De novo RNA synthesis catalyzed by HCV RNA-dependent RNA polymerase.
The 65 kDa RNA-dependent RNA polymerase (NS5B), encoded by the hepatitis C virus (HCV) genome, is a key component involved in viral replication. Here we provide the direct evidence that purified HCV polymerase catalyzed de novo RNA synthesis in a primer-independent manner using homopolymers and HCV RNA as templates. The enzyme could utilize both polyC and polyU as templates for de novo RNA synthesis, suggesting that NS5B specifically recognized pyrimidine bases for initiation. More importantly, NS5B also catalyzed de novo RNA synthesis with an HCV RNA template; the resulting nascent RNA products, smaller than the template used, contained ATP as the first nucleotide. These results indicate that the newly synthesized RNAs did not result from template self-priming and suggest that a replication initiation site in the HCV RNA genome is a uridylate.
Sun
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XL
Infectious Diseases Research, Lilly Research Laboratories, Indianapolis, Indiana 46285, USA.
Johnson
R B
RB
Hockman
M A
MA
Wang
Q M
QM
D020413:3' Untranslated Regions; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D011066:Poly C / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
D020413:3' Untranslated Regions; D012367:RNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D011072:Poly U; D011066:Poly C; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase