James H. McLinden

An 85-aa segment of the GB virus type C NS5A phosphoprotein inhibits HIV-1 replication in CD4+ Jurkat T cells

GB virus type C (GBV-C) is an apparently nonpathogenic virus that replicates in T and B lymphocytes and is a common cause of persistent human infection. Among HIV-1-infected individuals, persistent coinfection with GBV-C is associated with prolonged survival, and infection of blood mononuclear cells or CD4+ T cells with GBV-C and HIV in vitro results in significantly reduced HIV-1 replication. To date, the viral protein(s) that lead to HIV inhibition have not been identified. The GBV-C nonstructural phosphoprotein (NS5A) is predicted to have pleotropic effects on cells, including interactions with the IFN-induced dsRNA-activated protein kinase (PKR). We studied GBV-C NS5A to determine whether it is involved in inhibition of HIV replication. GBV-C NS5A protein from an isolate that was cleared by IFN therapy did not inhibit PKR, whereas NS5A from an isolate that was not cleared by IFN-inhibited PKR function in a yeast genetic system. Both of these GBV-C NS5A proteins were expressed in a CD4+ T cell line (Jurkat), and both induced a potent, dose-dependent inhibition of HIV-1 replication, thus the effect was independent of PKR inhibition. NS5A induced the release of the chemokine SDF-1 and decreased surface expression of the HIV coreceptor CXCR4, potentially explaining the HIV inhibition. Deletion mapping of the NS5A protein found that an 85-aa region between amino acids 152 and 237 inhibits HIV-1 replication. Thus, GBV-C NS5A protein alters the cellular milieu necessary for HIV-1 replication and may provide a previously undescribed therapeutic approach for anti-HIV therapy.

Viruses within the Flaviviridae Decrease CD4 Expression and Inhibit HIV Replication in Human CD4+ Cells

Viral infections alter host cell homeostasis and this may lead to immune evasion and/or interfere with the replication of other microbes in coinfected hosts. Two flaviviruses are associated with a reduction in HIV replication or improved survival in HIV-infected people (dengue virus (DV) and GB virus type C (GBV-C)). GBV-C infection and expression of the GBV-C nonstructural protein 5A (NS5A) and the DV NS5 protein in CD4+ T cells inhibit HIV replication in vitro. To determine whether the inhibitory effect on HIV replication is conserved among other flaviviruses and to characterize mechanism(s) of HIV inhibition, the NS5 proteins of GBV-C, DV, hepatitis C virus, West Nile virus, and yellow fever virus (YFV; vaccine strain 17D) were expressed in CD4+ T cells. All NS5 proteins inhibited HIV replication. This correlated with decreased steady-state CD4 mRNA levels and reduced cell surface CD4 protein expression. Infection of CD4+ T cells and macrophages with YFV (17D vaccine strain) also inhibited HIV replication and decreased CD4 gene expression. In contrast, mumps virus was not inhibited by the expression of flavivirus NS5 protein or by YFV infection, and mumps infection did not alter CD4 mRNA or protein levels. In summary, CD4 gene expression is decreased by all human flavivirus NS5 proteins studied. CD4 regulation by flaviviruses may interfere with innate and adaptive immunity and contribute to in vitro HIV replication inhibition. Characterization of the mechanisms by which flaviviruses regulate CD4 expression may lead to novel therapeutic strategies for HIV and immunological diseases.

GB Virus Type C Envelope Protein E2 Elicits Antibodies That React with a Cellular Antigen on HIV-1 Particles and Neutralize Diverse HIV-1 Isolates

Broadly neutralizing Abs to HIV-1 are well described; however, identification of Ags that elicit these Abs has proven difficult. Persistent infection with GB virus type C (GBV-C) is associated with prolonged survival in HIV-1–infected individuals, and among those without HIV-1 viremia, the presence of Ab to GBV-C glycoprotein E2 is also associated with survival. GBV-C E2 protein inhibits HIV-1 entry, and an antigenic peptide within E2 interferes with gp41-induced membrane perturbations in vitro, suggesting the possibility of structural mimicry between GBV-C E2 protein and HIV-1 particles. Naturally occurring human and experimentally induced GBV-C E2 Abs were examined for their ability to neutralize infectious HIV-1 particles and HIV-1–enveloped pseudovirus particles. All GBV-C E2 Abs neutralized diverse isolates of HIV-1 with the exception of rabbit anti-peptide Abs raised against a synthetic GBV-C E2 peptide. Rabbit anti–GBV-C E2 Abs neutralized HIV-1–pseudotyped retrovirus particles but not HIV-1–pseudotyped vesicular stomatitis virus particles, and E2 Abs immune-precipitated HIV-1 gag particles containing the vesicular stomatitis virus type G envelope, HIV-1 envelope, GBV-C envelope, or no viral envelope. The Abs did not neutralize or immune-precipitate mumps or yellow fever viruses. Rabbit GBV-C E2 Abs inhibited HIV attachment to cells but did not inhibit entry following attachment. Taken together, these data indicate that the GBV-C E2 protein has a structural motif that elicits Abs that cross-react with a cellular Ag present on retrovirus particles, independent of HIV-1 envelope glycoproteins. The data provide evidence that a heterologous viral protein can induce HIV-1–neutralizing Abs.

Characterization of a Peptide Domain within the GB Virus C NS5A Phosphoprotein that Inhibits HIV Replication

Background GBV-C infection is associated with prolonged survival in HIV-infected people and GBV-C inhibits HIV replication in co-infection models. Expression of the GBV-C nonstructural phosphoprotein 5A (NS5A) decreases surface levels of the HIV co-receptor CXCR4, induces the release of SDF-1 and inhibits HIV replication in Jurkat CD4+ T cell lines. Methodology/Principal Findings Jurkat cell lines stably expressing NS5A protein and peptides were generated and HIV replication in these cell lines assessed. HIV replication was significantly inhibited in all cell lines expressing NS5A amino acids 152–165. Substitution of an either alanine or glycine for the serine at position 158 (S158A or S158G) resulted in a significant decrease in the HIV inhibitory effect. In contrast, substituting a phosphomimetic amino acid (glutamic acid; S158E) inhibited HIV as well as the parent peptide. HIV inhibition was associated with lower levels of surface expression of the HIV co-receptor CXCR4 and increased release of the CXCR4 ligand, SDF-1 compared to control cells. Incubation of CD4+ T cell lines with synthetic peptides containing amino acids 152–167 or the S158E mutant peptide prior to HIV infection resulted in HIV replication inhibition compared to control peptides. Conclusions/Significance Expression of GBV-C NS5A amino acids 152–165 are sufficient to inhibit HIV replication in vitro, and the serine at position 158 appears important for this effect through either phosphorylation or structural changes in this peptide. The addition of synthetic peptides containing 152–167 or the S158E substitution to Jurkat cells resulted in HIV replication inhibition in vitro. These data suggest that GBV-C peptides or a peptide mimetic may offer a novel, cellular-based approach to antiretroviral therapy.

Characterization of an Immunodominant Antigenic Site on GB Virus C Glycoprotein E2 That Is Involved in Cell Binding

ABSTRACT GB virus type C (GBV-C) is a human flavivirus that may cause persistent infection, although most infected individuals clear viremia and develop antibodies to the envelope glycoprotein E2. To study GBV-C E2 antigenicity and cell binding, murine anti-E2 monoclonal antibodies (MAbs) were evaluated to topologically map immunogenic sites on GBV-C E2 and for the ability to detect or block recombinant E2 binding to various cell lines. Five competition groups of MAbs were identified. Groups I and II did not compete with each other. Group III competed with both groups I and II. Group IV did not compete with group I, II, or III. One MAb competed with all of the other MAbs, suggesting that the epitopes bound by these MAbs are intimately related. Individually, none of the MAbs competed extensively with polyclonal human convalescent antibody (PcAb); however, combinations of all five MAb groups completely blocked PcAb binding to E2, suggesting that the epitopes bound by these MAbs form a single, immunodominant antigenic site. Only group I and III MAbs detected purified recombinant E2 bound to cells in binding assays. In contrast, group II MAbs neutralized the binding of E2 to cells. Both PcAb and MAbs were conformation dependent, with the exception of one group II MAb (M6). M6 bound to a five-amino-acid sequence on E2 if the peptide included four C-terminal or eight N-terminal residues, suggesting that the GBV-C E2 protein contains a single immunodominant antigenic site which includes a complex epitope that is involved in specific cellular binding.

GB Virus C Particles Inhibit T Cell Activation via Envelope E2 Protein-Mediated Inhibition of TCR Signaling

Viruses enter into complex interactions within human hosts, leading to facilitation or suppression of each other’s replication. Upon coinfection, GB virus C (GBV-C) suppresses HIV-1 replication in vivo and in vitro, and GBV-C coinfection is associated with prolonged survival in HIV-infected people. GBV-C is a lymphotropic virus capable of persistent infection. GBV-C infection is associated with reduced T cell activation in HIV-infected humans, and immune activation is a critical component of HIV disease pathogenesis. We demonstrate that serum GBV-C particles inhibited activation of primary human T cells. T cell activation inhibition was mediated by the envelope glycoprotein E2, because expression of E2 inhibited TCR-mediated activation of Lck. The region on the E2 protein was characterized and revealed a highly conserved peptide motif sufficient to inhibit TCR-mediated signaling. The E2 region contained a predicted Lck substrate site, and substitution of an alanine or histidine for the tyrosine reversed TCR-signaling inhibition. GBV-C E2 protein and a synthetic peptide representing the inhibitory amino acid sequence were phosphorylated by Lck in vitro. The synthetic peptide also inhibited TCR-mediated activation of primary human CD4+ and CD8+ T cells. Extracellular microvesicles from GBV-C E2–expressing cells contained E2 protein and inhibited TCR signaling in bystander T cells not expressing E2. Thus, GBV-C reduced global T cell activation via competition between its envelope protein E2 and Lck following TCR engagement. This novel inhibitory mechanism of T cell activation may provide new approaches for HIV and immunoactivation therapy.

GB Virus C Envelope Protein E2 Inhibits TCR-Induced IL-2 Production and Alters IL-2–Signaling Pathways

GB virus type C (GBV-C) viremia is associated with reduced CD4+ T cell expansion following IL-2 therapy and with a reduction in T cell activation in HIV-infected individuals. The mechanism(s) by which GBV-C might alter T cell activation or IL-2 signaling have not been studied. In this study, we assess IL-2 release, IL-2R expression, IL-2 signaling, and cell proliferation in tet-off Jurkat cells expressing the GBV-C envelope glycoprotein (E2) following activation through the TCR. TCR activation was induced by incubation in anti-CD3/CD28 Abs. IL-2 release was measured by ELISA, STAT5 phosphorylation was assessed by immunoblot, and IL-2Rα (CD25) expression and cell proliferation were determined by flow cytometry. IL-2 and IL-2Rα steady-state mRNA levels were measured by real-time PCR. GBV-C E2 expression significantly inhibited IL-2 release, CD25 expression, STAT5 phosphorylation, and cellular proliferation in Jurkat cells following activation through the TCR compared with control cell lines. Reducing E2 expression by doxycycline reversed the inhibitory effects observed in the E2-expressing cells. The N-terminal 219 aa of E2 was sufficient to inhibit IL-2 signaling. Addition of purified recombinant GBV-C E2 protein to primary human CD4+ and CD8+ T cells inhibited TCR activation-induced IL-2 release and upregulation of IL-2Rα expression. These data provide evidence that the GBV-C E2 protein may contribute to the block in CD4+ T cell expansion following IL-2 therapy in HIV-infected individuals. Furthermore, the effects of GBV-C on IL-2 and IL-2–signaling pathways may contribute to the reduction in chronic immune activation observed in GBV-C/HIV–coinfected individuals.

Characterization of a peptide domain within the GB virus C envelope glycoprotein (E2) that inhibits HIV replication.

GB virus C (GBV-C) infection is associated with prolonged survival in HIV-infected cohorts, and GBV-C E2 protein inhibits HIV entry when added to CD4+ T cells. To further characterize E2 effects on HIV replication, stably transfected Jurkat cell lines expressing GBV-C E2 or control sequences were infected with HIV and replication was measured. HIV replication (all 6 isolates studied) was inhibited in all cell lines expressing a region of 17 amino acids of GBV-C E2, but not in cell lines expressing E2 without this region. In contrast, mumps and yellow fever virus replication was not inhibited by E2 protein expression. Synthetic GBV-C E2 17mer peptides did not inhibit HIV replication unless they were fused to a tat-protein-transduction-domain (TAT) for cellular uptake. These data identify the region of GBV-C E2 protein involved in HIV inhibition, and suggest that this GBV-C E2 peptide must gain entry into the cell to inhibit HIV.

Expression of the Dengue Virus Type 2 NS5 Protein in a CD4+ T Cell Line Inhibits HIV Replication

Human immunodeficiency virus (HIV) load is suppressed during dengue virus infection. The NS5A phosphoprotein of GB virus C (a related flavivirus) inhibits HIV replication in vitro. To determine whether the dengue virus NS5 protein inhibits HIV replication, CD4(+) T cell lines expressing this protein were generated. HIV replication in dengue virus NS5-expressing cells decreased by >90% compared with control cells (P < .01), and this was mediated in part by decreased HIV coreceptor (CXCR4) expression and increased production of SDF-1. Thus, the dengue virus NS5 protein inhibits HIV replication in vitro, potentially explaining the reduction in HIV load observed during acute dengue virus infection.

GBV-C Infection and Risk of NHL among U.S. Adults

Some retrospective studies suggest an association between infection with GB virus-C (GBV-C) and non-Hodgkin lymphoma (NHL). We evaluated this association prospectively in a nested case-control study within the U.S. Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial. Cases (N = 658) and controls (N = 1,316) were individually matched by age, sex, race/ethnicity, timing of study entry, and sample selection. Prediagnostic PLCO serum samples were tested for GBV-C RNA (as a measure of active infection) and E2 antibody (active or resolved infection). Logistic regression was used to estimate odds ratios (OR) for the association between GBV-C and NHL overall and NHL subtypes. Twelve cases (1.8%) and seven controls (0.5%) were GBV-C RNA-positive. GBV-C RNA positivity was associated with NHL overall [OR, 3.43; 95% confidence interval (CI), 1.35-8.71] and, based on small numbers, diffuse large B-cell lymphoma (OR, 5.31; 95% CI, 1.54-18.36). The association with NHL persisted when the interval between testing and selection was greater than 4 years (OR, 6.00; 95% CI, 1.21-29.73). In contrast, E2 antibody positivity was not associated with NHL risk (OR, 1.08; 95% CI, 0.74-1.58). Our study demonstrates that GBV-C infection precedes development of NHL. GBV-C infection may play an etiologic role in a small proportion of NHL cases, perhaps by causing chronic immune stimulation or impaired immunosurveillance.