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  • MARCH8 Ubiquitinates the Hepatitis C Virus Nonstructural 2 Protein and Mediates Viral Envelopment.

MARCH8 Ubiquitinates the Hepatitis C Virus Nonstructural 2 Protein and Mediates Viral Envelopment.

Cell reports (2019-02-14)
Sathish Kumar, Rina Barouch-Bentov, Fei Xiao, Stanford Schor, Szuyuan Pu, Elise Biquand, Albert Lu, Brett D Lindenbach, Yves Jacob, Caroline Demeret, Shirit Einav
ABSTRACT

The mechanisms that regulate envelopment of HCV and other viruses that bud intracellularly and/or lack late-domain motifs are largely unknown. We reported that K63 polyubiquitination of the HCV nonstructural (NS) 2 protein mediates HRS (ESCRT-0 component) binding and envelopment. Nevertheless, the ubiquitin signaling that governs NS2 ubiquitination remained unknown. Here, we map the NS2 interactome with the ubiquitin proteasome system (UPS) via mammalian cell-based screens. NS2 interacts with E3 ligases, deubiquitinases, and ligase regulators, some of which are candidate proviral or antiviral factors. MARCH8, a RING-finger E3 ligase, catalyzes K63-linked NS2 polyubiquitination in vitro and in HCV-infected cells. MARCH8 is required for infection with HCV, dengue, and Zika viruses and specifically mediates HCV envelopment. Our data reveal regulation of HCV envelopment via ubiquitin signaling and both a viral protein substrate and a ubiquitin K63-linkage of the understudied MARCH8, with potential implications for cell biology, virology, and host-targeted antiviral design.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
Sigma-Aldrich
Cycloheximide, from microbial, ≥94% (TLC)
Sigma-Aldrich
Normal Rabbit IgG, Normal Rabbit IgG Polyclonal Antibody control validated for use in Immunoprecipitation & Western Blotting.