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Updated: Mar 16, 2026
A Protocol for Analyzing Hepatitis C Virus Replication
Published on: June 26, 2014
Reconstitution and Functional Analysis of a Full-Length Hepatitis C Virus NS5B Polymerase on a Supported Lipid
Nam-Joon Cho1, Edward A Pham2, Rachel J Hagey3
1Department of Chemical Engineering, Stanford University, Palo Alto, California 94305, United States; Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Palo Alto, California 94305, United States.
Abstract:
Therapeutic targeting of membrane-associated viral proteins is complicated by the challenge of investigating their enzymatic activities in the native membrane-bound state. To permit functional characterization of these proteins, we hypothesized that the supported lipid bilayer (SLB) can support in situ reconstitution of membrane-associated viral protein complexes. As proof-of-principle, we selected the hepatitis C virus (HCV) NS5B polymerase which is essential for HCV genome replication, and determined that the SLB platform enables functional reconstitution of membrane protein activity. Quartz crystal microbalance with dissipation (QCM-D) monitoring enabled label-free detection of full-length NS5B membrane association, its interaction with replicase subunits NS3, NS5A, and template RNA, and most importantly its RNA synthesis activity. This latter activity could be inhibited by the addition of candidate small molecule drugs. Collectively, our results demonstrate that the SLB platform can support functional studies of membrane-associated viral proteins engaged in critical biological activities.
