Hepatitis C Virus NS5B Polymerase Exhibits Distinct Nucleotide Requirements for Initiation and Elongation

Eric Ferrari,Zhiqing He,Robert E Palermo,H.-C Huang

doi:10.1074/jbc.m803094200

Abstract

The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3'-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (K(app)(m) approximately 100-400 mum) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 10(3)- to 10(4)-fold, revealing a much reduced nucleotide requirement for elongation (K(app)(m) approximately 0.03-0.09 microm). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by <0.1 microm levels of NTP. De novo synthesis at high NTP concentrations was shown to be preferred over primer extension. On a dideoxycytidine-blocked synthetic RNA template derived from the 3'-end of the HCV(-)UTR, the addition of the corresponding initiating trinucleotide also dramatically reduced the NTP levels needed to achieve efficient RNA synthesis. Thus, distinct nucleotide requirements exist for initiation and elongation steps catalyzed by the HCV NS5B polymerase.

Highlights

Incorporation of 5Ј-end-labeled GAU into the template-length product was observed at 0.1 ␮M NTPs by denaturing PAGE analysis. These findings indicate that high concentrations of the first three incorporated nucleotides required for de novo initiation (Km app ϳ100 – 400 ␮M) could be surmounted by the presence of initiating trinucleotides, which presumably served to facilitate the transition from initiation to elongation, where the nucleotide requirement is significantly reduced (Km app ϳ 0.03– 0.09 ␮M)
A Heteropolymer RNA Template Derived from the hepatitis C virus (HCV)(Ϫ)UTR—We have focused the study of the nucleotide requirements for initiation on DCoH75ddC, a heterologous RNA template containing four unique nucleotides at its 3Ј-end
RNA synthesis catalyzed by the HCV NS5B polymerase is a multistep process involving initiation, elongation, and product dissociation, followed by reinitiation for another round of polymerization

Summary

EXPERIMENTAL PROCEDURES

Materials—␣-32P- and ␣-33P-labeled NTPs (10 mCi/ml, 3,000 Ci/mmol) and nucleoside 5Ј-triphosphates were purchased from Amersham Biosciences. [␥-32P]ATP and [32P]pCp (10 mCi/ml, 3,000 Ci/mmol) were from PerkinElmer Life Sciences. To determine the Km app of GTP under single-round elongation conditions, NS5B was preincubated with primer and template (each at 0.1 ␮M final concentration) at room temperature for 22 h before RdRp reactions were initiated with 1.5 ␮Ci of [␣-33P]CTP (at 20 nM), 500 ␮M each ATP and UTP, 0.2 mg/ml heparin, and GTP ranging from 10 nM to 500 ␮M. NS5B activity in the presence of heparin was followed by [␣-33P]CTP incorporation at 30 s, 1 min, 5 min, and 10 min, and the single round burst rate of RNA synthesis derived from the slope at each GTP concentration was obtained. RNA was cleaved by RNase T1 under the condition used, an end-labeled 30-mer RNA was shown to be completely digested but protected when it was preannealed to a complementary strand

RESULTS

Km app With initiator

DISCUSSION