Recombinant viral RdRps can initiate RNA synthesis from circular templates.
The crystal structure of the recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) revealed extensive interactions between the fingers and the thumb subdomains, resulting in a closed conformation with an established template channel that should specifically accept single-stranded templates. We made circularized RNA templates and found that they were efficiently used by the HCV RdRp to synthesize product RNAs that are significantly longer than the template, suggesting that RdRp could exist in an open conformation prior to template binding. RNA synthesis using circular RNA templates had properties similar to those previously documented for linear RNA, including a need for higher GTP concentration for initiation, usage of GTP analogs, sensitivity to salt, and involvement of active-site residues for product formation. Some products were resistant to challenge with the template competitor heparin, indicating that the elongation complexes remain bound to template and are competent for RNA synthesis. Other products were not elongated in the presence of heparin, indicating that the elongation complex was terminated. Lastly, recombinant RdRps from two other flaviviruses and from the Pseudomonas phage phi6 also could use circular RNA templates for RNA-dependent RNA synthesis, although the phi6 RdRp could only use circular RNAs made from the 3'-terminal sequence of the phi6 genome.
Ranjith-Kumar
C T
CT
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA. rkumar@tamu.edu
Kao
C C
CC
D020134:Catalytic Domain; D006160:Guanosine Triphosphate / Q000378:metabolism; D016174:Hepacivirus / Q000235:genetics; D006493:Heparin / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D000079962:RNA, Circular; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013698:Templates, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism
D000079962:RNA, Circular; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012313:RNA; D006160:Guanosine Triphosphate; D006493:Heparin; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase