NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication

ABSTRACT Induction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA vi-ruses. These altered membranes serve as scaffolds for the assembly of viral replication factories (RFs). We have recently shown that hepatitis C virus (HCV) infection induces endoplasmic reticulum-derived double-membrane vesicles (DMVs) representing the major constituent of the RF within the infected cell. RF formation requires the concerted action of nonstructural action of nonstructural protein (NS)3,-4A, protein (NS)3-4A,-4B,-5A, and-5B. Although the sole expression of NS5A is sufficient to in-duce DMV formation, its efficiency is very low. In this study, we dissected the determinants within NS5A responsible for DMV formation and found that RNA-binding domain 1 (D1) and the amino-terminal membrane anchor are indispensable for this process. In contrast, deletion of NS5A D2 or D3 did not affect DMV formation but disrupted RNA replication and virus assem-bly, respectively. To identify cis- and trans-acting factors of DMV formation, we established a trans cleavage assay. We found that induction of DMVs requires full-length NS3, whereas a helicase-lacking mutant was unable to trigger DMV formation in spite of efficient polyprotein cleavage. Importantly, a mutation accelerating cleavage kinetics at the NS4B-5A site diminished DMV formation, while the insertion of an internal ribosome entry site mimicking constitutive cleavage at this boundary com-pletely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible im-plications for our understanding of how RFs are formed in other positive-strand RNA viruses. IMPORTANCE Like all positive-strand RNA viruses, hepatitis C virus (HCV) extensively reorganizes intracellular membranes t