title: Reconstitution and Functional Analysis of a Full-Length Hepatitis C Virus NS5B Polymerase on a Supported Lipid Bilayer.
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.
authors: Department 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.
authors: Nam-Joon
authors: NJ
authors: Cho
authors: Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Palo Alto, California 94305, United States; Department of Microbiology and Immunology, Stanford University School of Medicine, Palo Alto, California 94305, United States.
authors: Edward A
authors: EA
authors: Pham
authors: Department of Microbiology and Immunology, Stanford University School of Medicine , Palo Alto, California 94305, United States.
authors: Rachel J
authors: RJ
authors: Hagey
authors: Virology Discovery, Hoffmann-La Roche Inc. , Nutley, New Jersey 07110, United States.
authors: Vincent J
authors: VJ
authors: Lévêque
authors: Virology Discovery, Hoffmann-La Roche Inc. , Nutley, New Jersey 07110, United States.
authors: Han
authors: H
authors: Ma
authors: Virology Discovery, Hoffmann-La Roche Inc. , Nutley, New Jersey 07110, United States.
authors: Klaus
authors: K
authors: Klumpp
authors: Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Palo Alto, California 94305, United States; Department of Microbiology and Immunology, Stanford University School of Medicine, Palo Alto, California 94305, United States; Veterans Administration Medical Center, Palo Alto, California 94304, United States.
authors: Jeffrey S
authors: JS
authors: Glenn