Abstract
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.
Highlights
About 3% of the world’s population is chronically infected with hepatitis C virus (HCV).1 Many of these chronically infected patients develop life threatening cirrhosis and hepatocellular carcinoma [1, 2]
We report that an RNA template with secondary structure is preferentially bound and more efficient at generating template-directed product via a template elongation mechanism compared with binding to a linear RNA with the same 3Ј region
We examine the interaction of truncated forms of non-structural protein 5B (NS5B) with a series of short related RNA templates, some able to form secondary structure
Summary
Protein Expression and Purification—The NS5B 1b-BK⌬21-His gene was constructed from hybridized DNA oligomers and PCR products, using standard molecular biology techniques. Labeling reactions were carried out at 37 °C for 30 min in a 50-l volume containing 50 pmol of RNA, 100 Ci of [␥-32P]ATP, and 20 units of T4 polynucleotide kinase (in buffer 70 mM Tris-HCl, 10 mM MgCl2, 5 mM DTT, pH 7.6). 2 M RNA template and 2.5 M NS5B in the above standard assay buffer were preincubated for 5 min at 23 °C and initiated by the addition of a mixture of 10 M pGpG and 200 M UTP, 2.5 Ci of [␣-32P]UTP (values are final concentrations). The reaction volume was 20 l in standard replication buffer with 1 M of 5Ј-labeled RNA template, 1.25 M NS5B, and 100 M cold UTP with/without 10 M pGpG primer. We obtained the dissociation constant value using, y ϭx ϩ
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