Binding and Sliding Dynamics of the Hepatitis C Virus Polymerase: Hunting the 3′ Terminus

The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase catalyzes the replication of the (+) single-stranded RNA genome of HCV. In vitro studies have shown that replication can be performed in the absence of a primer. However, the dynamics and mechanism by which NS5B locates the 3′-te...

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Bibliographic Details
Published in:	ACS infectious diseases Vol. 9; no. 8; pp. 1488 - 1498
Main Authors:	Gidi, Yasser, Robert, Anaïs, Tordo, Alix, Lovell, Terri C., Ramos-Sanchez, Jorge, Sakaya, Aya, Götte, Matthias, Cosa, Gonzalo
Format:	Journal Article
Language:	English
Published:	United States American Chemical Society 11-08-2023
Subjects:	Hepacivirus - genetics Hepacivirus - metabolism Hepatitis C Humans Hunting Nucleotidyltransferases RNA, Viral - genetics RNA-Dependent RNA Polymerase - metabolism Viral Nonstructural Proteins - metabolism sliding binding HCV single-molecule PIFE polymerase
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Summary:	The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase catalyzes the replication of the (+) single-stranded RNA genome of HCV. In vitro studies have shown that replication can be performed in the absence of a primer. However, the dynamics and mechanism by which NS5B locates the 3′-terminus of the RNA template to initiate de novo synthesis remain elusive. Here, we performed single-molecule fluorescence studies based on protein-induced fluorescence enhancement reporting on NS5B dynamics on a short model RNA substrate. Our results suggest that NS5B exists in a fully open conformation in solution wherefrom it accesses its binding site along RNA and then closes. Our results revealed two NS5B binding modes: an unstable one resulting in rapid dissociation, and a stable one characterized by a larger residence time on the substrate. We associate these bindings to an unproductive and productive orientation, respectively. Addition of extra mono (Na+)- and divalent (Mg2+) ions increases the mobility of NS5B along its RNA substrate. However, only Mg2+ ions induce a decrease in NS5B residence time. Dwell times of residence increase with the length of the single-stranded template, suggesting that NS5B unbinds its substrate by unthreading the template rather than by spontaneous opening.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	2373-8227 2373-8227
DOI:	10.1021/acsinfecdis.3c00048