GB virus C interactions with HIV: effects on immunoactivation and mechanisms of immunomodulation

GB virus C (GBV-C) is a lymphotropic human virus which was recently assigned to a new genus Pegivirus within the Flaviviridae family. GBV-C infection is found worldwide, and viremia prevalence is about 1% to 4% in healthy blood donors and up to 42% in HIV-infected individuals. In clinical studies, GBV-C coinfection is associated with prolonged survival of HIV-infected individuals. GBV-C infection modestly alters T cell homeostasis in vivo through various mechanisms, including modulation of chemokine and cytokine release and receptor expression, and by diminution of T cell activation, proliferation and apoptosis, all of which may contribute to improved HIV clinical outcomes. This thesis explores the interrelationship between GBV-C infection and immunoactivation and identifies potential mechanisms by which GBV-C reduces immunoactivation.

Chronic HIV infection is associated with persistent immunoactivation which contributes to the immune dysfunction. In particular, T cell activation supports HIV replication and correlates with HIV viral load (VL). Persistent immunoactivation also contributes to the depletion of uninfected bystander cells by mechanisms of activation induced cell death (AICD). Although treatment with combination antiretroviral therapy (cART) reduces HIV VL, T cell activation does not return to levels found in HIVuninfected individuals. Sustained immunoactivation is also associated with lower virological response to cART suggesting therapies to reduce immunoactivation in combination with cART may benefit HIV-infected individuals. Since GBV-C infection is associated with reduced immunoactivation, understanding mechanisms by which GBV-C modulates these signaling pathways may provide insights into novel approaches to treat HIV infection and chronic immunoactivation.

The effect of GBV-C infection on T cell activation and IL-2 signaling pathways were studied in a cohort of HIV-positive individuals. GBV-C viremic HIV positive individuals on cART have reduced T cell activation which was significantly associated with higher percentage of immunomodulatory CD3 +CD4-CD8-T cells. Ex vivo GBV-C infection was associated with reduced lymphocyte proliferation in response to IL-2, lower frequency of reactivation of latent HIV and protection against AICD. In vitro expression of GBV-C envelope glycoprotein E2 in CD4+ T cell lines inhibited T cell receptor (TCR) induced IL-2 secretion and inhibited IL-2 signaling pathways. This effect was mediated at least in part by reducing activation of lymphocyte specific tyrosine kinase (Lck). Through deletion mutagenesis, the inhibitory motif within the viral protein was mapped to a region that contains a predicted Lck substrate, a highly conserved tyrosine at position 87 (Y87). Lck phosphorylated GBV-C E2 protein in vitro and mutation of Y87 residue abolished the inhibitory effects of E2 protein. Synthetic peptides containing this inhibitory motif competed for Lck phosphorylation and inhibited TCR signaling in primary human T cells. The number of GBV-C infected T cells was found to be low in vivo, yet GBV-C infection reduced global TCR signaling. GBV-C RNA and E2 protein were detected in extracellular microvesicles purified from GBV-C infected human serum or the culture supernatant of E2 expressing cells, and these microvesicles inhibited TCR signaling in uninfected bystander T cells. Together, these data identify a novel mechanism by which GBV-C infection leads to global reduction in T cell activation and IL-2 signaling in the infected host, and provide a working model in which the viral envelope glycoprotein serves as a substrate for Lck and competes for Lck phosphorylation in the infected T cells and in uninfected bystander T cells.