The replication of avian leukosis virus (ALV), a simple retrovirus that induces neoplastic diseases in chickens, is regulated by host factors. Investigating the antiviral activity of these host factors and the underlying mechanisms could provide a theoretical foundation for the prevention and control of ALV infections. In this study, RNA sequencing revealed that ALV-A infection upregulates the expression of membrane-associated RING-CH 2 (MARCH2). Increasing MARCH2 levels inhibited ALV-A replication by 3.47- to 16.98-fold in viral titer, while conversely, knockout of MARCH2 promoted it by 5.04- to 10.70-fold. MARCH2 was found to interact with the viral gp85 protein, catalyzing its K27-linked ubiquitination and proteasomal degradation at lysine 282 (K282). Substituting K282 with arginine prevented degradation and enhanced ALV-A replication. Importantly, the K282 site was determined to be conserved across various ALV subgroups, with its mutation disrupting MARCH2-mediated degradation. Our findings not only identified MARCH2 as a robust defense mechanism against multiple ALV subgroups, functioning by targeting viral proteins, but also provide a potential target for novel strategies against avian leukosis.IMPORTANCEAvian leukosis virus (ALV), a simple retrovirus, not only causes chicken tumor disease and immunosuppression but, more importantly, can spread vertically through hatching eggs, affecting the quality of chicks and endangering the safety of poultry breeds. Currently, no vaccines or treatments are available, and the most effective strategy for preventing and controlling this disease in chicken flocks is the eradication of ALV. The MARCH protein family exhibits antiviral activity in mammals. Our research found that the MARCH protein family has antiviral functions in birds. Further, MARCH2 was determined to bind gp85 in different subgroups of ALVs, ubiquitinate and facilitate the degradation of gp85 via the K282 residue, and inhibit the replication of different ALV subgroups. This study significantly advances our understanding of the avian defense mechanisms against viral infections and offers new targets for developing novel ALV prevention and control strategies.

Keywords: MARCH2; avian leukosis virus; degradation; gp85; ubiquitination.