BIOCHEMICAL-PROPERTIES OF HEPATITIS-C VIRUS NS5B RNA-DEPENDENT RNA-POLYMERASE AND IDENTIFICATION OF AMINO-ACID-SEQUENCE MOTIFS ESSENTIAL FOR ENZYMATIC-ACTIVITY
V. Lohmann et al., BIOCHEMICAL-PROPERTIES OF HEPATITIS-C VIRUS NS5B RNA-DEPENDENT RNA-POLYMERASE AND IDENTIFICATION OF AMINO-ACID-SEQUENCE MOTIFS ESSENTIAL FOR ENZYMATIC-ACTIVITY, Journal of virology, 71(11), 1997, pp. 8416-8428
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.