Homology model of human interferon-alpha 8 and its receptor complex.

Human interferon-alpha 8 (HuIFN alpha 8), a type I interferon (IFN), is a cytokine belonging to the hematopoietic super-family that includes human growth hormone (HGH). Recent data identified two human type I IFN receptor components. One component (p40) was purified from human urine by its ability to bind to immobilized type I IFN. A second receptor component (IFNAR), consisting of two cytokine receptor-like domains (D200 and D200'), was identified by expression cloning. Murine cells transfected with a gene encoding this protein were able to produce an antiviral response to human IFN alpha 8. Both of these receptor proteins have been identified as members of the immunoglobulin superfamily of which HGH receptor is a member. The cytokine receptor-like structural motifs present in p40 and IFNAR were modeled based on the HGH receptor X-ray structure. Models of the complexes of HuIFN alpha 8 with the receptor subunits were built by superpositioning the conserved C alpha backbone of the HuIFN alpha 8 and receptor subunit models with HGH and its receptor complex. The HuIFN alpha 8 model was constructed from the C alpha coordinates of murine interferon-beta crystal structure. Electrostatic potentials and hydrophobic interactions appear to favor the model of HuIFN alpha 8 interacting with p40 at site 1 and the D200' domain of IFNAR at site 2 because there are regions of complementary electrostatic potential and hydrophobic interactions at both of the proposed binding interfaces. Some of the predicted receptor binding residues within HuIFN alpha 8 correspond to functionally important residues determined previously for human IFN alpha 1, IFN alpha 2, and IFN alpha 4 subtypes by site-directed mutagenesis studies. The models predict regions of interaction between HuIFN alpha 8 and each of the receptor proteins, and provide insights into interactions between other type I IFNs (IFN-alpha subtypes and IFN-beta) and their respective receptor components.     

Modifying effects of interferon and puromycin on cytogenetic damage induced by alkylating drug phopurine in human lymphocytes.

The induction of sister chromatid exchanges (SCEs) by the bifunctional alkylating antineoplastic drug phopurine (2-dimethyl-amino-6-diethyleneiminophosphamido-7-methylpurine) and its modification by human recombinant interferons alpha 2, beta and gamma (HuIFN alpha 2, HuIFN beta and HuIFN gamma) and puromycin (PM) were studied in human lymphocytes. Results demonstrated a striking similarity in the modifying action of HuIFN alpha 2 and PM: 1) both modifiers reduced SCE values induced by phopurine, 2) at high and low doses of phopurine the effect of both modifiers was minimal, and 3) both agents were able to convert DNA lesions from short-term to long-term.     

Characterization of human beta-interferon-binding sites on human cells

Radioiodinated recombinant human beta-interferon (rHuIFN beta Ser), with almost full (greater than 90%) biological activity, was used to study the binding of human beta-interferon to Daudi cells. Specific binding was not observed with less biologically active (less than or equal to 10%) radioiodinated interferon. The bound radioiodinated interferon was shown to compete with human beta-interferon (HuIFN beta), rHuIFN beta Ser, human alpha-interferon (HuIFN alpha) and with human gamma-interferon (HuIFN gamma). Scatchard plot analyses suggest the presence of about 10,000 binding sites for HuIFN beta/Daudi cell. About 6,600 of these sites can be blocked by HuIFN alpha and 3,700 sites can be blocked by HuIFN gamma. The apparent Kd for HuIFN beta is 2.7 nM. The apparent Kd values for HuIFN alpha and HuIFN gamma are 3.7 and 1.1 nM, respectively. It was possible to demonstrate the cross-linking of HuIFN beta to two macromolecular components of Mr = 128,000 and 103,000. We propose the existence of at least two binding sites for HuIFN beta in Daudi cells, one site recognizing both HuIFN beta and HuIFN gamma, the other site recognizing both HuIFN beta and HuIFN alpha. Each site is capable of recognizing only HuIFN gamma or HuIFN alpha.     

Effects of human interferon on cellular response to UV in UV-sensitive human cell strains

Cells of a human RSa cell line, with high sensitivity to UV killing and low capacity for DNA repair, when pretreated with 1-100 units/ml of human interferon (HuIFN) preparations for more than 12 h before irradiation, acquired an enhancement of UV-induced DNA-repair replication synthesis in association with recovery from inhibition of total cellular DNA synthesis and UV survival. Prompt and transient induction of plasminogen activator activities was also found within 5 min after UV irradiation in the cells pretreated with HuIFN but not in the cells non-pretreated with HuIFN. The enhancement and induction effects of HuIFN were observed, irrespective of the kind of HuIFN preparation used (alpha, beta or gamma, and natural or recombinant) and in other UV-sensitive fibroblast cells which were derived from Cockayne syndrome and xeroderma pigmentosum fibroblasts (XP1KY). However, all of the enhancement of DNA-repair synthesis and the induction of plasminogen activator activities by HuIFN was suppressed by treatment with cycloheximide immediately after UV irradiation.     

Chronic recurrent aphthous stomatitis: oral treatment with low-dose interferon alpha

Recombinant human interferon alfa-2a (HuIFN alpha) was administered orally once daily in a low concentration (1,200 IU/day) to nine patients with chronic recurrent aphthous stomatitis (RAS), and a placebo solution was given to 10 control chronic RAS patients in a double-blind study. All HuIFN alpha-treated patients had total remission of their aphthae within a 2-week period, while placebo control patients had no change in their condition. The 10 placebo control patients were then treated with HuIFN alpha in a manner identical to that used for the initial principal group. Within a 2-week period, all original placebo patients had complete remission of their aphthae. Eleven of the patients did not have a recurrence of RAS during a subsequent 6-month observation period. Eight patients had recurring aphthae; however, the lesions were resolved by retreating with oral HuIFN alpha for less than 1 week.     

Differential binding of human interferon-alpha subtypes to receptors on lymphoblastoid cells

Highly purified human interferon-alpha subtype A (HuIFN alpha A) was iodinated for use in direct ligand binding studies on human lymphoblastoid (Daudi) cells. Unlabelled preparations of HuIFN alpha subtypes A, C, D, and hybrid molecules AD (Bgl II), AD (Pvu II), and DA (Bgl II) showed different responses in competition experiments with labelled alpha A probe. Specifically, IFNs alpha D and alpha DA were unable to displace the probe, whereas IFNS alpha A, alpha C, and the hybrid alpha ADs showed similar competition curves. These results support a two-idiotope model of IFN recognition by its receptor. IFN effects on [3H]-thymidine incorporation and cell growth (long term effects) did not reflect the apparent affinities of HuIFN alpha subtypes for cell surface receptor.     

Cyclophilin A and TRIM5alpha independently regulate human immunodeficiency virus type 1 infectivity in human cells

Cyclophilin A (CypA), a cytoplasmic, human immunodeficiency virus type 1 (HIV-1) CA-binding protein, acts after virion membrane fusion with human cells to increase HIV-1 infectivity. HIV-1 CA is similarly greeted by CypA soon after entry into rhesus macaque or African green monkey cells, where, paradoxically, the interaction decreases HIV-1 infectivity by facilitating TRIM5alpha-mediated restriction. These observations conjure a model in which CA recognition by the human TRIM5alpha orthologue is precluded by CypA. Consistent with the model, selection of a human cell line for decreased restriction of the TRIM5alpha-sensitive, N-tropic murine leukemia virus (N-MLV) rendered HIV-1 transduction of these cells independent of CypA. Additionally, HIV-1 virus-like particles (VLPs) saturate N-MLV restriction activity, particularly when the CA-CypA interaction is disrupted. Here the effects of CypA and TRIM5alpha on HIV-1 restriction were examined directly. RNA interference was used to show that endogenous human TRIM5alpha does indeed restrict HIV-1, but the magnitude of this antiviral activity was not altered by disruption of the CA-CypA interaction or by elimination of CypA protein. Conversely, the stimulatory effect of CypA on HIV-1 infectivity was completely independent of human TRIM5alpha. Together with previous reports, these data suggest that CypA protects HIV-1 from an unknown antiviral activity in human cells. Additionally, target cell permissivity increased after loading with heterologous VLPs, consistent with a common saturable target that is epistatic to both TRIM5alpha and the putative CypA-regulated restriction factor.     

The LEM domain proteins emerin and LAP2alpha are dispensable for human immunodeficiency virus type 1 and murine leukemia virus infections

The human nuclear envelope proteins emerin and lamina-associated polypeptide 2alpha (LAP2alpha) have been proposed to aid in the early replication steps of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). However, whether these factors are essential for HIV-1 or MLV infection has been questioned. Prior studies in which conflicting results were obtained were highly dependent on RNA interference-mediated gene silencing. To shed light on these contradictory results, we examined whether HIV-1 or MLV could infect primary cells from mice deficient for emerin, LAP2alpha, or both emerin and LAP2alpha. We observed HIV-1 and MLV infectivity in mouse embryonic fibroblasts (MEFs) from emerin knockout, LAP2alpha knockout, or emerin and LAP2alpha double knockout mice to be comparable in infectivity to wild-type littermate-derived MEFs, indicating that both emerin and LAP2alpha were dispensable for HIV-1 and MLV infection of dividing, primary mouse cells. Because emerin has been suggested to be important for infection of human macrophages by HIV-1, we also examined HIV-1 transduction of macrophages from wild-type mice or knockout mice, but again we did not observe a difference in susceptibility. These findings prompted us to reexamine the role of human emerin in supporting HIV-1 and MLV infection. Notably, both viruses efficiently infected human cells expressing high levels of dominant-negative emerin. We thus conclude that emerin and LAP2alpha are not required for the early replication of HIV-1 and MLV in mouse or human cells.     

HIV-1 infection of human T lymphocytes results in enhanced alpha 5 beta 1 integrin expression.

Altered T cell adherence after human immunodeficiency virus 1 (HIV-1) infection may contribute to viral pathogenesis in the acquired immune deficiency syndrome. To address this hypothesis, we assessed mechanisms of T cell adherence to extracellular matrix proteins in vitro. We found that after HIV-1 infection, both chronically infected H9 CD4+ T cells and acutely infected primary peripheral blood lymphocytes acquired the ability to adhere to the extracellular matrix glycoprotein fibronectin, to a lesser extent to type IV collagen and laminin, but not to type I collagen. H9 cells chronically infected with two of the three HIV-1 strains studied showed approximately a sevenfold increase in attachment to fibronectin, while the same cells infected with the human retrovirus HIV-2 did not. Adhesion was accompanied by changes in morphology, including marked spreading and increased filopodia. These alterations were not blocked by the protein kinase C inhibitor H-7, which did inhibit TPA-induced T cell attachment to fibronectin. Monoclonal antibodies against both the alpha 5 and the beta 1 subunits of the classical fibronectin receptor as well as an Arg-Gly-Asp (RGD) peptide inhibited attachment, whereas anti-alpha 4 monoclonal antibodies and the CS1 peptide did not. Binding to collagen IV was also inhibited by the anti-beta 1 monoclonal antibody, but not the other antibodies. Cells metabolically labeled with [35S]methionine and analyzed by immunoprecipitation with polyclonal anti-beta 1 integrin antibody showed a 2.5-fold increase in integrin synthesis in infected cells compared to uninfected controls. This increase in synthesis was associated with an increase in cell surface expression of both alpha 5 and beta 1 integrins by FACS (registered trademark of Becton Dickinson for a fluorescence-activated cell sorter) analysis. Enhanced expression of integrins such as alpha 5 beta 1 may cause T cell adherence to a variety of tissues, where released viral gene products may induce some of the tissue-specific manifestations of HIV-1 infection.     

The effect of Trim5 polymorphisms on the clinical course of HIV-1 infection

The antiviral factor tripartite interaction motif 5alpha (Trim5alpha) restricts a broad range of retroviruses in a species-specific manner. Although human Trim5alpha is unable to block HIV-1 infection in human cells, a modest inhibition of HIV-1 replication has been reported. Recently two polymorphisms in the Trim5 gene (H43Y and R136Q) were shown to affect the antiviral activity of Trim5alpha in vitro. In this study, participants of the Amsterdam Cohort studies were screened for polymorphisms at amino acid residue 43 and 136 of the Trim5 gene, and the potential effects of these polymorphisms on the clinical course of HIV-1 infection were analyzed. In agreement with the reported decreased antiviral activity of Trim5alpha that contains a Y at amino acid residue 43 in vitro, an accelerated disease progression was observed for individuals who were homozygous for the 43Y genotype as compared to individuals who were heterozygous or homozygous for the 43H genotype. A protective effect of the 136Q genotype was observed but only after the emergence of CXCR4-using (X4) HIV-1 variants and when a viral load of 10(4.5) copies per ml plasma was used as an endpoint in survival analysis. Interestingly, naive CD4 T cells, which are selectively targeted by X4 HIV-1, revealed a significantly higher expression of Trim5alpha than memory CD4 T cells. In addition, we observed that the 136Q allele in combination with the -2GG genotype in the 5'UTR was associated with an accelerated disease progression. Thus, polymorphisms in the Trim5 gene may influence the clinical course of HIV-1 infection also underscoring the antiviral effect of Trim5alpha on HIV-1 in vivo.     

Cyclophilin A renders human immunodeficiency virus type 1 sensitive to Old World monkey but not human TRIM5 alpha antiviral activity

TRIM5alpha is an important mediator of antiretroviral innate immunity influencing species-specific retroviral replication. Here we investigate the role of the peptidyl prolyl isomerase enzyme cyclophilin A in TRIM5alpha antiviral activity. Cyclophilin A is recruited into nascent human immunodeficiency virus type 1 (HIV-1) virions as well as incoming HIV-1 capsids, where it isomerizes an exposed proline residue. Here we show that cyclophilin A renders HIV-1 sensitive to restriction by TRIM5alpha in cells from Old World monkeys, African green monkey and rhesus macaque. Inhibition of cyclophilin A activity with cyclosporine A, or reducing cyclophilin A expression with small interfering RNA, rescues TRIM5alpha-restricted HIV-1 infectivity. The effect of cyclosporine A on HIV-1 infectivity is dependent on TRIM5alpha expression, and expression of simian TRIM5alpha in permissive feline cells renders them able to restrict HIV-1 in a cyclosporine A-sensitive way. We use an HIV-1 cyclophilin A binding mutant (CA G89V) to show that cyclophilin A has different roles in restriction by Old World monkey TRIM5alpha and owl monkey TRIM-Cyp. TRIM-Cyp, but not TRIM5alpha, recruits its tripartite motif to HIV-1 capsid via cyclophilin A and, therefore, HIV-1 G89V is insensitive to TRIM-Cyp but sensitive to TRIM5alpha. We propose that cyclophilin A isomerization of a proline residue in the TRIM5alpha sensitivity determinant of the HIV-1 capsid sensitizes it to restriction by Old World monkey TRIM5alpha. In humans, where HIV-1 has adapted to bypass TRIM5alpha activity, the effects of cyclosporine A are independent of TRIM5alpha. We speculate that cyclophilin A alters HIV-1 sensitivity to a TRIM5alpha-independent innate immune pathway in human cells.     

Induction of protective immune responses against R5 human immunodeficiency virus type 1 (HIV-1) infection in hu-PBL-SCID mice by intrasplenic immunization with HIV-1-pulsed dendritic cells: possible involvement of a novel factor of human CD4(+) T-cell origin

The potential of a dendritic cell (DC)-based vaccine against human immunodeficiency virus type 1 (HIV-1) infection in humans was explored with SCID mice reconstituted with human peripheral blood mononuclear cells (PBMC). HIV-1-negative normal human PBMC were transplanted directly into the spleens of SCID mice (hu-PBL-SCID-spl mice) together with autologous mature DCs pulsed with either inactivated HIV-1 (strain R5 or X4) or ovalbumin (OVA), followed by a booster injection 5 days later with autologous DCs pulsed with the same respective antigens. Five days later, these mice were challenged intraperitoneally with R5 HIV-1(JR-CSF). Analysis of infection at 7 days postinfection showed that the DC-HIV-1-immunized hu-PBL-SCID-spl mice, irrespective of the HIV-1 isolate used for immunization, were protected against HIV-1 infection. In contrast, none of the DC-OVA-immunized mice were protected. Sera from the DC-HIV-1- but not the DC-OVA-immunized mice inhibited the in vitro infection of activated PBMC and macrophages with R5, but not X4, HIV-1. Upon restimulation with HIV-1 in vitro, the human CD4(+) T cells derived from the DC-HIV-1-immunized mice produced a similar R5 HIV-1 suppressor factor. Neutralizing antibodies against human RANTES, MIP-1alpha, MIP-1beta, alpha interferon (IFN-alpha), IFN-beta, IFN-gamma, interleukin-4 (IL-4), IL-10, IL-13, IL-16, MCP-1, MCP-3, tumor necrosis factor alpha (TNF-alpha), or TNF-beta failed to reverse the HIV-1-suppressive activity. These results show that inactivated HIV-1-pulsed autologous DCs can stimulate splenic resident human CD4(+) T cells in hu-PBL-SCID-spl mice to produce a yet-to-be-defined, novel soluble factor(s) with protective properties against R5 HIV-1 infection.     

Nitric oxide regulates MIP-1alpha expression in primary macrophages and T lymphocytes: implications for anti-HIV-1 response

BACKGROUND: Chemokines and chemokine receptors have been shown to play a critical role in HIV infection. Chemokine receptors have been identified as coreceptors for viral entry into susceptible target cells, and several members of the beta chemokine subfamily of cytokines, MIP-1alpha, MIP-1beta, and RANTES, have been identified as the major human immunodeficiency virus (HIV)-suppressive factors produced by activated CD8+ T lymphocytes. In macrophages, HIV-1 infection itself was shown to upregulate the production of MIP-1alpha and MIP-1beta. In the present study, we address the mechanisms by which HIV-1 infection regulates beta chemokine responses in macrophages and lymphocytes. MATERIAL AND METHODS: To address whether nitric oxide (NO), generated as a consequence of HIV-1 infection, regulates beta chemokine responses in monocyte/macrophages and/or macrophage-depleted peripheral blood mononuclear cells (PBMCs) these two cell populations were isolated from HIV seronegative donors, placed in culture, and infected with HIV-1 in either the presence or absence of exogenous activators (e.g. lipopolysaccharide, phytohemagglutinin), inhibitors of nitric oxide synthase (NOS), or chemical donors of NO. Cultures were analyzed for beta chemokine responses by ELISA and RNase protection. RESULTS: LPS-induced MIP-1alpha release is enhanced in HIV-1-infected, as compared to uninfected, monocyte/macrophage cultures, and this enhancing effect is partially blocked by the addition of inhibitors of NOS, and can be reproduced by chemical generators of NO even in the absence of HIV-1 infection. A similar strategy was used to demonstrate a role for NO in HIV-1-mediated induction of MIP-1alpha in unstimulated macrophage cultures. NOS inhibitors also decreased MIP-1alpha and MIP-1beta production by phytohemagglutinin-stimulated monocyte-depleted PBMC cultures. CONCLUSIONS: These results indicate that NO amplifies MIP-1alpha responses in activated macrophages and lymphocytes, and suggests that this pleiotropic molecule might function as an enhancing signal that regulates secretion of beta chemokines during HIV-1 infection. These findings reveal a novel mechanism by which NO might regulate the anti-HIV activity of immune cells.     

Genetic association of the antiviral restriction factor TRIM5alpha with human immunodeficiency virus type 1 infection

The innate antiviral factor TRIM5alpha restricts the replication of some retroviruses through its interaction with the viral capsid protein, leading to abortive infection. While overexpression of human TRIM5alpha results in modest restriction of human immunodeficiency virus type 1 (HIV-1), this inhibition is insufficient to block productive infection of human cells. We hypothesized that polymorphisms within TRIM5 may result in increased restriction of HIV-1 infection. We sequenced the TRIM5 gene (excluding exon 5) and the 4.8-kb 5' putative regulatory region in genomic DNA from 110 HIV-1-infected subjects and 96 exposed seronegative persons, along with targeted gene sequencing in a further 30 HIV-1-infected individuals. Forty-eight single nucleotide polymorphisms (SNPs), including 20 with allele frequencies of >1.0%, were identified. Among these were two synonymous and eight nonsynonymous coding polymorphisms. We observed no association between TRIM5 polymorphism in HIV-1-infected subjects and their set-point viral load after acute infection, although one TRIM5 haplotype was weakly associated with more rapid CD4(+) T-cell loss. Importantly, a TRIM5 haplotype containing the nonsynonymous SNP R136Q showed increased frequency among HIV-1-infected subjects relative to exposed seronegative persons, with an odds ratio of 5.49 (95% confidence interval = 1.83 to 16.45; P = 0.002). Nonetheless, we observed no effect of individual TRIM5alpha nonsynonymous mutations on the in vitro HIV-1 susceptibility of CD4(+) T cells. Therefore, any effect of TRIM5alpha polymorphism on HIV-1 infection in primary lymphocytes may depend on combinations of SNPs or on DNA sequences in linkage disequilibrium with the TRIM5alpha coding sequence.     

Structural and functional characterization of human CXCR4 as a chemokine receptor and HIV-1 co-receptor by mutagenesis and molecular modeling studies

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1alpha) and a co-receptor for the entry of specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1alpha or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-receptor. An approach combining protein structural modeling and site-directed mutagenesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1alpha and vMIP-II and HIV-1 envelope protein gp120. Hypothetical three-dimensional structures were proposed by molecular modeling studies of the CXCR4.SDF-1alpha complex, which rationalize extensive biological information on the role of CXCR4 in its interactions with HIV-1 envelope protein gp120. With site-directed mutagenesis, we have identified that the amino acid residues Asp (D20A) and Tyr (Y21A) in the N-terminal domain and the residue Glu (E268A) in extracellular loop 3 (ECL3) are involved in ligand binding, whereas the mutation Y190A in extracellular loop 2 (ECL2) impairs the signaling mediated by SDF-1alpha. As an HIV-1 co-receptor, we found that the N-terminal domain, ECL2, and ECL3 of CXCR4 are involved in HIV-1 entry. These structural and mutational studies provide valuable information regarding the structural basis for CXCR4 activity in chemokine binding and HIV-1 viral entry, and could guide the design of novel targeted inhibitors.     

HIV-1-infected macrophages induce astrogliosis by SDF-1alpha and matrix metalloproteinases

Brain macrophages/microglia and astrocytes are known to be involved in the pathogenesis of HIV-1-associated dementia (HAD). To clarify their interaction and contribution to the pathogenesis, HIV-1-infected or uninfected macrophages were used as a model of brain macrophages/microglia, and their effects on human astrocytes in vitro were examined. The culture supernatants of HIV-1-infected or uninfected macrophages induced significant astrocyte proliferation, which was annihilated with a neutralizing antibody to stromal cell-derived factor (SDF)-1alpha or a matrix metalloproteinase (MMP) inhibitor. In these astrocytes, CXCR4, MMP, and tissue inhibitors of matrix metalloproteinase mRNA expression and SDF-1alpha production were significantly up-regulated. The supernatants of infected macrophages were always more effective than those of uninfected cells. Moreover, the enhanced production of SDF-1alpha was suppressed by the MMP inhibitor. These results indicate that the activated and HIV-1-infected macrophages can indirectly induce astrocyte proliferation through up-regulating SDF-1alpha and MMP production, which implies a mechanism of astrogliosis in HAD.     

Removal of arginine 332 allows human TRIM5alpha to bind human immunodeficiency virus capsids and to restrict infection

Human TRIM5alpha (TRIM5alpha(hu)) only modestly inhibits human immunodeficiency virus type 1 (HIV-1) and does not inhibit simian immunodeficiency virus (SIV(mac)). Alteration of arginine 332 in the TRIM5alpha(hu) B30.2 domain to proline, the residue found in rhesus monkey TRIM5alpha, has been shown to create a potent restricting factor for both HIV-1 and SIV(mac.) Here we demonstrate that the potentiation of HIV-1 inhibition results from the removal of a positively charged residue at position 332 of TRIM5alpha(hu.) The increase in restricting activity correlated with an increase in the ability of TRIM5alpha(hu) mutants lacking arginine 332 to bind HIV-1 capsid complexes. A change in the cyclophilin A-binding loop of the HIV-1 capsid decreased TRIM5alpha(hu) R332P binding and allowed escape from restriction. The ability of TRIM5alpha(hu) to restrict SIV(mac) could be disrupted by the presence of any charged residue at position 332. Thus, charged residues in the v1 region of the TRIM5alpha(hu) B30.2 domain can modulate capsid binding and restriction potency. Therapeutic strategies designed to neutralize arginine 332 of TRIM5alpha(hu) might potentiate the innate resistance of human cells to HIV-1 infection.     

GCN2 Has Inhibitory Effect on Human Immunodeficiency Virus-1 Protein Synthesis and Is Cleaved upon Viral Infection

The reversible phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 (eIF2alpha) is a well-characterized mechanism of translational control in response to a wide variety of cellular stresses, including viral infection. Beside PKR, the eIF2alpha kinase GCN2 participates in the cellular response against viral infection by RNA viruses with central nervous system tropism. PKR has also been involved in the antiviral response against HIV-1, although this antiviral effect is very limited due to the distinct mechanisms evolved by the virus to counteract PKR action. Here we report that infection of human cells with HIV-1 conveys the proteolytic cleavage of GCN2 and that purified HIV-1 and HIV-2 proteases produce direct proteolysis of GCN2 in vitro, abrogating the activation of GCN2 by HIV-1 RNA. Transfection of distinct cell lines with a plasmid encoding an HIV-1 cDNA clone competent for a single round of replication resulted in the activation of GCN2 and the subsequent eIF2alpha phosphorylation. Moreover, transfection of GCN2 knockout cells or cells with low levels of phosphorylated eIF2alpha with the same HIV-1 cDNA clone resulted in a marked increase of HIV-1 protein synthesis. Also, the over-expression of GCN2 in cells led to a diminished viral protein synthesis. These findings suggest that viral RNA produced during HIV-1 infection activates GCN2 leading to inhibition of viral RNA translation, and that HIV-1 protease cleaves GCN2 to overcome its antiviral effect.