Tissue myeloid cells in SIV-infected primates acquire viral DNA through phagocytosis of infected T cells.

The viral accessory protein Vpx, expressed by certain simian and human immunodeficiency viruses (SIVs and HIVs), is thought to improve viral infectivity of myeloid cells. We infected 35 Asian macaques and African green monkeys with viruses that do or do not express Vpx and examined viral targeting of cells in vivo. While lack of Vpx expression affected viral dynamics in vivo, with decreased viral loads and infection of CD4⁺ T cells, Vpx expression had no detectable effect on infectivity of myeloid cells. Moreover, viral DNA was observed only within myeloid cells in tissues not massively depleted of CD4⁺ T cells. Myeloid cells containing viral DNA also showed evidence of T cell phagocytosis in vivo, suggesting that their viral DNA may be attributed to phagocytosis of SIV-infected T cells. These data suggest that myeloid cells are not a major source of SIV in vivo, irrespective of Vpx expression.

Defining the molecular mechanisms of HIV-1 Tat secretion: PtdIns(4,5)P2 at the epicenter.

The human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat) protein functions both intracellularly and extracellularly. Intracellularly, the main function is to enhance transcription of the viral promoter. However, this process only requires a small amount of intracellular Tat. The majority of Tat is secreted through an unconventional mechanism by binding to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2 ), a phospholipid in the inner leaflet of the plasma membrane that is required for secretion. This interaction is mediated by the basic domain of Tat (residues 48-57) and a conserved tryptophan (residue 11). After binding to PtdIns(4,5)P2 , Tat secretion diverges into multiple pathways, which we categorized as oligomerization-mediated pore formation, spontaneous translocation and incorporation into exosomes. Extracellular Tat has been shown to be neurotoxic and toxic to other cells of the central nervous system (CNS) and periphery, able to recruit immune cells to the CNS and cerebrospinal fluid, and alter the gene expression and morphology of uninfected cells. The effects of extracellular Tat have been examined in HIV-1-associated neurocognitive disorders (HAND); however, only a small number of studies have focused on the mechanisms underlying Tat secretion. In this review, the molecular mechanisms of Tat secretion will be examined in a variety of biologically relevant cell types.

Real-time visualization of chromatin modification in isolated nuclei.

Chromatin modification is traditionally assessed in biochemical assays that provide average measurements of static events given that the analysis requires components from many cells. Microscopy can visualize single cells, but the cell body and organelles can hamper staining and visualization of the nucleus. Normally, chromatin is visualized by immunostaining a fixed sample or by expressing exogenous fluorescently tagged proteins in a live cell. Alternative microscopy tools to observe changes of endogenous chromatin in real-time are needed. Here, we isolated transcriptionally competent nuclei from cells and used antibody staining without fixation to visualize changes in endogenous chromatin. This method allows the real-time addition of drugs and fluorescent probes to one or more nuclei while under microscopy observation. A high-resolution map of 11 endogenous nuclear markers of the histone code, transcription machinery and architecture was obtained in transcriptionally active nuclei by performing confocal and structured illumination microscopy. We detected changes in chromatin modification and localization at the single-nucleus level after inhibition of histone deacetylation. Applications in the study of RNA transcription, viral protein function and nuclear architecture are presented. This article has an associated First Person interview with the first author of the paper.

Destabilization of the gut microbiome marks the end-stage of simian immunodeficiency virus infection in wild chimpanzees.

Enteric dysbiosis is a characteristic feature of progressive human immunodeficiency virus type 1 (HIV-1) infection but has not been observed in simian immunodeficiency virus (SIVmac)-infected macaques, including in animals with end-stage disease. This has raised questions concerning the mechanisms underlying the HIV-1 associated enteropathy, with factors other than virus infection, such as lifestyle and antibiotic use, implicated as playing possible causal roles. Simian immunodeficiency virus of chimpanzees (SIVcpz) is also associated with increased mortality in wild-living communities, and like HIV-1 and SIVmac, can cause CD4+ T cell depletion and immunodeficiency in infected individuals. Given the central role of the intestinal microbiome in mammalian health, we asked whether gut microbial constituents could be identified that are indicative of SIVcpz status and/or disease progression. Here, we characterized the gut microbiome of SIVcpz-infected and -uninfected chimpanzees in Gombe National Park, Tanzania. Subjecting a small number of fecal samples (N = 9) to metagenomic (shotgun) sequencing, we found bacteria of the family Prevotellaceae to be enriched in SIVcpz-infected chimpanzees. However, 16S rRNA gene sequencing of a larger number of samples (N = 123) failed to show significant differences in both the composition and diversity (alpha and beta) of gut bacterial communities between infected (N = 24) and uninfected (N = 26) chimpanzees. Similarly, chimpanzee stool-associated circular virus (Chi-SCV) and chimpanzee adenovirus (ChAdV) identified by metagenomic sequencing were neither more prevalent nor more abundant in SIVcpz-infected individuals. However, fecal samples collected from SIVcpz-infected chimpanzees within 5 months before their AIDS-related death exhibited significant compositional changes in their gut bacteriome. These data indicate that SIVcpz-infected chimpanzees retain a stable gut microbiome throughout much of their natural infection course, with a significant destabilization of bacterial (but not viral) communities observed only in individuals with known immunodeficiency within the last several months before their death. Am. J. Primatol. 80:e22515, 2018. © 2015 Wiley Periodicals, Inc.

Exosomes from HIV-1-infected Cells Stimulate Production of Pro-inflammatory Cytokines through Trans-activating Response (TAR) RNA.

HIV-1 infection results in a chronic illness because long-term highly active antiretroviral therapy can lower viral titers to an undetectable level. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under highly active antiretroviral therapy frequently develop various metabolic disorders, neurocognitive abnormalities, and cardiovascular diseases. We have previously shown that exosomes containing trans-activating response (TAR) element RNA enhance susceptibility of undifferentiated naive cells to HIV-1 infection. This study indicates that exosomes from HIV-1-infected primary cells are highly abundant with TAR RNA as detected by RT-real time PCR. Interestingly, up to a million copies of TAR RNA/µl were also detected in the serum from HIV-1-infected humanized mice suggesting that TAR RNA may be stable in vivo. Incubation of exosomes from HIV-1-infected cells with primary macrophages resulted in a dramatic increase of proinflammatory cytokines, IL-6 and TNF-ß, indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. The intact TAR molecule was able to bind to PKR and TLR3 effectively, whereas the 5' and 3' stems (TAR microRNAs) bound best to TLR7 and -8 and none to PKR. Binding of TAR to PKR did not result in its phosphorylation, and therefore, TAR may be a dominant negative decoy molecule in cells. The TLR binding through either TAR RNA or TAR microRNA potentially can activate the NF-κB pathway and regulate cytokine expression. Collectively, these results imply that exosomes containing TAR RNA could directly affect the proinflammatory cytokine gene expression and may explain a possible mechanism of inflammation observed in HIV-1-infected patients under cART.

The inhibition of microRNAs by HIV-1 Tat suppresses beta catenin activity in astrocytes.

BACKGROUND: Long term infection with HIV-1, even in the context of therapy, leads to chronic health problems including an array of neurocognitive dysfunctions. The viral Tat protein has previously been implicated in neuropathogenesis through its effect on astrocytes. Tat has also been shown to inhibit the biogenesis of miRNAs by inhibiting the activity of the cellular Dicer protein in an RNA dependent fashion. Whether there is a mechanistic connection between the ability of HIV-1 Tat to alter miRNAs and its observed effects on cells of the central nervous system has not been well examined. RESULTS: Here, we examined the ability of HIV-1 Tat to bind to and inhibit the production of over 300 cellular miRNAs. We found that the Tat protein only binds to and inhibits a fraction of the total cellular miRNAs. By mapping the downstream targets of these miRNAs we have determined a possible role for Tat alterations of miRNAs in the development of neuropathogenesis. Specifically, this work points to suppression of miRNAs function as the mechanism for Tat suppression of ß-catenin activity. CONCLUSIONS: The discovery that HIV-1 Tat inhibits only a fraction of miRNAs opens new areas of research regarding changes in cellular pathways through suppression of RNA interference. Our initial analysis strongly suggests that these pathways may contribute to HIV-1 disruption of the central nervous system.

Activation of HIV-1 from latent infection via synergy of RUNX1 inhibitor Ro5-3335 and SAHA.

A major barrier to the elimination of HIV-1 infection is the presence of a pool of long-lived, latently infected CD4+ memory T-cells. The search for treatments to re-activate latent HIV to aid in clearance is hindered by the incomplete understanding of the mechanisms that lead to transcriptional silencing of viral gene expression in host cells. Here we identify a previously unknown role for RUNX1 in HIV-1 transcriptional latency. The RUNX proteins, in combination with the co-factor CBF-ß, are critical transcriptional regulators in T-cells. RUNX1 strongly modulates CD4 expression and contributes to CD4+ T-cell function. We show that RUNX1 can bind DNA sequences within the HIV-1 LTR and that this binding represses transcription. Using patient samples we show a negative correlation between RUNX1 expression and viral load. Furthermore, we find that pharmacologic inhibition of RUNX1 by a small molecule inhibitor, Ro5-3335, synergizes with the histone deacetylase (HDAC) inhibitor SAHA (Vorinostat) to enhance the activation of latent HIV-1 in both cell lines and PBMCs from patients. Our findings indicate that RUNX1 and CBF-ß cooperate in cells to modulate HIV-1 replication, identifying for the first time RUNX1 as a cellular factor involved in HIV-1 latency. This work highlights the therapeutic potential of inhibitors of RUNX1 to re-activate virus and aid in clearance of HIV-1.

The cellular autophagy pathway modulates human T-cell leukemia virus type 1 replication.

Autophagy, a general homeostatic process for degradation of cytosolic proteins or organelles, has been reported to modulate the replication of many viruses. The role of autophagy in human T-cell leukemia virus type 1 (HTLV-1) replication has, however, been uncharacterized. Here, we report that HTLV-1 infection increases the accumulation of autophagosomes and that this accumulation increases HTLV-1 production. We found that the HTLV-1 Tax protein increases cellular autophagosome accumulation by acting to block the fusion of autophagosomes to lysosomes, preventing the degradation of the former by the latter. Interestingly, the inhibition of cellular autophagosome-lysosome fusion using bafilomycin A increased the stability of the Tax protein, suggesting that cellular degradation of Tax occurs in part through autophagy. Our current findings indicate that by interrupting the cell's autophagic process, Tax exerts a positive feedback on its own stability.

The extent of sequence complementarity correlates with the potency of cellular miRNA-mediated restriction of HIV-1.

MicroRNAs (miRNAs) are 22-nt non-coding RNAs involved in the regulation of cellular gene expression and potential cellular defense against viral infection. Using in silico analyses, we predicted target sites for 22 human miRNAs in the HIV genome. Transfection experiments using synthetic miRNAs showed that five of these miRNAs capably decreased HIV replication. Using one of these five miRNAs, human miR-326 as an example, we demonstrated that the degree of complementarity between the predicted viral sequence and cellular miR-326 correlates, in a Dicer-dependent manner, with the potency of miRNA-mediated restriction of viral replication. Antagomirs to miR-326 that knocked down this cell endogenous miRNA increased HIV-1 replication in cells, suggesting that miR-326 is physiologically functional in moderating HIV-1 replication in human cells.

Adult Human Brain Tissue Cultures to Study NeuroHIV.

HIV-associated neurocognitive disorders (HAND) persist under antiretroviral therapy as a complex pathology that has been difficult to study in cellular and animal models. Therefore, we generated an ex vivo human brain slice model of HIV-1 infection from surgically resected adult brain tissue. Brain slice cultures processed for flow cytometry showed >90% viability of dissociated cells within the first three weeks in vitro, with parallel detection of astrocyte, myeloid, and neuronal populations. Neurons within brain slices showed stable dendritic spine density and mature spine morphologies in the first weeks in culture, and they generated detectable activity in multi-electrode arrays. We infected cultured brain slices using patient-matched CD4+ T-cells or monocyte-derived macrophages (MDMs) that were exposed to a GFP-expressing R5-tropic HIV-1 in vitro. Infected slice cultures expressed viral RNA and developed a spreading infection up to 9 days post-infection, which were significantly decreased by antiretrovirals. We also detected infected myeloid cells and astrocytes within slices and observed minimal effect on cellular viability over time. Overall, this human-centered model offers a promising resource to study the cellular mechanisms contributing to HAND (including antiretroviral toxicity, substance use, and aging), infection of resident brain cells, and new neuroprotective therapeutics.

MicroRNAs and HIV-1: complex interactions.

RNAi plays important roles in many biological processes, including cellular defense against viral infection. Components of the RNAi machinery are widely conserved in plants and animals. In mammals, microRNAs (miRNAs) represent an abundant class of cell encoded small noncoding RNAs that participate in RNAi-mediated gene silencing. Here, findings that HIV-1 replication in cells can be regulated by miRNAs and that HIV-1 infection of cells can alter cellular miRNA expression are reviewed. Lessons learned from and questions outstanding about the complex interactions between HIV-1 and cellular miRNAs are discussed.

Retrovirus infected cells contain viral microRNAs.

The encoding of microRNAs in retroviral genomes has remained a controversial hypothesis despite significant supporting evidence in recent years. A recent publication demonstrating the production of functional miRNAs from the retrovirus bovine leukemia virus adds further credence to the fact that retroviruses do indeed encode their own miRNAs. Here we comment on the importance of this paper to the field, as well as examine the other known examples of miRNAs encoded by RNA viruses.

Absence of DICER in monocytes and its regulation by HIV-1.

MicroRNAs (miRNAs) are a class of small RNA molecules that function to control gene expression and restrict viral replication in host cells. The production of miRNAs is believed to be dependent upon the DICER enzyme. Available evidence suggests that in T lymphocytes, HIV-1 can both suppress and co-opt the host's miRNA pathway for its own benefit. In this study, we examined the state of miRNA production in monocytes and macrophages as well as the consequences of viral infection upon the production of miRNA. Monocytes in general express low amounts of miRNA-related proteins, and DICER in particular could not be detected until after monocytes were differentiated into macrophages. In the case where HIV-1 was present prior to differentiation, the expression of DICER was suppressed. MicroRNA chip results for RNA isolated from transfected and treated cells indicated that a drop in miRNA production coincided with DICER protein suppression in macrophages. We found that the expression of DICER in monocytes is restricted by miR-106a, but HIV-1 suppressed DICER expression via the viral gene Vpr. Additionally, analysis of miRNA expression in monocytes and macrophages revealed evidence that some miRNAs can be processed by both DICER and PIWIL4. Results presented here have implications for both the pathology of viral infections in macrophages and the biogenesis of miRNAs. First, HIV-1 suppresses the expression and function of DICER in macrophages via a previously unknown mechanism. Second, the presence of miRNAs in monocytes lacking DICER indicates that some miRNAs can be generated by proteins other than DICER.

Human T-lymphotropic virus type 1 transcription and chromatin-remodeling complexes.

Human T-lymphotropic virus type 1 (HTLV-1) encodes the viral protein Tax, which is believed to act as a viral transactivator through its interactions with a variety of transcription factors, including CREB and NF-kappaB. As is the case for all retroviruses, the provirus is inserted into the host DNA, where nucleosomes are deposited to ensure efficient packaging. Nucleosomes act as roadblocks in transcription, making it difficult for RNA polymerase II (Pol II) to proceed toward the 3' end of the genome. Because of this, a variety of chromatin remodelers can act to modify nucleosomes, allowing for efficient transcription. While a number of covalent modifications are known to occur on histone tails in HTLV-1 infection (i.e., histone acetyltransferases [HATs], histone deacetylases [HDACs], and histone methyltransferases [HMTs]), evidence points to the use of chromatin remodelers that use energy from ATP hydrolysis to remodel nucleosomes. Here we confirm that BRG1, which is the core subunit of eight chromatin-remodeling complexes, is essential not only for Tax transactivation but also for viral replication. This is especially evident when wild-type infectious clones of HTLV-1 are used. BRG1 associates with Tax at the HTLV-1 long terminal repeat (LTR), and coexpression of BRG1 and Tax results in increased rates of transcription. The interaction of BRG1 with Tax additionally recruits the basal transcriptional machinery and removes some of the core histones from the nucleosome at the start site (Nuc 1). When using the BRG1-deficient cell lines SW13, C33A, and TSUPR1, we observed little viral transcription and no viral replication. Importantly, while these three cell lines do not express detectable levels of BRG1, much of the SWI/SNF complex remains assembled in the cells. Knockdown of BRG1 and associated SWI/SNF subunits suggests that the BRG1-utilizing SWI/SNF complex PBAF is responsible for HTLV-1 nucleosome remodeling. Finally, HTLV-1 infection of cell lines with a knockdown in BRG1 or the PBAF complex results in a significant reduction in viral production. Overall, we concluded that BRG1 is required for Tax transactivation and HTLV-1 viral production and that the PBAF complex appears to be responsible for nucleosome remodeling.

CD8+ T-Cell Mediated Control of HIV-1 in a Unique Cohort With Low Viral Loads.

A unique population of HIV-1 infected individuals can control infection without antiretroviral therapy. These individuals fall into a myriad of categories based on the degree of control (low or undetectable viral load), the durability of control over time and the underlying mechanism (i.e., possession of protective HLA alleles or the absence of critical cell surface receptors). In this study, we examine a cohort of HIV-1 infected individuals with a documented history of sustained low viral loads in the absence of therapy. Through in vitro analyses of cells from these individuals, we have determined that infected individuals with naturally low viral loads are capable of controlling spreading infection in vitro in a CD8+ T-cell dependent manner. This control is lost when viral load is suppressed by antiretroviral therapy and correlates with a clinical CD4:CD8 ratio of <1. Our results support the conclusion that HIV-1 controllers with low, but detectable viral loads may be controlling the virus due to an effective CD8+ T-cell response. Understanding the mechanisms of control in these subjects may provide valuable understanding that could be applied to induce a functional cure in standard progressors.

Alprazolam Prompts HIV-1 Transcriptional Reactivation and Enhances CTL Response Through RUNX1 Inhibition and STAT5 Activation.

The HIV-1 pandemic is a significant challenge to the field of medicine. Despite advancements in antiretroviral (ART) development, 38 million people worldwide still live with this disease without a cure. A significant barrier to the eradication of HIV-1 lies in the persistently latent pool that establishes early in the infection. The "shock and kill" strategy relies on the discovery of a latency-reversing agent (LRA) that can robustly reactivate the latent pool and not limit immune clearance. We have found that a benzodiazepine (BDZ), that is commonly prescribed for panic and anxiety disorder, to be an ideal candidate for latency reversal. The BDZ Alprazolam functions as an inhibitor of the transcription factor RUNX1, which negatively regulates HIV-1 transcription. In addition to the displacement of RUNX1 from the HIV-1 5'LTR, Alprazolam potentiates the activation of STAT5 and its recruitment to the viral promoter. The activation of STAT5 in cytotoxic T cells may enable immune activation which is independent of the IL-2 receptor. These findings have significance for the potential use of Alprazolam in a curative strategy and to addressing the neuroinflammation associated with neuroHIV-1.

The identification of unique serum proteins of HIV-1 latently infected long-term non-progressor patients.

BACKGROUND: The search for disease biomarkers within human peripheral fluids has become a favorable approach to preventative therapeutics throughout the past few years. The comparison of normal versus disease states can identify an overexpression or a suppression of critical proteins where illness has directly altered a patient's cellular homeostasis. In particular, the analysis of HIV-1 infected serum is an attractive medium with which to identify altered protein expression due to the ease and non-invasive methods of collecting samples as well as the corresponding insight into the in vivo interaction of the virus with infected cells/tissue. The utilization of proteomic techniques to globally identify differentially expressed serum proteins in response to HIV-1 infection is a significant undertaking that is complicated due to the innate protein profile of human serum. RESULTS: Here, the depletion of 12 of the most abundant serum proteins, followed by two-dimensional gel electrophoresis coupled with identification of these proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, has allowed for the identification of differentially expressed, low abundant serum proteins. We have analyzed and compared serum samples from HIV-1 infected subjects who are being treated using highly active antiretroviral therapy (HAART) to those who are latently infected but have not progressed to AIDS despite the absence of treatment, i.e. long term non-progressors (LTNPs). Here we have identified unique serum proteins that are differentially expressed in LTNP HIV-1 patients and may contribute to the ability of these patients to combat HIV-1 infection in the absence of HAART. We focused on the cdk4/6 cell cycle inhibitor p16INK4A and found that the treatment of HIV-1 latently infected cell lines with p16INK4A decreases viral production despite it not being expressed endogenously in these cells. CONCLUSIONS: Identification of these unique proteins may serve as an indication of altered viral states in response to infection as well as a natural phenotypic variability in response to HIV-1 infection in a given population.

Visualizing Chromatin Modifications in Isolated Nuclei.

Modifications in chromatin structure are traditionally monitored by biochemical assays that provide average measurements of static events in a population of cells. Microscopy provides a method by which single cells or nuclei can be observed. Traditionally, microscopy has been used to image the nucleus by the application of immunostaining to chemically fixed samples or the use of exogenously expressed fluorescent proteins. This method represents an approach to observe changes in endogenous proteins relating to chromatin structure in real time. Here we describe a method for isolating transcriptionally and enzymatically active nuclei from live cells and visualizing events using fluorescently labeled antibodies. This method allows the observation of real time changes in chromatin architecture and can be used to observe the effects of drugs on nuclei while under microscopic observation.

ROR1 regulates chemoresistance in Breast Cancer via modulation of drug efflux pump ABCB1.

Chemoresistance is one of the leading causes of mortality in breast cancer (BC). Understanding the molecules regulating chemoresistance is critical in order to combat chemoresistant BC. Drug efflux pump ABCB1 is overexpressed in chemoresistant neoplasms where it effluxes various chemotherapeutic agents from cells. Because it is expressed in normal and cancerous cells alike, attempts at targeting ABCB1 directly have failed due to low specificity and disruption of normal tissue. A proposed method to inhibit ABCB1 is to target its cancer-specific, upstream regulators, mitigating damage to normal tissue. Few such cancer-specific upstream regulators have been described. Here we characterize ROR1 as an upstream regulator of ABCB1. ROR1 is highly expressed during development but not expressed in normal adult tissue. It is however highly expressed in several cancers. ROR1 is overexpressed in chemoresistant BC where it correlates with poor therapy response and tumor recurrence. Our data suggests, ROR1 inhibition sensitizes BC cells to chemo drugs. We also show ROR1 regulates ABCB1 stability and transcription via MAPK/ERK and p53. Validating our overall findings, inhibition of ROR1 directly correlated with decreased efflux of chemo-drugs from cells. Overall, our results highlight ROR1's potential as a therapeutic target for multidrug resistant malignancies.