PMID:26152585

Citation	Romero-Brey, I, Berger, C, Kallis, S, Kolovou, A, Paul, D, Lohmann, V and Bartenschlager, R (2015) NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication. MBio 6:e00759
Abstract	Induction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA viruses. 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 induce 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 assembly, 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 completely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible implications for our understanding of how RFs are formed in other positive-strand RNA viruses.
Links	PubMed PMC4488949 Online version:10.1128/mBio.00759-15
Keywords	Cell Line; Cytoplasmic Vesicles/metabolism; Cytoplasmic Vesicles/virology; DNA Mutational Analysis; Hepacivirus/genetics; Hepacivirus/physiology; Humans; Mutant Proteins/genetics; Mutant Proteins/metabolism; Polyproteins/metabolism; Protein Processing, Post-Translational; Protein Structure, Tertiary; Proteolysis; Viral Nonstructural Proteins/genetics; Viral Nonstructural Proteins/metabolism; Virus Replication
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
HCVH:POLG	part_of	GO:0039714: cytoplasmic viral factory	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete

References

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