Prospective characterization of full-length hepatitis C virus NS5A quasispecies during induction and combination antiviral therapy

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been contro versially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies, In this study, the relationship betw een NS5A mutations and selection pressures before and during antiviral ther apy and virologic response to therapy were investigated. Full-length NS5A c lones were sequenced from 20 HCV genotype 1-infected patients in a prospect ive, randomized clinical trial of IFN induction (daily) therapy and IFN plu s ribavirin combination therapy. Pretreatment NS5A nucleotide and amino aci d phylogenies did not correlate with clinical IFN responses and domains inv olved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR237-276) seq uence associated with IFN resistance was not found, although the presence o f Ala(245) within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P < 0.01), There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatm ent isolates from responders versus nonresponders in both HCTr-1a- and HCV- 1b-infected patients (P < 0.05), In HCV-1a patients, more amino acid change s were observed in isolates from IPN-sensitive patients (P < 0.001), and th e mutations appeared to be concentrated in two variable regions in the C te rminus of NS5A that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasi species was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not ch ange in either patient response group. These data suggest that host-mediate d selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences ou tside of the putative ISDR, Mechanistic studies are needed to address the r ole of the C terminus of NS5A in HCV replication and antiviral resistance.