Specific interaction of hepatitis C virus protease/helicase NS3 with the 3'-terminal sequences of viral positive- and negative-strand RNA

The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be i nvolved in viral RNA replication. We have expressed and purified recombinan t NS3 (protease and helicase domains) and Delta pNS3 (helicase domain only) and examined their abilities to interact with the 3'-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were ch osen because initiation of RNA synthesis is likely to occur at or near the 3' untranslated region (UTR). The results presented here demonstrate that N S3 (and Delta pNS3) interacts efficiently and specifically with the 3' term inal sequences of both positive- and negative strand RNA but not with the c orresponding complementary 5'-terminal RNA sequences. The interaction of NS 3 with the S'-terminal negative strand [called 3'(-) UTR127] was specific i n that only homologous land not heterologous) RNA competed efficiently in t he binding reaction. A predicted stem-loop structure present at the 3' term inus (nucleotides 5 to 20 from the 3' end) of the negative-strand RNA appea rs to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and Delta pNS3) bindin g. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggest ed that both a double-stranded structure and the 3'-proximal guanosine resi dues in the stem were important determinants of protein binding. In contras t to the relatively stringent requirement for 3'(-) UTR binding, specific i nteraction of NS3 (or Delta pNS3) with the 3'-terminal sequences of the pos itive-strand RNA [3'(+) UTR] appears to require the entire 3'(+) UTR of HCV . Deletion of either the 98-nucleotide 3'-terminal conserved region or the 5' half sequence containing the variable region and the poly(U) and/or poly (UC) stretch significantly impaired RNA-protein interaction. The implicatio n of NS3 binding to the 3'-terminal sequences of viral positive- and negati ve-strand RNA in viral replication is discussed.