Expression of hepatitis C virus proteins interferes with the antiviral action of interferon independently of PKR-mediated control of protein synthesis

Hepatitis C virus (HCV) of genotype 1 is the most resistant to interferon ( IFN) therapy. Were, we have analyzed the response to IFN of the human cell line UHCV-11 engineered to inducibly express the entire HCV genotype la pol yprotein. IFN-treated, induced UHCV cells were found to better support the growth of encephalomyocarditis virus (EMCV) than IFN-treated, uninduced cel ls. This showed that expression of the HCV proteins allowed the development of a partial resistance to the antiviral action of IFN. The nonstructural 5A (NS5A) protein of HCV has been reported to inhibit PKR, an HFN-induced k inase involved in the antiviral action of IFN, at the level of control of p rotein synthesis through the phosphorylation of the initiation factor eIF2 alpha (M, Gale, Jr., C. M. Blakely, B, Kwieciszewski, S. L. Tan, M. Dossett , N. M. Tang, M. J. Korth, S. J. Polyak, D. R Gretch, and M. G. Katze, Mel. Cell. Biol, 18:5208-5218, 1998), Accordingly, cell lines inducibly express ing NS5A were found to rescue EMCV growth (S. J. Polyak, D. M. Paschal, S. McArdle, M. J. Gale, Jr., D. Moradpour, and D. R. Gretch, Hepatology 29:126 2-1271, 1999). In the present study we analyzed whether the resistance of U HCV-11 cells to IFN could also be attributed to inhibition of PKR, Confocal laser scanning microscopy showed no colocalization of PKR, which is diffus e throughout the cytoplasm, and the induced HCV proteins, which localize ar ound the nucleus within the endoplasmic reticulum. The effect of expression of HCV proteins on PKR activity was assayed in a reporter assay and by dir ect analysis of the in vivo phosphorylation of eIF2 alpha after treatment o f cells with poly(I)-poly(C). We found that neither PKR activity nor eIP2 a lpha phosphorylation was affected by coexpression of the HCV proteins. In c onclusion, expression of HCV proteins in their biological context interfere s with the development of the antiviral action of IFN, Although the possibi lity that some inhibition of PKR (by either NS5A or another viral protein) occurs at a very localized level cannot be excluded, the resistance to IFN, resulting from the expression of the HCV proteins, cannot be explained sol ely by inhibition of the negative control of translation by PKR.