Albendazole Suppresses SARS‐CoV‐2 Replication by Enhancing HERC5‐Mediated ISGylation of Nucleocapsid Protein

Interferons constitute the host's primary antiviral defense system. It causes the expression of interferon-stimulated genes (ISGs), including ISG15, which modulate viral replication. Here, we identify the E3 ligase HERC5-a crucial mediator of ISGylation-as an antiviral factor against SARS-CoV-2. Analysis using mass spectrometry showed that the viral nucleocapsid (N) protein is directly bound by HERC5, which then catalyzes ISGylation occurs at four lysine residues-K61, K65, K102, and K355-found in the NTD and CTD. Functional assays confirmed that ISGylation at these sites disrupts the N protein's capacity for RNA binding and oligomerization. Conversely, the papain-like protease (PLpro) of SARS-CoV-2 cleaves ISG15 conjugates, thereby reversing N protein ISGylation. To discover small molecules that enhance the "HERC5-N" interaction, we developed a NanoLuc luciferase-based screening platform and evaluated 1815 FDA-approved drugs. Albendazole emerged as a potent enhancer of "HERC5-N" binding, further promoted N protein ISGylation, and inhibited viral replication. In vivo, Albendazole treatment reduced viral load and alleviated pulmonary inflammation in a transgenic mouse model of K18-hACE2. Together, these findings reveal the crucial functionality of N protein ISGylation in SARS-CoV-2 replication and highlight HERC5-mediated ISGylation as a promising therapeutic target.