miR-124 and 3'-UTR of both MECP2 and STAT3. Gene silencing of MECP2 and DNMT1 and overexpression of miR-124 blocked HIV-1 Tat-mediated downregulation of miR-124 and microglial activation. In vitro findings were also confirmed in the basal ganglia of SIV-infected rhesus macaques (both sexes). In summary, our findings demonstrate a novel mechanism of HIV-1 Tat-mediated activation of microglia via downregulation of miR-124, leading ultimately to increased MECP2 and STAT3 signaling.
SIGNIFICANCE STATEMENT
Despite the effectiveness of combination antiretroviral therapy in controlling viremia, the CNS continues to harbor viral reservoirs. The persistence of low-level virus replication leads to the accumulation of early viral proteins including HIV-1 Tat protein. Understanding the epigenetic/molecular mechanism(s) by which viral proteins such as HIV-1 Tat can activate microglia is thus of paramount importance. This study demonstrated HIV-1 Tat-mediated DNA methylation of the miR-124 promoter leads to its downregulation with a concomitant upregulation of the MECP2-STAT3-IL6 resulting in microglial activation. These findings reveal an unexplored epigenetic/molecular mechanism(s) underlying HIV-1 Tat-mediated microglial activation, thereby providing a potential target for the development of therapeutics aimed at ameliorating microglial activation and neuroinflammation in the context of HIV-1 infection.

We report a case of an adolescent who presented at our emergency department with acute abdominal pain. While the initial diagnosis was acute appendicitis, a secondary and coincidental diagnosis of primary HIV-1 infection was made. Concurrent and subsequent clinical and molecular biology findings form the basis of our argument that primary HIV-1 infection was the cause of acute appendicitis in this individual.

The most immediate and evident effect of mucosal exposure to semen in vivo is a local release of proinflammatory mediators accompanied by an influx of leukocytes into the female genital mucosa (FGM). The implication of such response in HIV-1 transmission has never been addressed due to limitations of currently available experimental models. Using human tissue explants from the uterine cervix, we developed a system of mucosal exposure to seminal plasma (SP) that supports HIV-1 replication. Treatment of ectocervical explants with SP resulted in the upregulation of inflammatory and growth factors, including IL-6, TNF, CCL5, CCL20, CXCL1, and CXCL8, and IL1A, CSF2, IL7, PTGS2, as evaluated by measuring protein levels in explant conditioned medium (ECM) and gene expression in tissue. SP treatment was also associated with increased recruitment of monocytes and neutrophils, as observed upon incubation of peripheral blood leukocytes with ECM in a transwell system. To evaluate the impact of the SP-mediated response on local susceptibility to HIV-1, we infected ectocervical explants with the CCR5-tropic variant HIV-1BaL either in the presence of SP, or after explant pre-incubation with SP. In both experimental settings SP enhanced virus replication as evaluated by HIV-1 p24gag released in explant culture medium over time, as well as by HIV-1 DNA quantification in explants infected in the presence of SP. These results suggest that a sustained inflammatory response elicited by SP soon after coitus may promote HIV-1 transmission to the FGM. Nevertheless, ectocervical tissue explants did not support the replication of transmitted/founder HIV-1 molecular clones, regardless of SP treatment. Our system offers experimental and analytical advantages over traditional models of HIV-1 transmission for the study of SP immunoregulatory effect on the FGM, and may provide a useful platform to ultimately identify new determinants of HIV-1 infection at this site.

HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection.

During sexual intercourse, HIV-1 crosses epithelial barriers composing the genital mucosa, a poorly understood feature that requires an HIV-1-infected cell vectoring efficient mucosal HIV-1 entry. Therefore, urethral mucosa comprising a polarized epithelium and a stroma composed of fibroblasts and macrophages were reconstructed in vitro. Using this system, we demonstrate by live imaging that efficient HIV-1 transmission to stromal macrophages depends on cell-mediated transfer of the virus through virological synapses formed between HIV-1-infected CD4+ T cells and the epithelial cell mucosal surface. We visualized HIV-1 translocation through mucosal epithelial cells via transcytosis in regions where virological synapses occurred. In turn, interleukin-13 is secreted and HIV-1 targets macrophages, which develop a latent state of infection reversed by lipopolysaccharide (LPS) activation. The live observation of virological synapse formation reported herein is key in the design of vaccines and antiretroviral therapies aimed at blocking HIV-1 access to cellular reservoirs in genital mucosa.

HIV-1 integration into the host genome favors actively transcribed genes. Prior work indicated that the nuclear periphery provides the architectural basis for integration site selection, with viral capsid-binding host cofactor CPSF6 and viral integrase-binding cofactor LEDGF/p75 contributing to selection of individual sites. Here, by investigating the early phase of infection, we determine that HIV-1 traffics throughout the nucleus for integration. CPSF6-capsid interactions allow the virus to bypass peripheral heterochromatin and penetrate the nuclear structure for integration. Loss of interaction with CPSF6 dramatically alters virus localization toward the nuclear periphery and integration into transcriptionally repressed lamina-associated heterochromatin, while loss of LEDGF/p75 does not significantly affect intranuclear HIV-1 localization. Thus, CPSF6 serves as a master regulator of HIV-1 intranuclear localization by trafficking viral preintegration complexes away from heterochromatin at the periphery toward gene-dense chromosomal regions within the nuclear interior.

A safe and effective human immunodeficiency virus type 1 (HIV-1) vaccine is urgently needed, but remains elusive. While HIV-1 live-attenuated vaccine can provide potent protection as demonstrated in rhesus macaque-simian immunodeficiency virus model, the potential pathogenic consequences associated with the uncontrolled virus replication preclude such vaccine from clinical applications. We investigated a novel approach to address this problem by controlling live-attenuated HIV-1 replication through an unnatural genetic switch that was based on the amber suppression strategy. Here we report the construction of all-in-one live-attenuated HIV-1 mutants that contain genomic copy of the amber suppression system. This genetic modification resulted in viruses that were capable of multicycle replication in vitro and could be switched on and off using an unnatural amino acid as the cue. This stand-alone, replication-controllable attenuated HIV-1 virus represents an important step toward the generation of a safe and efficacious live-attenuated HIV-1 vaccine. The strategy reported in this work can be adopted for the development of other live-attenuated vaccines.

We have recently developed the first microscopy-based strategy that enables simultaneous multiplex detection of viral RNA (vRNA), viral DNA (vDNA), and viral protein. Here, we used this approach to study the kinetics of latency reactivation in cells infected with the human immunodeficiency virus (HIV). We showed the transcription of nascent vRNA from individual latently integrated and reactivated vDNA sites appearing earlier than viral protein. We further demonstrated that this method can be used to quantitatively assess the efficacy of a variety of latency reactivating agents. Finally, this microscopy-based strategy was augmented with a flow-cytometry-based approach, enabling the detection of transcriptional reactivation of large numbers of latently infected cells. Hence, these approaches are shown to be suitable for qualitative and quantitative studies of HIV-1 latency and reactivation.