Fluorescence resonance energy transfer-based intracellular assay for the conformation of hepatitis C virus drug target NS5A.

ABSTRACT: Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and assembly and is a critical drug target. Biochemical data suggest large parts of NS5A are unfolded as an isolated protein, but little is known about its folded state in the cell. We used fluorescence resonance energy transfer (FRET) to probe whether or not different regions of NS5A are in close proximity within the cell. Twenty-three separate reporter constructs were created by inserting one or more fluorophores into different positions throughout the three domains of NS5A. FRET efficiency was maximal when donor and acceptor fluorophores were positioned next to each other but also could be observed when the two fluorophores flanked NS5A domain 1 or domain 3. Informatic and biochemical analysis suggests

SUBMITTER: Bhattacharya D 

PROVIDER: S-EPMC3421644 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature