Function of HCV NS5A protein and the mechanism of interferon resistance

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The full-length or truncated NS5A cDNA with different ISDR sequences was cloned into a yeast or mammalian expression vector to form a fusion protein consisting of the GAL4 DNA-binding domain and NS5A protein. Following transfection, the transcriptional activities of these constructs were determined using β-galactosidase (yeast) or chloramphenicol acetyltransferase (mammalian cell) reporter gene expression under the control of GAL4 binding sites. The results suggest that the ISDR has a transcriptional activity and it is enhanced by amino acid mutations, that are also related to decreased viral load and increased interferon sensitivity. The possible association between transcriptional activation and interferon sensitivity or viral replication should be studied further.Seventy-five patients with chronic HCV-2 infection (40 with HCV-2a and 35 with HCV-2b) were treated with interferon-alpha for 6 months with a total dose of 468-860 (mean±SD, 739±159) million units. Pretreatment NS5A sequenc … More es were determined by direct sequencing of RT-PCR products. The results indicate that interferon sensitivity is associated with amino acid variations in the NS5A protein in HCV-2a infection, like HCV-1b.Sequential changes in ISDR quasispecies were compared before and just after interferon therapy (total dose 240-880 million units) in 22 patients with chronic hepatitis caused by HCV-1b. Eighteen patients were non-responders with positive serum HCV-RNA after the cessation of interferon therapy. The other four patients were complete responders with no evidence of serum HCV-RNA for 6 months after therapy. Amino acid sequences of predominant quasispecies were determined by direct sequencing of the HCV genome amplified by polymerase chain reaction (PCR), and compared with that of the prototype HCV-1b. Populational changes of ISDR quasispecies were also evaluated by cloning and sequencing of PCR products. HCV detected in sera after interferon therapy have fewer amino acid substitutions in ISDR than those detected before therapy. Such interferon-resistant HCV are already present before therapy as minor quasispecies in non-responders, and are selected by interferon resulting in therapeutic failure. Less