High Resolution Footprinting of the Hepatitis C Virus Polymerase NS5B in Complex with RNA

Claudia M D'Abramo,Suzanne Mccormick,Sonja Hess,Dimitrios Coutsinos,Zhuojun Zhao,Jérôme Deval,Mamuka Kvaratskhelia,Matthias Götte

doi:10.1074/jbc.m701973200

Claudia M D'Abramo, Suzanne Mccormick + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m701973200

Copy DOI

Abstract

The nucleic acid binding channel of the hepatitis C virus RNA polymerase remains to be defined. Here we employed complementary footprinting techniques and show that the enzyme binds to a newly synthesized duplex of approximately seven to eight base pairs. Comparative analysis of surface topologies of free enzyme versus the nucleoprotein complex revealed certain lysines and arginines that are protected from chemical modification upon RNA binding. The protection pattern helps to define the trajectory of the nucleic acid substrate. Lys(81), Lys(98), Lys(100), Lys(106), Arg(158), Arg(386), and Arg(394) probably interact with the bound RNA. The selective protection of amino acids of the arginine-rich region in helix T points to RNA-induced conformational rearrangements. Together, these findings suggest that RNA-protein interaction through the entire substrate binding channel can modulate intradomain contacts at the C terminus.

Highlights

The nucleic acid binding channel of the hepatitis C virus RNA polymerase remains to be defined
The structure of Hepatitis C virus (HCV) NS5B is reminiscent of a human right hand, in contrast to many other DNA or RNA polymerases with a similar fold, extensive interaction between the “thumb” and the tips of the “fingers” subdomains encircle the active site of NS5B (8 –10)
RNA footprinting revealed that the NS5B enzyme is in contact with approximately seven to eight newly synthesized residues, and mass spectrometric nucleoprotein footprinting enabled us to identify specific lysines and arginines that are involved in RNA binding

Summary

EXPERIMENTAL PROCEDURES

Chemicals and Nucleic Acid—The RNA template sequences used in this study were 5Ј-AACAGUUUCCUUUUCUCUCC-3Ј (T20-RNA), and 5Ј-AACTTTTAGTCTTTTTTTTTTTTCTCC-3Ј (T27-DNA), with the underlined base as the unique site of chain termination. RNA Synthesis and Rescue Assay—Standard chain termination reaction mixtures consisted of 1 ␮M RNA template, 1 ␮M HCV NS5B, and 0.25 ␮M radiolabeled GG primer in 40 mM HEPES, pH 8, buffer containing 10 mM NaCl, 1 mM dithiothreitol, and 0.2 mM MnCl2. Ribonuclease H (RNase H) Protection Assay—RNA/DNA duplexes were generated by mixing together 1 ␮M HCV NS5B and 0.25 ␮M radiolabeled GG primer in 40 mM HEPES, pH 8, buffer containing 10 mM NaCl, 1 mM dithiothreitol, and 0.2 mM MnCl2. Surface accessibility of lysine residues was monitored using 100 ␮M NHS-biotin in 40 mM HEPES, pH 8, buffer containing 10 mM NaCl, 1 mM dithiothreitol, and 0.2 mM MnCl2.

RESULTS

DISCUSSION