HERC5-mediated ISGylation of SARS-CoV-2 nsp8 facilitates its degradation and inhibits viral replication.

Severe acute respiratory syndrome coronavirus 2 non-structural protein 8 (SARS-CoV-2 nsp8) is a multifunctional protein essential for viral replication and immune evasion. However, the host factors that regulate nsp8 stability and function remain unclear. In this study, we identify HECT and RCC-like domain-containing protein 5 (HERC5) as an essential E3 ligase that regulates nsp8 stability through ISGylation, a ubiquitin-like post-translational modification that facilitates proteasome-dependent degradation. HERC5 overexpression significantly enhances nsp8 degradation in an enzymatic activity-dependent manner, whereas SARS-CoV-2 papain-like protease (PLpro) counteracts this process by deconjugating interferon-stimulated gene 15 (ISG15) from nsp8-thereby preventing its degradation and facilitating viral replication. Mass spectrometry and mutational analyses revealed that the N2 domain of nsp8 is indispensable for ISGylation, with multiple lysine residues acting as primary modification sites. Additionally, we demonstrated that the ISGylation system, including HERC5, ubiquitin-like modifier activating enzyme 7 (UBA7), and ISG15, effectively suppresses SARS-CoV-2 replication across multiple variants, including Omicron BA.5 and XBB.1.5.15. These findings provide novel insights into the role of ISGylation in host antiviral defense and highlight the interplay between HERC5 and PLpro in modulating viral replication. This study establishes a foundation for developing therapeutic strategies targeting HERC5 or PLpro to inhibit SARS-CoV-2 replication.