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.