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.

Fig 1. HCV serum particles inhibit T cell receptor (TCR) signaling in primary human T lymphocytes.

Healthy donor PBMCs were incubated with serum obtained from HCV positive (HCV+) humans infected with various genotypes and subtypes (GT; 1, 1a, 1b, 2, 2b, and 3) or HCV negative control serum (C1-C4) and IL-2 release (A) and CD69 surface expression (B) were measured following TCR stimulation with anti-CD3/CD28. CD69 MFI represents the average of four HCV negative and six HCV-positive sera samples. TCR-induced IL-2 release by human PBMCs incubated with various doses of pooled HCV positive or HCV negative serum (C). IL-2 release by purified primary human CD3+ T cells incubated with HCV-positive sera from genotypes (GT; 1, 1a, 1b, 2, 2b, and 3) or HCV negative serum (C1-C4) (D). US = unstimulated cells. MFI = mean fluorescent intensity. Data represent the average of three technical replicates and the standard deviation is shown. Each experiment was independently performed with three different donors with similar results. *P< 0.05; **P< 0.01.

Fig 2. HCV serum derived extracellular vesicles (EV) inhibit T cell receptor (TCR) signaling in primary human T lymphocytes.

Healthy donor PBMCs were incubated with pooled serum extracellular vesicles (EV) obtained from HCV-infected patient sera (HCV+ EV; GT 1, 1a, 1b, 2, 2b and 3). TCR induced IL-2 (A) and CD69 surface expression (B) was measured. TCR induced IL-2 release by human T cells incubated with HCV-positive or negative serum for two hours at 37°C or 4°C (C). Analysis of CFSE positive serum EV (D) and uptake of CFSE-positive EV by primary human T cells (E) as determined by flow cytometry. HCV RNA was detected using RT-PCR in EVs from HCV-positive serum, and in human PBMCs incubated with HCV-positive but not HCV RNA negative serum (F). HCV RNA US = unstimulated cells. MFI = mean fluorescent intensity. Data represent the average of three technical replicates, and the standard deviation is shown. Each experiment was independently performed with three different donors with similar results. *P< 0.05; **P< 0.01.

Fig 3. HCV cell culture derived particles (HCVcc) and HCV envelope pseudotyped retrovirus particles (HCVpp) inhibit T cell receptor (TCR) signaling in primary human T cells.

HCVcc produced in Huh7.5 cells inhibited TCR-mediated IL-2 release (A) and CD69 surface expression (B) in human peripheral blood mononuclear cells compared to cells incubated in mock-transfected Huh7.5 cell culture supernatant fluids following TCR stimulation with anti-CD3/CD28. Similarly, HCVpp’s inhibited TCR-mediated IL-2 release (C) and CD69 surface expression (D) compared to cells incubated in retrovirus GAG particles in a dose-related manner. US = unstimulated cells. MFI = mean fluorescent intensity. Data represent the average of three technical replicates, and the standard deviation is shown. Each experiment was independently performed with three different donors with similar results. *P< 0.05; **P< 0.01.

Fig 4. HCV envelope protein E2 inhibits T cell receptor (TCR)-mediated signaling.

Jurkat control cells (JC) or Jurkat cells stably expressing HCV E2 protein were stimulated with anti-CD3 and anti-CD28. Twenty-four hours later, IL-2 release (A) was measured. Phosphorylation and activation of the lymphocyte specific tyrosine kinase (Lck Y394; B), the zeta-chain-associated protein kinase (ZAP)-70 (Y319; C) and linker for activation of T cells (LAT, Y226; D) was analyzed in HCV E2 expressing Jurkat cells compared to the controls (JC) following TCR activation using anti-CD3. IL-2 released in truncated or substitution mutant HCV E2 proteins expressing Jurkat cells are shown in panel E. The amino acid numbers relate to their location on the HCV polyprotein. Phospho-blots for Lck, ZAP-70 and LAT were performed at least three times with consistent results. Data represent the average of three technical replicates and the standard deviation is shown. Each experiment was independently performed three times with consistent results. *P< 0.01, ns = not significant.

Fig 5. HCV envelope (E2) coding RNA is sufficient to inhibit proximal T cell receptor (TCR) signaling.

Jurkat cells were generated that stably expressed HCV envelope (E2) RNA (coding aa 384–703) with a frame-shift mutation to abolish protein expression from isolates belonging to genotype (GT) 2a and GT3, or the GT 2a sequence in which four cytidine residues were changed to alanine residues. TCR induced IL-2 release from these Jurkat cells were measured after 24 hour stimulation with anti-CD3/CD28 (A). Activation of lymphocyte specific tyrosine kinase (Lck) was measured by immunoblotting for phosphoY394 following anti-CD3 stimulation (B). Total Lck served as the loading control. The RNA sequence of the HCV E2 (aa 603–619) coding region from the different HCV genotypes (GT) and mutants are shown in panel (C). Conserved sequences are underlined and mutations introduced into the GT 2a sequence noted by * (C). Small RNAs were amplified following 3’linker ligation and specific cDNA synthesis. Small RNAs were cloned and sequenced, and the HCV E2 region encoding (aa 590–621) was detected in Jurkat cells expressing HCV E2 protein. Panel (D) demonstrates the partial sequence of the plasmid (pCR2.1) and HCV E2 RNA amplification product, followed by the oligonucleotide linker sequence. *P< 0.01; ns = not significant.

Fig 6. HCV E2 RNA inhibits protein tyrosine phosphatase receptor type E (PTPRE) expression.

Sequence alignment of two sites within PTPRE 3’ untranslated region (UTR) predicted to bind to HCV E2 RNA (aa 603–619) region (A). Immunoblot analysis of PTPRE protein levels in control, HCV E2 RNA or HCV E2 mutant RNA expressing Jurkat cells, or Huh7 cells expressing full length HCV replicon (FL) or non-structural protein (NS) expressing replicon. The upper band represents full-length PTPRE with transmembrane domain (isoform-1) and lower band represents cytoplasmic PTPRE (isoform-2). GAPDH serves as a loading control (B). GFP expression by HEK 293 cells co-transfected with 1μg of plasmid DNA encoding GFP alone or GFP with PTPRE 3’UTR sequence shown in panel A and 5 μg of plasmid DNA encoding HCV E2 (C) or incubated with HCV-positive serum (D) and GFP expression measured after 72 hours. Data represent the average of three technical replicates and each experiment was repeated at least three times with consistent results. The region of HCV E2 targeting PTPRE was replaced with sequences targeting the cellular chemokine receptor CXCR4 (E), and a Jurkat cell line stably expressing this sequence was generated. CXCR4 was reduced in Jurkat cells expressing this HCV E2 sequence targeting CXCR4, but not Jurkat cells expressing the native HCV E2 RNA sequence (F). *P<0.01.

Fig 7. HCV E2 protein inhibits distal T cell receptor (TCR) signaling.

PMA + Ionomycin (P+I) mediated IL-2 release by Jurkat cells expressing full-length or various truncated or tyrosine 613 mutant HCV E2 protein fragments as indicated (A). Recombinant HCV E2 protein was phosphorylated by Lck in an in vitro kinase reaction, and was dephosphorylated by the CD45 phosphatase (B). HCV E2 protein (native, or Y613A mutant) expressed in Jurkat cells was precipitated before (-) or after (+) TCR stimulation with anti-CD3. E2 and phospho-E2 were detected by immunoblot with E2 specific antibody or anti-phosphotyrosine antibody respectively (C). P+I mediated IL-2 release control Jurkat cells (JC) or HCV E2-expressing Jurkat cells (384–747) which had been incubated in 100μg/ml Lck inhibitor or the vehicle control (DMSO) (D). Data represent the average of three technical replicates and the standard deviation is shown. Each experiment was repeated at least three times with consistent results. *P< 0.01.

Fig 8. HCV E2 protein inhibits NFAT nuclear translocation.

Dephosphorylation (A) and nuclear translocation (B) of the nuclear factor of activated T cells (NFAT) in Jurkat control cells or HCV E2 expressing cells as determined by immunoblot. The nuclear transcription factor Yin Yang 1 (YY1) served as the loading control for nuclear localization. P+I mediated IL-2 was release from primary healthy donor peripheral blood mononuclear cells (PBMCs) incubated with serum obtained from HCV positive (HCV+) humans infected with genotype (GT; 1, 1a, 1b, 2, 2b, and 3) HCV negative (HCV-) human subjects (C1-C4) (C) or cell-culture derived HCV particles (HCVcc) and retroviral particles pseudotyped with HCV envelope (E1-E2; HCVpps) or with HCV envelope containing the Y613F mutation (HCVpp Y613F) (D). US = unstimulated, and S = stimulated (no serum). Data represent the average of three technical replicates and the standard deviation is shown. Each experiment was repeated with three donors with consistent results *P< 0.01.

Fig 9. Proposed model for inhibition of T cell receptor (TCR) signaling during HCV infection.

HCV infection of hepatocytes results in the release of progeny HCV virions and extracellular vesicles containing HCV RNA and/or E2 protein. Viral RNA and/or E2 protein is released into T cells during particle interactions. HCV envelope RNA is processed into small RNA that inhibits protein tyrosine phosphatase E (PTPRE) expression, which results into impaired Lck activation following TCR engagement and defect in proximal TCR signaling. HCV E2 protein competes for Lck-mediated phosphorylation and phosphorylated HCV E2 at Y613 inhibits NFAT nuclear translocation, inhibiting distal TCR signaling. Inhibition of proximal and distal TCR signaling by HCV E2 RNA and protein contributes to impaired T cell function during HCV infection.