Pestivirus NS3 (p80) protein possesses RNA helicase activity.
The pestivirus bovine viral diarrhea virus (BVDV) p80 protein (referred to here as the NS3 protein) contains amino acid sequence motifs predictive of three enzymatic activities: serine proteinase, nucleoside triphosphatase, and RNA helicase. We have previously demonstrated that the former two enzymatic activities are associated with this protein. Here, we show that a purified recombinant BVDV NS3 protein derived from baculovirus-infected insect cells possesses RNA helicase activity. BVDV NS3 RNA helicase activity was specifically inhibited by monoclonal antibodies to the p80 protein. The activity was dependent on the presence of nucleoside triphosphate and divalent cation, with a preference for ATP and Mn2+. Hydrolysis of the nucleoside triphosphate was necessary for strand displacement. The helicase activity required substrates with an un-base-paired region on the template strand 3' of the duplex region. As few as three un-base-paired nucleotides were sufficient for efficient oligonucleotide displacement. However, the enzyme did not act on substrates having a single-stranded region only to the 5' end of the duplex or on substrates lacking single-stranded regions altogether (blunt-ended duplex substrates), suggesting that the directionality of the BVDV RNA helicase was 3' to 5' with respect to the template strand. The BVDV helicase activity was able to displace both RNA and DNA oligonucleotides from RNA template strands but was unable to release oligonucleotides from DNA templates. The possible role of this activity in pestivirus replication is discussed.
Warrener
P
P
PathoGenesis Corporation, Seattle, Washington 98119.
Collett
M S
MS
D004247:DNA / Q000378:metabolism; D001908:Diarrhea Viruses, Bovine Viral / Q000201:enzymology*; D009711:Nucleotides / Q000378:metabolism; D010447:Peptide Hydrolases; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D011994:Recombinant Proteins; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D004247:DNA; D009711:Nucleotides; D010447:Peptide Hydrolases; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C086730:p80 protein, bovine viral diarrhea virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases