Gag ubiquitination facilitates ALIX-mediated lentiviral RNA packaging, counteracted by USP8

Lentiviral Gag protein plays an integral role in viral assembly. In host cells, Gag multimerizes, recruits host budding factors, and buds from the plasma membrane. Additionally, it binds to viral genomic RNA (vRNA) and facilitates its packaging into viral particles. However, the mechanisms regulating Gag-vRNA interactions remain poorly understood. Previous studies have suggested that Gag ubiquitination influences viral infectivity. In this study, we elucidated a new mechanism by which Gag-vRNA interactions are regulated via Gag ubiquitination. Using an HIV-1-derived lentiviral vector system, we found that ubiquitin-specific protease 8 (USP8), a deubiquitinating enzyme involved in antiviral responses, reduced the vRNA content in virions through its catalytic activity. USP8 deubiquitinates Gag, and mutations at the Gag ubiquitination sites similarly impair vRNA packaging. We hypothesized that Gag ubiquitination enhances its interaction with RNA-binding proteins, thereby facilitating Gag-vRNA binding. We focused on ALG-2-interacting protein X (ALIX), which interacts with Gag, ubiquitin, and RNA. Mechanistic analyses revealed that Gag ubiquitination promotes its interaction with ALIX, which in turn binds to vRNA. In ALIX knockout (KO) cells, both Gag-vRNA binding and vRNA packaging were reduced, confirming the role of ALIX in this process. Furthermore, the deubiquitinating activity of USP8 suppressed Gag-vRNA binding, and this effect was abolished by mutation of the Gag ubiquitination site or ALIX KO. Collectively, these results reveal a new regulatory mechanism for lentiviral assembly, in which Gag ubiquitination facilitates ALIX-mediated vRNA packaging and is counteracted by USP8. Significance StatementLentiviral Gag-vRNA binding and vRNA packaging are essential for viral particle formation; however, their regulation remains unclear. We identified a USP8-Gag-ALIX axis that controls this process. USP8, a host deubiquitinating enzyme, suppresses Gag-vRNA binding by removing ubiquitin from Gag. Gag ubiquitination promotes ALIX interaction, which facilitates Gag-vRNA binding. Although ALIX is known to play a role in viral budding, we uncovered a new role for ALIX in vRNA recruitment. These findings advance our understanding of lentiviral assembly and identify USP8 and ALIX as potential targets for antiviral therapy. They may also improve lentiviral vector production by providing a molecular basis for reducing transgene-deficient particles and enhancing vector quality.