BIOCHEMICAL-PROPERTIES OF HEPATITIS-C VIRUS NS5B RNA-DEPENDENT RNA-POLYMERASE AND IDENTIFICATION OF AMINO-ACID-SEQUENCE MOTIFS ESSENTIAL FOR ENZYMATIC-ACTIVITY

The NS5B protein of the hepatitis C virus (HCV) is an RNA-dependent RN A polymerase (RdRp) (S.-E. Behrens, L. Tomei, and R. De Francesco, EMB O J. 15:12-22, 1996) that is assumed to be required for replication of the viral genome, To further study the biochemical and structural pro perties of this enzyme, an NS5B-hexahistidine fusion protein was expre ssed with recombinant baculoviruses in insect cells and purified to ne ar homogeneity, The enzyme was found to have a primer-dependent RdRp a ctivity that was able to copy a complete in vitro-transcribed HCV geno me in the absence of additional viral or cellular factors, Filter bind ing assays and competition experiments showed that the purified enzyme binds RNA with no clear preference for HCV 3'-end sequences, Binding to homopolymeric RNAs was also examined, and the following order of sp ecificity was observed: poly(U) > poly(G) > poly(A) > poly(C). An inve rse order was found for the RdRp activity, which used poly(C) most eff iciently as a template but was inactive on poly(U) and poly(G), sugges ting that a high binding affinity between polymerase and template inte rferes with processivity, By using a mutational analysis, four amino a cid sequence motifs crucial for RdRp activity were identified, While m ost substitutions of conserved residues within these motifs severely r educed the enzymatic activities, a single substitution in motif D whic h enhanced the RdRp activity by about 50% was found. Deletion studies indicate that amino acid residues at the very termini, in particular t he amino terminus, are important for RdRp activity but not for RNA bin ding, Finally, we found a terminal transferase activity associated wit h the purified enzyme, However, this activity was also detected with N S5B proteins with an inactive RdRp, with an NS4B protein purified in t he same way, and with wild-type baculovirus, suggesting that it is not an inherent activity of NS5B.