Elongation of synthetic RNA templates by hepatitis C virus NS5B polymerase

Here we examine the ability of seven, 3'-related, short synthetic RNAs to serve as templates for the hepatitis C virus (HCV) polymerase, non-structural protein 5B (NS5B). These RNAs, termed HL, range from 8 to 16 nucleotides in length, each with ACC at the 3' terminus. Interestingly HL12 and longer templates have a predicted secondary structure. Those with one or two unpaired adenylates at the 5'-end of a stem were increased in size by one or two nucleotides, respectively, following incubation with NS5B and UTP. Using labeled template RNA and cold UTP, extension in size could be inhibited by addition of non-labeled template of the same size. This template elongation was not inhibited by cold linear HL10 template unless pGpG was added. Fluorescence anisotropy demonstrated HL14, a template with secondary structure, bound with an apparent K(d) of 22 nm. A linear template, HL10, plus pGpG primer was bound by NS5B with a K(d) of 45 nm, whereas HL10 alone bound with an apparent K(d) of 182 nm. The amplitude of the template extension product was increased by a brief preincubation at 4 degrees C followed by incubation at 23 or 30 degrees C. The nucleotide-mediated increase in size occurred for both templates that required a mismatch or bulge at the 3'-end as well as for those without the mismatch. These results suggest an NS5B active site pocket can readily accommodate short templates with four or five base stems and initiate copy-back replication in the presence of a one nucleotide mismatch.