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J Biol Chem. 2014 May 16;289(20):14399-411. doi: 10.1074/jbc.M113.529743. Epub 2014 Apr 1.

Dynamics of hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B in complex with RNA.

Author information

1
From the Department of Chemistry and.
2
the Department of Microbiology and Immunology, McGill University, Montréal, Quebec H3A 2B4, Canada.
3
From the Department of Chemistry and the Department of Biochemistry, McGill University, Montréal, Quebec H3G 1Y6, Canada and the Department of Medicine, Division of Experimental Medicine, McGill University, Montréal, Quebec H3A 1A3, Canada matthias.gotte@mcgill.ca.
4
From the Department of Chemistry and the Centre for Self-Assembled Chemical Structures (CSACS/CRMAA), McGill University, Montréal, Quebec H3A 2K6, Canada, gonzalo.cosa@mcgill.ca.

Abstract

The hepatitis C virus (HCV) non-structural protein 5B (NS5B) is an RNA-dependent RNA polymerase that is essentially required for viral replication. Although previous studies revealed important properties of static NS5B-RNA complexes, the nature and relevance of dynamic interactions have yet to be elucidated. Here, we devised a single molecule Förster Resonance Energy Transfer (SM-FRET) assay to monitor temporal changes upon binding of NS5B to surface immobilized RNA templates. The data show enzyme association-dissociation events that occur within the time resolution of our setup as well as FRET-fluctuations in association with stable binary complexes that extend over prolonged periods of time. Fluctuations are shown to be dependent on the length of the RNA substrate, and enzyme concentration. Mutations in close proximity to the template entrance (K98E, K100E), and in the center of the RNA binding channel (R394E), reduce both the population of RNA-bound enzyme and the fluctuations associated to the binary complex. Similar observations are reported with an allosteric nonnucleoside NS5B inhibitor. Our assay enables for the first time the visualization of association-dissociation events of HCV-NS5B with RNA, and also the direct monitoring of the interaction between HCV NS5B, its RNA template, and finger loop inhibitors. We observe both a remarkably low dissociation rate for wild type HCV NS5B, and a highly dynamic enzyme-RNA binary complex. These results provide a plausible mechanism for formation of a productive binary NS5B-RNA complex, here NS5B slides along the RNA template facilitating positioning of its 3' terminus at the enzyme active site.

KEYWORDS:

Antiviral Agents; Drug Resistance; Fluorescence Resonance Energy Transfer (FRET); Hepatitis c Virus; RNA Polymerase

PMID:
24692556
PMCID:
PMC4022906
DOI:
10.1074/jbc.M113.529743
[Indexed for MEDLINE]
Free PMC Article

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