The RNA-dependent RNA polymerases of different members of the family Flaviviridae exhibit similar properties in vitro.
The virus-encoded RNA-dependent RNA polymerase (RdRp), which is required for replication of the positive-strand RNA genome, is a key enzyme of members of the virus family Flaviviridae. By using heterologously expressed proteins, we demonstrate that the 77 kDa NS5B protein of two pestiviruses, bovine viral diarrhoea virus and classical swine fever virus, and the 100 kDa NS5 protein of the West Nile flavivirus possess RdRp activity in vitro. As originally shown for the RdRp of hepatitis C virus, RNA synthesis catalysed by the pestivirus and flavivirus enzymes is strictly primer-dependent in vitro. Accordingly, initiation of RNA polymerization on homopolymeric RNAs and heteropolymeric templates, the latter with a blocked 3'-hydroxyl group, was found to be dependent on the presence of complementary oligonucleotide primer molecules. On unblocked heteropolymeric templates, including authentic viral RNAs, the RdRps were shown to initiate RNA synthesis via intramolecular priming at the 3'-hydroxyl group of the template and 'copy-back' transcription, thus yielding RNase-resistant hairpin molecules. Taken together, the RdRps of different members of the Flaviviridae were demonstrated to exhibit a common reactivity profile in vitro, typical of nucleic acid-polymerizing enzymes.
Steffens
Sabine
S
Institut für Virologie (FB Veterin ärmedizin), Justus-Liebig-Universit ät Giessen, Frankfurter Str. 107, D- 35392 Giessen, Germany1.
Thiel
Heinz-Jürgen
HJ
Institut für Virologie (FB Veterin ärmedizin), Justus-Liebig-Universit ät Giessen, Frankfurter Str. 107, D- 35392 Giessen, Germany1.
Behrens
Sven-Erik
SE
Institut für Virologie (FB Veterin ärmedizin), Justus-Liebig-Universit ät Giessen, Frankfurter Str. 107, D- 35392 Giessen, Germany1.
D000818:Animals; D002417:Cattle; D002460:Cell Line; D006692:Classical Swine Fever Virus / Q000201:enzymology; D001908:Diarrhea Viruses, Bovine Viral / Q000201:enzymology; D018067:Flaviviridae / Q000201:enzymology*; D015870:Gene Expression; D012324:RNA-Dependent RNA Polymerase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014902:West Nile virus / Q000201:enzymology
C096041:NS5 protein, flavivirus; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase