Development of a robust cell-based high-throughput screening assay to identify targets of HIV-1 viral protein R dimerization

Targeting protein-protein interactions (PPI) is an emerging field in drug discovery. Dimerization and PPI are essential properties of human immunodeficiency virus (HIV)-1 proteins, their mediated functions, and virus biology. Additionally, dimerization is required for the functional interaction of HIV-1 proteins with many host cellular components. In this study, a bimolecular fluorescence complementation (BiFC)-based screening assay was developed that can quantify changes in dimerization, using HIV-1 viral protein R (Vpr) dimerization as a "proof of concept." Results demonstrated that Venus Vpr (generated by BiFC Vpr constructs) could be competed off in a dose-dependent manner using untagged, full-length Vpr as a competitor molecule. The change in signal intensity was measured quantitatively through flow cytometry and fluorescence microscopy in a high content screening assay. High content imaging was used to screen a library of small molecules for an effect on Vpr dimerization. Among the tested molecules, a few of the small molecules demonstrate an effect on Vpr dimerization in a dose-dependent manner

A comprehensive analysis of the naturally occurring polymorphisms in HIV-1 Vpr: Potential impact on CTL epitopes

The enormous genetic variability reported in HIV-1 has posed problems in the treatment of infected individuals. This is evident in the form of HIV-1 resistant to antiviral agents, neutralizing antibodies and cytotoxic T lymphocytes (CTLs) involving multiple viral gene products. Based on this, it has been suggested that a comprehensive analysis of the polymorphisms in HIV proteins is of value for understanding the virus transmission and pathogenesis as well as for the efforts towards developing anti-viral therapeutics and vaccines. This study, for the first time, describes an in-depth analysis of genetic variation in Vpr using information from global HIV-1 isolates involving a total of 976 Vpr sequences. The polymorphisms at the individual amino acid level were analyzed. The residues 9, 33, 39, and 47 showed a single variant amino acid compared to other residues. There are several amino acids which are highly polymorphic. The residues that show ten or more variant amino acids are 15, 16, 28, 36, 37, 48, 55, 58, 59, 77, 84, 86, 89, and 93. Further, the variant amino acids noted at residues 60, 61, 34, 71 and 72 are identical. Interestingly, the frequency of the variant amino acids was found to be low for most residues. Vpr is known to contain multiple CTL epitopes like protease, reverse transcriptase, Env, and Gag proteins of HIV-1. Based on this, we have also extended our analysis of the amino acid polymorphisms to the experimentally defined and predicted CTL epitopes. The results suggest that amino acid polymorphisms may contribute to the immune escape of the virus. The available data on naturally occurring polymorphisms will be useful to assess their potential effect on the structural and functional constraints of Vpr and also on the fitness of HIV-1 for replication

HIV-1 Infection of DC: Evidence for the Acquisition of Virus Particles from Infected T Cells by Antigen Uptake Mechanism

Dendritic cells (DC) play a pivotal role in transmission and dissemination of HIV-1. Earlier studies reported that DC present at the site of infection trap virus particles via DC-SIGN and transfer the virus to the interacting naïve T cells. This prompted us to ask the question whether DC could acquire virus from infected T cells during DC-T cell interaction. To address this, we investigated the likely transfer of virus from HIV-1 infected T cells to DC and the underlying mechanisms involved. Results indicate that DC acquire virus from infected T cells via antigen uptake mechanism and this results in infection of DC with expression of proteins directed by viral DNA. Further studies with HIV-1 lacking the Env protein also resulted in infection of DC. The use of antibodies against DC-SIGN and DC-SIGN-R ruled out a role for receptor in the infection of DC. Additional data show that DC infection is directly correlated with the ability of DC to take up antigen from infected T cells. Overall, these studies provide evidence to suggest that HIV-1, besides infecting immune cells, also utilizes immunological mechanism(s) to acquire and disseminate virus

Comparative Expression Profile of miRNA and mRNA in Primary Peripheral Blood Mononuclear Cells Infected with Human Immunodeficiency Virus (HIV-1)

Host cells respond to exogenous infectious agents such as viruses, including HIV-1. Studies have evaluated the changes associated with virus infection at the transcriptional and translational levels of the cellular genes involved in specific pathways. While this approach is useful, in our view it provides only a partial view of genome-wide changes. Recently, technological advances in the expression profiling at the microRNA (miRNA) and mRNA levels have made it possible to evaluate the changes in the components of multiple pathways. To understand the role of miRNA and its interplay with host cellular gene expression (mRNA) during HIV-1 infection, we performed a comparative global miRNA and mRNA microarray using human PBMCs infected with HIV-1. The PBMCs were derived from multiple donors and were infected with virus generated from the molecular clone pNL4-3. The results showed that HIV-1 infection led to altered regulation of 21 miRNAs and 444 mRNA more than 2-fold, with a statistical significance of p<0.05. Furthermore, the differentially regulated miRNA and mRNA were shown to be associated with host cellular pathways involved in cell cycle/proliferation, apoptosis, T-cell signaling, and immune activation. We also observed a number of inverse correlations of miRNA and mRNA expression in infected PBMCs, further confirming the interrelationship between miRNA and mRNA regulation during HIV-1 infection. These results for the first time provide evidence that the miRNA profile could be an early indicator of host cellular dysfunction induced by HIV-1

Temporal transcriptional response to latency reversing agents identifies specific factors regulating HIV-1 viral transcriptional switch

Background: Latent HIV-1 reservoirs are identified as one of the major challenges to achieve HIV-1 cure. Currently available strategies are associated with wide variability in outcomes both in patients and CD4+ T cell models. This underlines the critical need to develop innovative strategies to predict and recognize ways that could result in better reactivation and eventual elimination of latent HIV-1 reservoirs. Results and discussion: In this study, we combined genome wide transcriptome datasets post activation with Systems Biology approach (Signaling and Dynamic Regulatory Events Miner, SDREM analyses) to reconstruct a dynamic signaling and regulatory network involved in reactivation mediated by specific activators using a latent cell line. This approach identified several critical regulators for each treatment, which were confirmed in follow-up validation studies using small molecule inhibitors. Results indicate that signaling pathways involving JNK and related factors as predicted by SDREM are essential for virus reactivation by suberoylanilide hydroxamic acid. ERK1/2 and NF-κB pathways have the foremost role in reactivation with prostratin and TNF-aα, respectively. JAK-STAT pathway has a central role in HIV-1 transcription. Additional evaluation, using other latent J-Lat cell clones and primary T cell model, also confirmed that many of the cellular factors associated with latency reversing agents are similar, though minor differences are identified. JAK-STAT and NF-κB related pathways are critical for reversal of HIV-1 latency in primary resting T cells. Conclusion: These results validate our combinatorial approach to predict the regulatory cellular factors and pathways responsible for HIV-1 reactivation in latent HIV-1 harboring cell line models. JAK-STAT have a role in reversal of latency in all the HIV-1 latency models tested, including primary CD4+ T cells, with additional cellular pathways such as NF-κB, JNK and ERK 1/2 that may have complementary role in reversal of HIV-1 latency

Neuronal apoptosis by HIV-1 Vpr: contribution of proinflammatory molecular networks from infected target cells

Background: Human immunodeficiency virus type 1 (HIV-1) induces neuronal dysfunction through host cellular factors and viral proteins including viral protein R (Vpr) released from infected macrophages/microglia. Vpr is important for infection of terminally differentiated cells such as macrophages. The objective of this study was to assess the effect of Vpr in the context of infectious virus particles on neuronal death through proinflammatory cytokines released from macrophages.Methods: Monocyte-derived macrophages (MDM) were infected with either HIV-1 wild type (HIV-1wt), Vpr deleted mutant (HIV-1{increment}Vpr) or mock. Cell lysates and culture supernatants from MDMs were analyzed for the expression and release of proinflammatory cytokines by quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay respectively. Mitogen-activated protein kinases (MAPK) were analyzed in activated MDMs by western blots. Further, the effect of Vpr on neuronal apoptosis was examined using primary neurons exposed to culture supernatants from HIV-1wt, HIV-1{increment}Vpr or mock-infected MDMs by Annexin-V staining, MTT and Caspase - Glo® 3/7 assays. The role of interleukin (IL)-1β, IL-8 and tumor necrosis factor (TNF)-α on neuronal apoptosis was also evaluated in the presence or absence of neutralizing antibodies against these cytokines.Results: HIV-1{increment}Vpr-infected MDMs exhibited reduced infection over time and specifically a significant downregulation of IL-1β, IL-8 and TNF-α at the transcriptional and/or protein levels compared to HIV-1wt-infected cultures. This downregulation was due to impaired activation of p38 and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) in HIV-1{increment}Vpr-infected MDMs. The association of SAPK/JNK and p38 to IL-1β and IL-8 production was confirmed by blocking MAPKs that prevented the elevation of IL-1β and IL-8 in HIV-1wt more than in HIV-1{increment}Vpr-infected cultures. Supernatants from HIV-1{increment}Vpr-infected MDMs containing lower concentrations of IL-1β, IL-8 and TNF-α as well as viral proteins showed a reduced neurotoxicity compared to HIV-1wt-infected MDM supernatants. Reduction of neuronal death in the presence of anti-IL-1β and anti-IL-8 antibodies only in HIV-1wt-infected culture implies that the effect of Vpr on neuronal death is in part mediated through released proinflammatory factors.Conclusion: Collectively, these results demonstrate the ability of HIV-1{increment}Vpr to restrict neuronal apoptosis through dysregulation of multiple proinflammatory cytokines in the infected target cells either directly or indirectly by suppressing viral replication. © 2012 Guha et al.; licensee BioMed Central Ltd

Human immunodeficiency virus (HIV) type 1 Vpr induces differential regulation of T cell costimulatory molecules: Direct effect of Vpr on T cell activation and immune function

AbstractHuman immunodeficiency virus type 1 (HIV-1) viral proteins disrupt the normal host cellular immune pathways thus exploiting the cellular machinery for replication, survival and to escape host immune attack. Here we evaluated the direct effects of HIV-1 Vpr-mediated immune modulation of infected T cells. Vpr specifically downregulated the expression of CD28 and increased the expression of CTLA-4, whereas no significant difference in the expression of CD25 and HLA-DR was observed. Interferon gamma (IFN-γ) production in T cells was evaluated as a measure of the downstream effector functions. Results indicate that Vpr significantly inhibited IFN-γ production and this may, in part, due to Vpr's ability to inhibit the nuclear translocation of NF-κB, and its transcriptional regulation. Together these results support that HIV-1 Vpr selectively dysregulates the immune functions at multiple levels and exerts its inhibitory effects in the presence of other viral proteins

Human immunodeficiency virus (HIV-1) infection selectively downregulates PD-1 expression in infected cells and protects the cells from early apoptosis in vitro and in vivo

AbstractProgrammed Death-1 (PD-1), a member of T cell costimulatory molecules is expressed in high levels on antigen specific T cells during chronic viral infection, whereas PD-1 expression in the context of HIV-1 infected CD4+ T cells is not known. Here we report that productively infected CD4+ T cells lose PD-1, whereas bystander cells were unaffected. Additionally, p24+/PD-1 negative cells are less susceptible to apoptosis compared to bystander cells in the same infected milieu. Similar results were observed in vivo, as infected T cells isolated from HIV-1+ individuals have significantly low level of PD-1 and the observed loss of PD-1 in vivo is independent of viral load, CD4 count, and/or antiviral treatment. Together these results indicate that productively infected cells are resistant to early apoptosis by downregulating PD-1, whereas PD-1 enhances the susceptibility of effector T cells to apoptosis suggesting a dual role for PD-1 during HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1)-mediated neuroinflammation dysregulates neurogranin and induces synaptodendritic injury

Abstract Background Human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorder (HAND) is a common outcome of a majority of HIV-1-infected subjects and is associated with synaptodendritic damage. Neurogranin (Ng), a postsynaptic protein, and calmodulin (CaM) are two important players of synaptic integrity/functions. The biological role of Ng in the context of HAND is unknown. Methods We compared the expression of Ng in frontal cortex (FC) tissues from control and HIV-1-positive subjects with and without HAND by immunohistochemistry, western blot, and qRT-PCR. The interaction between Ng and CaM was analyzed by co-immunoprecipitation. Ng, microtubule-associated protein 2 (MAP2), CaM, CaM-dependent protein kinase II (CaMKII), CREB, synaptophysin (Syp), and synapsin I (Syn I) expressions were evaluated by western blot using FC tissue lysates and differentiated SH-SY5Y (dSH-SY5Y) cells. Identification of inflammatory factors related to Ng loss was accomplished by exposing dSH-SY5Y cells to HIV-1 and mock-infected monocyte-derived macrophage (MDM) supernatants or HIV-1 NLYU2 pseudotyped with VSV-G-Env. Levels of interleukin (IL)-1β, IL-8, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, MCP-2, and CXCL5 in MDM supernatants were measured by ELISA. Association of IL-1β and IL-8 to Ng expression in context of HIV-1 infection was evaluated in the presence or absence of neutralizing antibodies against these cytokines. Results Expression level of Ng was reduced significantly in FC of HAND-positive (HAND+) patients compared to uninfected individuals. Although no difference was found in CaM expression, interaction between Ng and CaM was reduced in HAND+ patients, which was associated with decreased level of CaMKII, a downstream signaling molecule of CaM pathway. This in turn resulted in reduction of synaptic markers, Syp and Syn I. HIV-1 infection directly had no considerable effect on dysregulation of Ng expression in dSH-SY5Y cells, whereas high amount of pro-inflammatory IL-1β and IL-8 in HIV-1-infected MDM supernatants was associated with significant reduction in Ng expression. Conclusions Synaptic damage in HAND+ patients could be a result of abrogation of Ng through HIV-1-induced inflammation that dysregulates Ng-CaM interaction and downstream signaling cascades associated with synaptodendritic functions. This is the first study evaluating the potential role of Ng in the context of HIV-1 neuropathogenesis