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13:04
A Protocol for Analyzing Hepatitis C Virus Replication
Published on: June 26, 2014
Ligand-induced changes in hepatitis C virus NS5B polymerase structure.
Karen Rigat1, Yi Wang, Thomas W Hudyma
1Department of Virology, Bristol-Myers Squibb Co., Research & Development, Wallingford, CT 06492, USA. karen.rigat@bms.com
Antiviral Research
|September 4, 2010
Summary
Hepatitis C virus NS5B polymerase inhibitors and RNA binding induce conformational changes. Limited trypsin digestion reveals that template binding displaces the Δ1 loop, while primer addition reverses this, impacting viral replication.
Area of Science:
- Biochemistry
- Virology
- Structural Biology
Background:
- Hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B) is crucial for viral replication.
- Crystal structures show NS5B's finger and thumb domains encircle the active site.
- P495-resistant inhibitors disrupt NS5B structure by binding the thumb-finger interface and displacing the Δ1 finger loop.
Purpose of the Study:
- To investigate the impact of inhibitors and substrates on NS5B's Δ1 loop movement using an alternative method.
- To study NS5B conformations in solution under conditions supporting enzymatic activity.
Main Methods:
- Limited trypsin protease digestion assay.
- Investigated conformational changes induced by P495-site inhibitors, RNA template, and primer binding to NS5B.
- Correlated trypsin hypersensitivity with Δ1 loop movement using known inhibitors and mutant polymerases.
Main Results:
- Inhibitor binding induced specific trypsin hypersensitivity at lysine residues 50 and 51, linked to Δ1 loop movement.
- RNA template binding caused similar trypsin hypersensitivity.
- Primer addition to the NS5B-template complex abolished this hypersensitivity.
Conclusions:
- NS5B Δ1 loop displacement from the thumb occurs upon template binding.
- Primer or NTP addition reverses this displacement, suggesting dynamic conformational changes.
- The trypsin digestion assay provides a rapid method to study NS5B dynamics under functional conditions, complementing co-crystal studies.
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