Concanamycin A counteracts HIV-1 Nef to enhance immune clearance of infected primary cells by cytotoxic T lymphocytes.

Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.

Class 1-Selective Histone Deacetylase (HDAC) Inhibitors Enhance HIV Latency Reversal while Preserving the Activity of HDAC Isoforms Necessary for Maximal HIV Gene Expression.

Combinations of drugs that affect distinct mechanisms of HIV latency aim to induce robust latency reversal leading to cytopathicity and elimination of the persistent HIV reservoir. Thus far, attempts have focused on combinations of protein kinase C (PKC) agonists and pan-histone deacetylase inhibitors (HDIs) despite the knowledge that HIV gene expression is regulated by class 1 histone deacetylases. We hypothesized that class 1-selective HDIs would promote more robust HIV latency reversal in combination with a PKC agonist than pan-HDIs because they preserve the activity of proviral factors regulated by non-class 1 histone deacetylases. Here, we show that class 1-selective agents used alone or with the PKC agonist bryostatin-1 induced more HIV protein expression per infected cell. In addition, the combination of entinostat and bryostatin-1 induced viral outgrowth, whereas bryostatin-1 combinations with pan-HDIs did not. When class 1-selective HDIs were used in combination with pan-HDIs, the amount of viral protein expression and virus outgrowth resembled that of pan-HDIs alone, suggesting that pan-HDIs inhibit robust gene expression induced by class 1-selective HDIs. Consistent with this, pan-HDI-containing combinations reduced the activity of NF-κB and Hsp90, two cellular factors necessary for potent HIV protein expression, but did not significantly reduce overall cell viability. An assessment of viral clearance from in vitro cultures indicated that maximal protein expression induced by class 1-selective HDI treatment was crucial for reservoir clearance. These findings elucidate the limitations of current approaches and provide a path toward more effective strategies to eliminate the HIV reservoir.IMPORTANCE Despite effective antiretroviral therapy, HIV evades eradication in a latent form that is not affected by currently available drug regimens. Pharmacologic latency reversal that leads to death of cellular reservoirs has been proposed as a strategy for reservoir elimination. Because histone deacetylases (HDACs) promote HIV latency, HDAC inhibitors have been a focus of HIV cure research. However, many of these inhibitors broadly affect multiple classes of HDACs, including those that promote HIV gene expression (class 1 HDACs). Here, we demonstrate that targeted treatment with class 1-selective HDAC inhibitors induced more potent HIV latency reversal than broadly acting agents. Additionally, we provide evidence that broadly acting HDIs are limited by inhibitory effects on non-class 1 HDACs that support the activity of proviral factors. Thus, our work demonstrates that the use of targeted approaches to induce maximum latency reversal affords the greatest likelihood of reservoir elimination.

An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients.

The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for "anti-latency" therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.

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.

Hematopoietic Stem and Progenitor Cells (HSPCs).

Cord blood is a readily available source of hematopoietic stem and progenitor cells (HSPCs) which can be infected with HIV-1 in vitro to produce inducible latently infected cells for reactivation studies. Infected HSPCs can also be found in the setting of clinically undetectable viremia in vivo. Here we describe an in vitro infection model utilizing cord blood derived HSPCs, as well as methods for isolating and characterizing provirus from bone marrow HSPCs from suppressed patients.

Hematopoietic Stem and Progenitor Cells Are a Distinct HIV Reservoir that Contributes to Persistent Viremia in Suppressed Patients.

Long-lived reservoirs of persistent HIV are a major barrier to a cure. CD4+ hematopoietic stem and progenitor cells (HSPCs) have the capacity for lifelong survival, self-renewal, and the generation of daughter cells. Recent evidence shows that they are also susceptible to HIV infection in vitro and in vivo. Whether HSPCs harbor infectious virus or contribute to plasma virus (PV) is unknown. Here, we provide strong evidence that clusters of identical proviruses from HSPCs and their likely progeny often match residual PV. A higher proportion of these sequences match residual PV than proviral genomes from bone marrow and peripheral blood mononuclear cells that are observed only once. Furthermore, an analysis of near-full-length genomes isolated from HSPCs provides evidence that HSPCs harbor functional HIV proviral genomes that often match residual PV. These results support the conclusion that HIV-infected HSPCs form a distinct and functionally significant reservoir of persistent HIV in infected people.

Vpr overcomes macrophage-specific restriction of HIV-1 Env expression and virion production.

The HIV-1 accessory protein Vpr enhances infection of primary macrophages through unknown mechanisms. Recent studies demonstrated that Vpr interactions with the cellular DCAF1-DDB1-CUL4 E3 ubiquitin ligase complex limit activation of innate immunity and interferon (IFN) induction. We describe a restriction mechanism that targets the HIV-1 envelope protein Env, but is overcome by Vpr and its interaction with DCAF1. This restriction is active in the absence of Vpr in HIV-1-infected primary macrophages and macrophage-epithelial cell heterokaryons, but not epithelial cell lines. HIV-1-infected macrophages lacking Vpr express more IFN following infection, target Env for lysosomal degradation, and produce fewer Env-containing virions. Conversely, Vpr expression reduces IFN induction, rescues Env expression, and enhances virion release. Addition of IFN or silencing DCAF1 reduces the amount of cell-associated Env and virion production in wild-type HIV-1-infected primary macrophages. These findings provide insight into an IFN-stimulated macrophage-specific restriction pathway targeting HIV-1 Env that is counteracted by Vpr.

Suberoylanilide hydroxamic acid induces limited changes in the transcriptome of primary CD4(+) T cells.

OBJECTIVE: To assess the off-target effects of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) in human primary CD4 T cells. DESIGN: A pharmacologically relevant concentration (340 nmol/l) of SAHA was shown to significantly increase histone hyperacetylation by 24 h and this length of treatment was selected to determine its impact on gene expression in primary CD4 T cells. METHODS: Illumina Beadchips for microarray gene expression analysis were used to analyze differential gene expression between cells treated or not with SAHA with a paired analysis using multivariate permutation tests. Gene ontology, biological pathway and protein interaction network analyses were used to identify the higher order biological processes affected by SAHA treatment. RESULTS: Modest modulation by SAHA was observed for 1847 genes with 80% confidence level of no more than 10% false positives. A thousand genes were upregulated by SAHA and 847 downregulated. Pathways and gene ontologies overrepresented in the list of differentially expressed genes included Glycolysis/Gluconeogenesis, tRNA Modification, and the Histone Acetyltransferase Complex. Protein interaction network analysis revealed that transcription factor c-Myc, which was downregulated by SAHA treatment at the mRNA level, interacts with a number of SAHA-responsive genes. CONCLUSIONS: The effects on transcription by SAHA were sufficiently modest to support trials to activate HIV replication as part of an eradication strategy. SAHA did not appear to modulate proliferative or apoptotic processes to a great extent, which might impact the ability of patients to eradicate the virus reservoir following activation by HDACi treatment.

Highly precise measurement of HIV DNA by droplet digital PCR.

Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR (long terminal repeat) circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it.

Microwell devices with finger-like channels for long-term imaging of HIV-1 expression kinetics in primary human lymphocytes.

A major obstacle in the treatment of human immunodeficiency virus type 1 (HIV-1) is a sub-population of latently infected CD4(+) T lymphocytes. The cellular and viral mechanisms regulating HIV-1 latency are not completely understood, and a promising technique for probing the regulation of HIV-1 latency is single-cell time-lapse microscopy. Unfortunately, CD4(+) T lymphocytes rapidly migrate on substrates and spontaneously detach, making them exceedingly difficult to track, hampering single-cell level studies. To overcome these problems, we built microdevices with a three-level architecture. The devices contain arrays of finger-like microchannels to "corral" T-lymphocyte migration, round wells that are accessible to pipetting, and microwells connecting the microchannels with the round wells. T lymphocytes that are loaded into a well first settle into the microwells and then to microchannels by gravity. Within the microchannels, T lymphocytes are in favorable culture conditions because they are in physical contact with each other, under no mechanical stress, and fed from a large reservoir of fresh medium. Most importantly, T lymphocytes in the microchannels are not exposed to any flow and their random migration is restricted to a nearly one-dimensional region, greatly facilitating long-term tracking of multiple cells in time-lapse microscopy. The devices have up to nine separate round wells, making it possible to test up to nine different cell lines or medium conditions in a single experiment. Activated primary CD4(+) T lymphocytes, resting primary CD4(+) T lymphocytes, and THP-1 monocytic leukemia cells loaded into the devices maintained viability over multiple days. The devices were used to track the fluorescence level of individual primary CD4(+) T lymphocytes expressing green fluorescent protein (GFP) for up to 60 hours (h) and to quantify single-cell gene-expression kinetics of four different HIV-1 variants. The kinetics of GFP expression from the lentiviruses in the primary CD4(+) T lymphocytes agree with previous measurements of these lentiviral vectors in the immortalized Jurkat T lymphocyte cell line. The proposed devices offer a simple, robust approach to long-term single-cell studies of environmentally sensitive primary lymphocytes.

CD44 microbeads accelerate HIV-1 infection in T cells.

Super-paramagnetic CD44 MicroBeads (Miltenyi) designed for the isolation of infectious HIV-1 from dilute or difficult biological samples dramatically enhance the infectivity of bound HIV virions, even if the original viral suspension is merely incubated with beads. Infection of the CEM T cell line with the NL4-3 virus clone or primary human CD4 T cells with X4- and R5-tropic clones and a clade C primary virus isolate all showed accelerated p24 production and larger fractions of infected target cells. Effects could be detected very early; incubation of virus with the CD44 MicroBeads promoted higher levels of viral integration within the first infection cycle. In summary, CD44 MicroBeads provide the means not only to concentrate dilute viral samples, but also to directly facilitate within days rather than weeks the in vitro expansion of patient isolates independent of coreceptor usage and the performance of HIV replication assays that require a large fraction of infected primary T cells.