Terminal nucleotidyl transferase activity of recombinant Flaviviridae RNA-dependent RNA polymerases: implication for viral RNA synthesis.
Recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was reported to possess terminal transferase (TNTase) activity, the ability to add nontemplated nucleotides to the 3' end of viral RNAs. However, this TNTase was later purported to be a cellular enzyme copurifying with the HCV RdRp. In this report, we present evidence that TNTase activity is an inherent function of HCV and bovine viral diarrhea virus RdRps highly purified from both prokaryotic and eukaryotic cells. A change of the highly conserved GDD catalytic motif in the HCV RdRp to GAA abolished both RNA synthesis and TNTase activity. Furthermore, the nucleotides added via this TNTase activity are strongly influenced by the sequence near the 3' terminus of the viral template RNA, perhaps accounting for the previous discrepant observations between RdRp preparations. Last, the RdRp TNTase activity was shown to restore the ability to direct initiation of RNA synthesis in vitro on an initiation-defective RNA substrate, thereby implicating this activity in maintaining the integrity of the viral genome termini.
Ranjith-Kumar
C T
CT
Department of Biology, Indiana University, Bloomington, Indiana 47405, USA.
Gajewski
J
J
Gutshall
L
L
Maley
D
D
Sarisky
R T
RT
Kao
C C
CC
D000818:Animals; D002460:Cell Line; D003568:Cytidine Monophosphate / Q000378:metabolism; D001908:Diarrhea Viruses, Bovine Viral / Q000201:enzymology* / Q000235:genetics; D018067:Flaviviridae / Q000201:enzymology / Q000235:genetics; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D018411:Spodoptera; D013698:Templates, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
C096041:NS5 protein, flavivirus; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012316:RNA Nucleotidyltransferases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; D003568:Cytidine Monophosphate