INTERFERON RESISTANCE OF HEPATITIS-C VIRUS GENOTYPE 1B - RELATIONSHIPTO NONSTRUCTURAL 5A GENE QUASI-SPECIES MUTATIONS

A 40-amino-acid sequence located in the nonstructural 5A (NS5A) protei n of hepatitis C virus genotype Ib (HCV-lb) was recently suggested to be the interferon sensitivity-determining region (ISDR), because HCV-l b strains with an ISDR amino acid sequence identical to that of the pr ototype strain HCV-J were found to be resistant to alpha interferon (I FN-alpha) whereas strains vc;with amino acid substitutions were Pound to be sensitive (N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Mu rakami, C. Yamamoto, N. Izumi, F. Marumo, and C. Sato, J. Clin. Invest , 96:223-230, 1995; N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, Y. Ogura, N. Izumi, F. Marumo, and C. Sate, N. Engl. J. Med. 334:77-81, 1996). We used single-strand conformation po lymorphism (SSCP) analysis, combined with cloning and sequencing strat egies, to characterize NS5A quasispecies in HCV-1b-infected patients a nd determine the relationships between pre-and posttreatment NS5A quas ispecies mutations and the IFN-alpha sensitivity of HCV-1b. The serine residues involved in phosphorylation of NS5A protein were highly cons erved both in the various patients and in quasispecies in a given pati ent, suggesting that phosphorylation is important in NS5A protein func tion. A hot spot for amino acid substitutions was found at positions 2 217 to 2218; it could be the result of either strong selection pressur e or tolerance to these amino acid replacements. The proportion of syn onymous mutations was significantly higher than the proportion of nons ynonymous mutations, suggesting that genetic variability in the region studied was the result of high mutation rates and viral replication k inetics rather than of positive selection. Sustained HCV RNA clearance was associated with low viral load and low nucleotide sequence entrop y, suggesting (i) that the replication kinetics when treatment is star ted plays a critical role in HCV-1b sensitivity to IFN-a: and (ii) tha t HCV-1b resistance to IFN-alpha could be conferred by numerous and/or related mutations that could be patient specific and located at diffe rent positions throughout the viral genome and could allow escape vari ants to be selected by IFN-ol-stimulated immune responses. No NS5A seq uence appeared to be intrinsically resistant or sensitive to IFN-alpha , but the HCV-J sequence was significantly more frequent in nonrespond er quasispecies than in sustained virological responder quasispecies, suggesting that the balance between NS5A quasispecies sequences in inf ected patients could have a subtle regulatory influence on HCV replica tion.