Journal
ACS CENTRAL SCIENCE
Volume 2, Issue 7, Pages 456-466Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acscentsci.6b00112
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Funding
- NIAID NIH HHS [U19 AI109662, R01 AI087917, T32 AI007328, R01 AI099245] Funding Source: Medline
- NIDDK NIH HHS [F30 DK099017, R01 DK064223] Funding Source: Medline
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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.
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