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The emergence of drug resistant strains of Mycobacterium tuberculosis (M. tuberculosis) together with reports of co-infections with the human immunodeficiency virus (HIV) has renewed interest to better understand the intricate mechanisms prevalent during co-infections. In this study we report a synergistic effect of M. tuberculosis and HIV-1, and their antigens Rv3416 and Nef, respectively, in inhibiting apoptosis of macrophages. This inhibition involves the TLR2 pathway and second messengers that play complementing and contrasting roles in regulating apoptosis. Interestingly, the route of calcium influx into cells differentially regulates apoptosis during antigenic co-stimulation. While calcium released from intracellular stores was anti-apoptotic, calcium influx from the external milieu was pro-apoptotic. Further, molecular sensors of intracellular calcium release aid in antigen mediated inhibition of apoptosis. A cross-regulation between oxidative burst and differential routing of calcium influx governed apoptosis. Interestingly, the HIV-1 Nef supported anti-apoptotic responses in macrophages whereas Vpu had no significant effect. These results point to a synergistic liaison between M. tuberculosis and HIV-1 in regulating macrophage apoptosis. [ABSTRACT FROM AUTHOR]

The viral protein U (Vpu) encoded by HIV-1 has been shown to assist in the detachment of virion particles from infected cells. Vpu forms cation-specific ion channels in host cells, and has been proposed as a potential drug target. An understanding of the mechanism of ion transport through Vpu is desirable, but remains limited because of the unavailability of an experimental structure of the channel. Using a structure of the pentameric form of Vpu – modeled and validated based on available experimental data – umbrella sampling molecular dynamics simulations (cumulative simulation time of more than 0.4 µs) were employed to elucidate the energetics and the molecular mechanism of ion transport in Vpu. Free energy profiles corresponding to the permeation of Na+ and K+ were found to be similar to each other indicating lack of ion selection, consistent with previous experimental studies. The Ser23 residue is shown to enhance ion transport via two mechanisms: creating a weak binding site, and increasing the effective hydrophilic length of the channel, both of which have previously been hypothesized in experiments. A two-dimensional free energy landscape has been computed to model multiple ion permeation, based on which a mechanism for ion conduction is proposed. It is shown that only one ion can pass through the channel at a time. This, along with a stretch of hydrophobic residues in the transmembrane domain of Vpu, explains the slow kinetics of ion conduction. The results are consistent with previous conductance studies that showed Vpu to be a weakly conducting ion channel. [ABSTRACT FROM AUTHOR]

Background: The surrogate markers of HIV/AIDS progression include CD4 T cell count and plasma viral load. But, their reliability has been questioned in patients on anti-retroviral therapy (ART). Five microRNAs (miRNAs) - miR-16, miR-146b-5p, miR-150, miR-191 and miR-223 in peripheral blood mononuclear cells (PBMCs) were earlier found to assign HIV/AIDS patients into groups with varying CD4 T cell counts and viral loads. In this pilot study, we profiled the expression of these five miRNAs in PBMCs, and two of these miRNAs (miR-146b-5p and miR-150) in the plasma of HIV/AIDS patients, including those on ART and those who developed ART resistance, to evaluate if these are biomarkers of disease progression and therapy. Results: We quantified miRNA levels by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using RNA isolated from PBMCs and plasma of healthy persons or HIV-infected patients who were (1) asymptomatic; (2) symptomatic and ART naïve; (3) on ART; and (4) failing ART. Our results show miR-150 (p<0.01) and to a lesser extent miR-146b-5p (p<0.05) levels in PBMCs to reliably distinguish between ART-naïve AIDS patients, those on ART, and those developing drug resistance and failing ART. The plasma levels of these two miRNAs also varied significantly between patients in these groups and between patients and healthy controls (p values <0.05). Conclusions: We report for the first time that PBMC and plasma levels of miR-150 and miR-146b-5p are predictive of HIV/AIDS disease progression and therapy. [ABSTRACT FROM AUTHOR]

Background and Aims: Patients with liver disease infected with the human immunodeficiency virus (HIV) exhibit accelerated progression of hepatic fibrosis and liver cirrhosis compared to uninfected individuals. We studied the effects of soluble factors secreted by HIV-infected peripheral blood mononuclear cells (PBMCs) on hepatic stellate cells (HSCs), which are central mediators of liver fibrosis. Methods: An in vitro model was used in which a HSC line, LX2, was treated with culture supernatants of human PBMCs infected with macrophage tropic (R5) or T cell tropic (X4) strains of HIV-1. Quantitative reverse transcription PCR (qRT-PCR) and western blotting were used to assess the expression of fibrogenic and proinflammatory markers; LX2 proliferation and intracellular signaling pathways were also studied. A qRT-PCR based miRNome array was used for comparative miRNA profiling of LX2 cells treated with infected PBMC culture supernatants. Results: Pro-fibrogenic, angiogenic and proinflammatory markers, and proliferation of LX2 cells were increased following exposure to culture supernatants from HIV-1 infected PBMCs. The profiling of miRNAs in LX2 cells treated with culture supernatants from HIV-1 R5- or X4-infected PBMCs showed 66 and 22 miRNAs respectively, to be significantly altered compared to mock-treated LX2 cells. While different sets of miRNAs were altered in the two cases, bioinformatics analyses predicted these to be associated with common pathways, including TGF-β signaling and extracellular matrix receptor interaction pathways. Conclusions: HIV infection creates a favorable milieu for the activation of hepatic stellate cells and increased hepatic fibrosis. We identify some regulatory molecules important for these effects. [ABSTRACT FROM AUTHOR]

There is faster progression to fibrosis in persons with liver injury who are also infected with HIV. Other reports have suggested that HIV can directly infect and activate stellate cells, and the viral Tat and gp160 proteins also induce profibrogenic factors from peripheral blood mononuclear cells (PBMCs). We tested the role of HIV-1 Vpu accessory protein in promoting profibrogenic activation of hepatic stellate cells. Human stellate LX2 cells were cocultured with human monocytic U937 cells stably expressing the Vpu protein or latently infected U1 cells knocked down for Vpu expression, LX2 cells were also cultured with the supernatants from these cells. The expression of profibrogenic markers was evaluated in LX2 cells usingquantitative reverse transcription polymerase chain reaction (qRT-PCR),western blotting, immunofluorescence,flow cytometry and ELISA were used to confirm and quantitate protein expression. Monocytic cells expressing Vpu increased the expression of profibrogenic markers in LX2 cells. The culture supernatants of these cells contained increased levels of transforming growth factor beta (TGF-β), which correlated with increased activity of the AP-1 transcription factor. Antibodies against TGF-β or a TGF-β receptor inhibitor (SB431452) reversed Vpu-mediated profibrogenic activation of LX2 cells, suggesting that TGF-β mediated these effects. The cytokine macrophage migration inhibitory factor (MIF) attenuated Vpu-mediated TGF-β secretion and profibrogenic effects on LX2 cells. Besides its other roles in pathogenesis, Vpu is likely to contribute to hepatic fibrosis through this hitherto unknown mechanism. [ABSTRACT FROM AUTHOR]

The HIV-1 accessory protein Nef is an important virulence factor. It associates with cellular membranes and modulates the endocytic machinery and signaling pathways. Nef also increases the proliferation of multivesicular bodies (MVBs), which are sites for virus assembly and budding in macrophages. The RNA interference (RNAi) pathway proteins Ago2 and GW182 localize to MVBs, suggesting these to be sites for assembly and turnover of the miRNA-induced silencing complex (miRISC). While RNAi affects HIV replication, it is not clear if the virus encodes a suppressor activity to overcome this innate host response. Here we show that Nef colocalizes with MVBs and binds Ago2 through two highly conserved Glycine-Tryptophan (GW) motifs, mutations in which abolish Nef binding to Ago2 and reduce virus yield and infectivity. Nef also inhibits the slicing activity of Ago2 and disturbs the sorting of GW182 into exosomes resulting in the suppression of miRNA-induced silencing. Thus, besides its other activities, the HIV-1 Nef protein is also proposed to function as a viral suppressor of RNAi (VSR). [ABSTRACT FROM AUTHOR]

Background: Although HIV causes immune deficiency by infection and depletion of immunocytes, metabolic alterations with clinical manifestations are also reported in HIV/AIDS patients. Here we aimed to profile metabolite changes in the plasma, urine, and saliva of HIV/AIDS patients, including those on anti-retroviral therapy (ART). Methods: Metabolic profiling of biofluids collected from treatment naïve HIV/AIDS patients and those receiving ART was done with solution-state nuclear magnetic resonance (NMR) spectroscopy followed by statistical analysis and annotation. Results: In Principal Component Analysis (PCA) of the NMR spectra, Principal Component 1 (PC1) alone accounted for 99.3%, 87.2% and 78.8% variations in plasma, urine, and saliva, respectively. Partial least squares discriminant analysis (PLS-DA) was applied to generate three-component models, which showed plasma and urine to be better than saliva in discriminating between patients and healthy controls, and between ART-naïve patients and those receiving therapy. Twenty-six metabolites were differentially altered in any or two types of samples. Our results suggest that urinary Neopterin, and plasma Choline and Sarcosine could be used as metabolic biomarkers of HIV/AIDS infection. Pathway analysis revealed significant alternations in 12 metabolic pathways. Conclusions: This study catalogs differentially regulated metabolites in biofluids, which helped classify subjects as healthy controls, HIV/AIDS patients, and those on ART. It also underscores the importance of further studying the consequences of HIV infection on host metabolism and its implications for pathogenesis. [ABSTRACT FROM AUTHOR]

The hepatitis E virus (HEV) is a small RNA virus and the cause of acute viral hepatitis E. The open reading frame 3 protein (pORF3) of HEV appears to be a pleiotropic regulatory protein that helps in the establishment, propagation and progression of viral infection. However, the global cellular effects of this protein remain to be explored. In the absence of traditional in vitro viral infection systems or efficient replicon systems, we made an adenovirus based ORF3 protein expression system to study its effects on host cell gene expression. We infected Huh7 hepatoma cells with recombinant adenoviruses expressing pORF3 and performed microarray-based gene expression analyses. Several genes down regulated in pORF3-expressing cells were found to be under regulation of the liver-enriched hepatocyte nuclear factor 4 (HNF4), which regulates hepatocytespecific gene expression. While HNF4 localizes to the nucleus, its phosphorylation results in impaired nuclear localization of HNF4. Here we report that pORF3 increases HNF4 phosphorylation through the ERK and Akt kinases, which results in impaired nuclear translocation of HNF4 and subsequently the down modulation of HNF4-responsive genes in pORF3- expressing cells. We propose that modulation of several hepatocyte specific genes by pORF3 will create an environment favorable for viral replication and pathogenesis. [ABSTRACT FROM AUTHOR]

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is reported to cause apoptosis of infected cells and several of its proteins including the 3a accessory protein, are pro-apoptotic. Since the 3a protein localizes to the endoplasmic reticulum (ER)-Golgi compartment, its role in causing ER stress was investigated in transiently transfected cells. Cells expressing the 3a proteins showed ER stress based on activation of genes for the ER chaperones GRP78 and GRP94. Since ER stress can cause differential modulation of the unfolded protein response (UPR), which includes the inositol-requiring enzyme 1 (IRE-1), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) pathways, these were individually tested in 3a-expressing cells. Only the PERK pathway was found to be activated in 3a-expressing cells based on (1) increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) and inhibitory effects of a dominant-negative form of eIF2α on GRP78 promoter activity, (2) increased translation of activating transcription factor 4 (ATF4) mRNA, and (3) ATF4- dependent activation of the C/EBP homologous protein (CHOP) gene promoter. Activation of PERK affects innate immunity by suppression of type 1 interferon (IFN) signaling. The 3a protein was found to induce serine phosphorylation within the IFN alpha-receptor subunit 1 (IFNAR1) degradation motif and to increase IFNAR1 ubiquitination. Confocal microscopic analysis showed increased translocation of IFNAR1 into the lysosomal compartment and flow cytometry showed reduced levels of IFNAR1 in 3a-expressing cells. These results provide further mechanistic details of the pro-apoptotic effects of the SARS-CoV 3a protein, and suggest a potential role for it in attenuating interferon responses and innate immunity. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus type I (HIV-1) Vpu protein is 81 residues long and has two cytoplasmic and one transmembrane (TM) helical domains. The TM domain oligomerizes to form a monovalent cation selective ion channel and facilitates viral release from host cells. Exactly how many TM domains oligomerize to form the pore is still not understood, with experimental studies indicating the existence of a variety of oligomerization states. In this study, molecular dynamics (MD) simulations were performed to investigate the propensity of the Vpu TM domain to exist in tetrameric, pentameric, and hexameric forms. Starting with an idealized α-helical representation of the TM domain, a thorough search for the possible orientations of the monomer units within each oligomeric form was carried out using replica-exchange MD simulations in an implicit membrane environment. Extensive simulations in a fully hydrated lipid bilayer environment on representative structures obtained from the above approach showed the pentamer to be the most stable oligomeric state, with interhelical van der Waals interactions being critical for stability of the pentamer. Atomic details of the factors responsible for stable pentamer structures are presented. The structural features of the pentamer models are consistent with existing experimental information on the ion channel activity, existence of a kink around the Ile17, and the location of tetherin binding residues. Ser23 is proposed to play an important role in ion channel activity of Vpu and possibly in virus propagation. [ABSTRACT FROM AUTHOR]