Conserved Motifs within Hepatitis C Virus Envelope (E2) RNA and Protein Independently Inhibit T Cell Activation

Abstract

T cell receptor (TCR) signaling is required for T-cell activation, proliferation, differentiation, and effector function. Hepatitis C virus (HCV) infection is associated with impaired T-cell function leading to persistent viremia, delayed and inconsistent antibody responses, and mild immune dysfunction. Although multiple factors appear to contribute to T-cell dysfunction, a role for HCV particles in this process has not been identified. Here, we show that incubation of primary human CD4+ and CD8+ T-cells with HCV RNA-containing serum, HCV-RNA containing extracellular vesicles (EVs), cell culture derived HCV particles (HCVcc) and HCV envelope pseudotyped retrovirus particles (HCVpp) inhibited TCR-mediated signaling. Since HCVpp’s contain only E1 and E2, we examined the effect of HCV E2 on TCR signaling pathways. HCV E2 expression recapitulated HCV particle-induced TCR inhibition. A highly conserved, 51 nucleotide (nt) RNA sequence was sufficient to inhibit TCR signaling. Cells expressing the HCV E2 coding RNA contained a short, virus-derived RNA predicted to be a Dicer substrate, which targeted a phosphatase involved in Src-kinase signaling (PTPRE). T-cells and hepatocytes containing HCV E2 RNA had reduced PTPRE protein levels. Mutation of 6 nts abolished the predicted Dicer interactions and restored PTPRE expression and proximal TCR signaling. HCV RNA did not inhibit distal TCR signaling induced by PMA and Ionomycin; however, HCV E2 protein inhibited distal TCR signaling. This inhibition required lymphocyte-specific tyrosine kinase (Lck). Lck phosphorylated HCV E2 at a conserved tyrosine (Y613), and phospho-E2 inhibited nuclear translocation of NFAT. Mutation of Y613 restored distal TCR signaling, even in the context of HCVpps. Thus, HCV particles delivered viral RNA and E2 protein to T-cells, and these inhibited proximal and distal TCR signaling respectively. These effects of HCV particles likely aid in establishing infection and contribute to viral persistence.

Extracellular vesicles and viruses: Are they close relatives?

“…In vivo, EVs can interact with viruses and with each other either directly or via modulation of host responses, thus participating in a "War and Peace" between viruses and host (49,50). Some viruses induce the infected cells to release modified EVs that facilitate infection by increasing the pool of susceptible target cells (e.g., by increasing the number of activated cells) or their susceptibility to viral infection or by serving as decoys that absorb antiviral antibodies, thereby compromising antiviral immunity.…”

Hepatitis C virus infection inhibits a Src-kinase regulatory phosphatase and reduces T cell activation in vivo

“…These plasmids and the parent GFP expression plasmid were used to generate human embryonic kidney (HEK) 293 cell lines stably expressing GFP as previously described [11]. Each cell line was transfected with a synthetic, genome-length, HCV RNA transcript or the transfection reagent.…”

“…Fire and Parameswaran)[11]. Since PBMC PTPRE levels varied somewhat among HCV-infected individuals (as in Fig 7B), we sequenced the HCV E2 RNA present in serum obtained from ten subjects to determine if there is a relationship between PTPRE expression and HCV sequence diversity (Fig 8A, underlined) [31].…”

“…Expression of HCV vsRNA is sufficient to reduce PTPRE protein levels in Jurkat cells, and mutation of conserved residues in the HCV E2 short RNA region restore PTPRE levels and TCR-mediated Lck activation [11]. PTPRE specificity was confirmed by placing the PTPRE 3’UTR sequences after GFP, and showing that HCV E2 expression regulated GFP expression in this system.…”

Graft Versus Host Disease After Liver Transplantation in Adults

“…26 It is possible that the immune dysregulation in the recipient that originally led to the development of HCC, or alterations in the immune system caused by HCC, may predispose to alloreactivity and development of GVHD after LT. 27, 28 HCV is known to inhibit T cell receptor-mediated signaling required for activation and effector functions of T cells. 29 Whether HCV demonstrates the same effect on donor T-lymphocytes, thereby decreasing the incidence of GVHD, is unclear.…”