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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