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

Summary The mechanisms that regulate envelopmentof 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 ubiquitinsignaling that governs NS2 ubiquitinationremained unknown. Here, we map the NS2 interactomewith the ubiquitinproteasome 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-fingerE3 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 envelopmentvia ubiquitinsignaling and both a viral proteinsubstrate and a ubiquitinK63-linkage of the understudied MARCH8, with potential implications for cell biology, virology, and host-targeted antiviral design.