Role of g5Rp in African swine fever virus replication: disruption of host translation and autophagy

African swine fever (ASF), caused by the African swine fever virus (ASFV), is one of the most severe viral diseases affecting swine. ASFV employs sophisticated strategies to subvert host immune responses; however, the function of the viral protein g5Rp in viral pathogenesis remains incompletely defined. In this study, we demonstrate that g5Rp plays a critical role in viral replication by impairing host translation and Autophagy. Overexpression of g5Rp enhanced viral replication and increased p30 protein levels, whereas siRNA-mediated knockdown of g5Rp suppressed both, underscoring its essential proviral function. Proteomic profiling of infected porcine macrophages (3D4/21 cells) revealed that g5Rp dysregulated 122 host proteins, predominantly involved in translation, Autophagy, and Apoptosis pathways. Mechanistically, g5Rp directly interacted with eIF5A and RPS15, disrupting their complex formation and thereby inhibiting translation initiation and autophagic flux. Structural analyses identified key residues (SER¹¹⁸, SER²⁰⁶, and ASN⁶¹) critical for this interference. Mutation of these residues abrogated g5Rp activity. Furthermore, virtual screening identified 9″-methyl salvianolate B as a potent g5Rp inhibitor, which restored eIF5A hypusination, promoted Autophagy, and suppressed ASFV replication in vitro. Collectively, our findings establish g5Rp as a pivotal regulator of ASFV pathogenesis and a promising target for Antiviral drug development.

Importance: ASFV has caused significant economic losses to the global pork industry, and no effective treatment or prevention currently exists. In this study, the interaction of g5Rp with the host proteins eIF5A and RPS15 was identified for the first time, and its crucial role in the viral life cycle was clarified. Resolving the crystal structure of g5Rp revealed its binding site to the host protein, which provides a new target for developing Antiviral strategies against g5Rp. Additionally, the screened 9″-methyl salvianolate B, a small-molecule inhibitor, has shown the potential to effectively reduce viral replication and restore host protein synthesis. These findings not only deepen our understanding of the mechanism of ASFV Infection but also lay the foundation for developing effective anti-ASFV treatment strategies in the future, which has important scientific implications.

9″-methyl salvianolate B; ASFV; RPS15; eIF5A; g5Rp.