leupeptins and HIV-Infections

Induction of the detoxifying enzyme heme oxygenase-1 (HO-1) is a critical protective host response to cellular injury associated with inflammation and oxidative stress. We previously found that HO-1 protein expression is reduced in brains of HIV-infected individuals with HIV-associated neurocognitive disorders (HAND) and in HIV-infected macrophages, where this reduction associates with enhanced glutamate release and neurotoxicity. Because HIV-infected macrophages are a small component of the cellular content of the brain, the reduction of macrophage HO-1 expression likely accounts for a small portion of brain HO-1 loss in HIV infection. We therefore investigated the contribution of astrocytes, the major pool of brain HO-1. We identified immunoproteasome-mediated HO-1 degradation in astrocytes as a second possible mechanism of brain HO-1 loss in HIV infection. We demonstrate that prolonged exposure of human fetal astrocytes to interferon-gamma (IFNγ), an HIV-associated CNS immune activator, selectively reduces expression of HO-1 protein without a concomitant reduction in HO-1 RNA, increases expression of immunoproteasome subunits, and decreases expression of constitutive proteasome subunits, consistent with a shift towards increased immunoproteasome activity. In HIV-infected brain HO-1 protein reduction also associates with increased HO-1 RNA expression and increased immunoproteasome expression. Finally, we show that IFNγ treatment of astrocytic cells reduces HO-1 protein half-life in a proteasome-dependent manner. Our data thus suggest unique causal links among HIV infection, IFNγ-mediated immunoproteasome induction, and enhanced HO-1 degradation, which likely contribute to neurocognitive impairment in HAND. Such IFNγ-mediated HO-1 degradation should be further investigated for a role in neurodegeneration in inflammatory brain conditions.. Kovacsics et al. identify immunoproteasome degradation of heme oxygenase-1 (HO-1) in interferon gamma-stimulated astrocytes as a plausible mechanism for the observed loss of HO-1 protein expression in the brains of HIV-infected individuals, which likely contributes to the neurocognitive impairment in HIV-associated neurocognitive disorders.

Topics: Antiviral Agents; Astrocytes; Cells, Cultured; Cohort Studies; Cysteine Proteinase Inhibitors; Female; Fetus; Heme Oxygenase-1; HIV Infections; Humans; Interferon-gamma; Leupeptins; Lipopolysaccharides; Male; NAD(P)H Dehydrogenase (Quinone); Prefrontal Cortex; Proteasome Endopeptidase Complex; RNA; Time Factors

HIV-1 primarily infects activated CD4+ T cells and macrophages. Quiescent CD4+ T cells, however, possess cellular factors that limit HIV-1 infection at different postentry steps of the viral life cycle. Here, we show that the previously reported immune regulator monocyte chemotactic protein-induced protein 1 (MCPIP1) restricts HIV-1 production in CD4+ T cells. While the ectopic expression of MCPIP1 in cell lines abolished the production of HIV-1, silencing of MCPIP1 enhanced HIV-1 production. Subsequent analysis indicated that MCPIP1 imposes its restriction by decreasing the steady levels of viral mRNA species through its RNase domain. Remarkably, common T-cell stimuli induced the rapid degradation of MCPIP1 in both T-cell lines and quiescent human CD4+ T cells. Lastly, blocking the proteosomal degradation of MCPIP1 by MG132 abrogated HIV-1 production in phorbol 12-myristate 13-acetate/ionomycin-stimulated human CD4+ T cells isolated from healthy donors. Overall, MCPIP1 poses a potent barrier against HIV-1 infection at a posttranscriptional stage. Although the observed HIV restriction conferred by MCPIP1 does not seem to be overcome by any viral protein, it is removed during cellular stimulation. These findings provide insights into the mechanisms of cellular activation-mediated HIV-1 production in CD4+ T cells.

Topics: Blotting, Northern; CD4-Positive T-Lymphocytes; HEK293 Cells; HIV Infections; Humans; Immunoblotting; Leupeptins; Lymphocyte Activation; Proteolysis; Real-Time Polymerase Chain Reaction; Ribonucleases; RNA, Small Interfering; Transcription Factors

Autoantibodies with enzymic activities (abzymes) are a distinctive feature of autoimmune diseases. It was interesting whether Abs from patients with viral diseases can hydrolyze viral proteins. Electrophoretically and immunologically homogeneous IgGs were isolated from sera of AIDS patients by chromatography on several affinity sorbents. We present evidence showing that 89.5% IgGs purified from the sera of HIV-infected patients using several affinity resins including Sepharose with immobilized integrase specifically hydrolyze only HIV integrase (IN) but not many other tested proteins. Several rigid criteria have been applied to show that the IN-hydrolyzing activity is an intrinsic property of AIDS IgGs but not from healthy donors. Similar to autoimmune proteolytic abzymes, IN-hydrolyzing IgGs from some patients were inhibited by specific inhibitors of serine and metal-dependent proteases but a significant inhibition of the activity by specific inhibitors of acidic- and thiol-like proteases was observed for the first time. Although HIV infection leads to formation of Abs to many viral and human antigens, no possible biological role for most of them is known. Since anti-IN IgG can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded. In addition, detection of IN-hydrolyzing activity can be useful for diagnostic purposes and for estimation of the immune status in AIDS patients.

Topics: Adolescent; Adult; Antibodies, Catalytic; Chromatography, Affinity; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Female; HIV Infections; HIV Integrase; Humans; Hydrolysis; Leupeptins; Male; Pepstatins; Protease Inhibitors; Sulfones; Young Adult

Although APOBEC3G protein is a potent and innate anti-HIV-1 cellular factor, HIV-1 Vif counteracts the effect of APOBEC3G by promoting its degradation through proteasome-mediated proteolysis. Thus, any means that could prevent APOBEC3G degradation could potentially enhance its anti-viral effect. The UBA2 domain has been identified as an intrinsic stabilization signal that protects protein from proteasomal degradation. In this pilot study, we tested whether APOBEC3G, when it is fused with UBA2, can resist Vif-mediated proteasomal degradation and further inhibit HIV-1 infection.. APOBEC3G-UBA2 fusion protein is indeed more resistant to Vif-mediated degradation than APOBEC3G. The ability of UBA2 domain to stabilize APOBEC3G was diminished when polyubiquitin was over-expressed and the APOBEC3G-UBA2 fusion protein was found to bind less polyubiquitin than APOBEC3G, suggesting that UBA2 stabilizes APOBEC3G by preventing ubiquitin chain elongation and proteasome-mediated proteolysis. Consistently, treatment of cells with a proteasome inhibitor MG132 alleviated protein degradation of APOBEC3G and APOBEC3G-UBA2 fusion proteins. Analysis of the effect of APOBEC3G-UBA2 fusion protein on viral infectivity indicated that infection of virus packaged from HEK293 cells expressing APOBEC3G-UBA2 fusion protein is significantly lower than those packaged from HEK293 cells over-producing APOBEC3G or APOBEC3G-UBA2 mutant fusion proteins.. Fusion of UBA2 to APOBEC3G can make it more difficult to be degraded by proteasome. Thus, UBA2 could potentially be used to antagonize Vif-mediated APOBEC3G degradation by preventing polyubiquitination. The stabilized APOBEC3G-UBA2 fusion protein gives stronger inhibitory effect on viral infectivity than APOBEC3G without UBA2.

Topics: APOBEC-3G Deaminase; Cell Line; Cysteine Proteinase Inhibitors; Cytidine Deaminase; Gene Expression; HIV Infections; HIV-1; Humans; Leupeptins; Polyubiquitin; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Ubiquitin-Activating Enzymes; Ubiquitination; vif Gene Products, Human Immunodeficiency Virus; Virus Replication

Recent clinical trials have shown that the presence of a robust human immunodeficiency virus type 1 (HIV-1)-specific T-cell response may not be sufficient to prevent or control HIV-1 infection. Studies of antigen processing in the context of infectious HIV-1 are therefore warranted. Envelope-specific, major histocompatibility complex class II-restricted murine T-cell hybridomas were tested for responsiveness to splenic antigen-presenting cells exposed to HIV-1-infected GHOST cells. Interleukin-2 assays showed that the presence of a peptide within HIV-1 did not ensure the reactivation of peptide-specific T cells. Further experiments defined the impact of gamma interferon-induced thiol reductase and cysteine proteases on the processing of HIV-1 peptides. The results highlight potential influences of peptide context on T-cell reactivation by HIV-1 and encourage the continued study of antigen processing as support for improved vaccine design.

Topics: Animals; Antigen Presentation; Cysteine Endopeptidases; env Gene Products, Human Immunodeficiency Virus; Erythrocyte Membrane; Histocompatibility Antigens Class II; HIV Infections; HIV-1; Humans; Hybridomas; Interferon-gamma; Interleukin-2; Leupeptins; Lymphocyte Activation; Mice; Oxidoreductases; Oxidoreductases Acting on Sulfur Group Donors; T-Lymphocytes, Helper-Inducer

Prior work has implicated viral protein R (Vpr) in the arrest of human immunodeficiency virus type 1 (HIV-1)-infected cells in the G2 phase of the cell cycle, associated with increased viral replication and host cell apoptosis. We and others have recently shown that virion infectivity factor (Vif ) also plays a role in the G2 arrest of HIV-1-infected cells. Here, we demonstrate that, paradoxically, at early time points postinfection, Vif expression blocks Vpr-mediated G2 arrest, while deletion of Vif from the HIV-1 genome leads to a marked increase in G2 arrest of infected CD4 T-cells. Consistent with this increased G2 arrest, T-cells infected with Vif-deleted HIV-1 express higher levels of Vpr protein than cells infected with wild-type virus. Further, expression of exogenous Vif inhibits the expression of Vpr, associated with a decrease in G2 arrest of both infected and transfected cells. Treatment with the proteasome inhibitor MG132 increases Vpr protein expression and G2 arrest in wild-type, but not Vif-deleted, NL4-3-infected cells, and in cells cotransfected with Vif and Vpr. In addition, Vpr coimmunoprecipitates with Vif in cotransfected cells in the presence of MG132. This suggests that inhibition of Vpr by Vif is mediated at least in part by proteasomal degradation, similar to Vif-induced degradation of APOBEC3G. Together, these data show that Vif mediates the degradation of Vpr and modulates Vpr-induced G2 arrest in HIV-1-infected T-cells.

Topics: Apoptosis; Blotting, Western; CD4-Positive T-Lymphocytes; Cell Proliferation; Cells, Cultured; Cysteine Proteinase Inhibitors; G2 Phase; Gene Expression Regulation, Viral; HIV Infections; HIV-1; Humans; Leupeptins; Transfection; vif Gene Products, Human Immunodeficiency Virus; Virus Replication; vpr Gene Products, Human Immunodeficiency Virus

CD8+ cells from healthy HIV-infected individuals can suppress HIV replication in infected CD4(+) cells without killing the cells. This CD8+ cell noncytotoxic antiviral response (CNAR), observed by coculture of CD8+ cells with infected CD4+ cells, is associated with secretion of a CD8+ cell antiviral factor (CAF). In attempts to identify CAF, we discovered that certain protease inhibitors, particularly leupeptin, can block, by up to 95%, the anti-HIV activity in CD8+ cell culture fluids as well as inhibit CNAR. The effect is dose-dependent and is observed in up to 70% of the CAF and CNAR assays by using fluids and cells from several different subjects. Pretreatment of CD8+ cells with leupeptin reduces CNAR, further supporting an inhibitory effect on a CD8+ cell product. This inhibitory activity of protease inhibitors does not affect cell growth, expression of activation antigens, or viability of either CD8+ cells or the infected CD4+ cells. The results suggest that a part of the CD8+ cell noncytotoxic response involves the activity of a protease or a protein that interacts with protease inhibitors. Proteolysis of a CD8+ cell product(s) may be involved. This observation offers a promising approach for identifying the mechanism of CNARCAF activity.

Topics: Antiviral Agents; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; HIV; HIV Infections; Humans; In Vitro Techniques; Leupeptins; Protease Inhibitors; Virus Replication