Nudix protein D250R promotes African swine fever virus infection by disrupting host ubiquitination machinery

Numerous viruses have evolved diverse strategies to counteract host antiviral responses. African swine fever virus (ASFV), a large double-stranded DNA virus that threatens the global swine industry and food security, is thought to employ highly complex yet poorly understood mechanisms to facilitate infection. Here, we identify a previously uncharacterized strategy by which ASFV evades host antiviral defenses and maintains virulence. We show that ASFV infection progressively impairs the assembly of ubiquitin conjugates in host cells and that the ASFV-encoded Nudix protein D250R acts as a potent suppressor of cellular ubiquitination. Expression of D250R disrupts the host ubiquitination machinery in a manner dependent on its intact structure. Mechanistically, D250R directly binds ATP via its Nudix domain and inhibits ATP-dependent, E1-mediated ubiquitin activation, thereby suppressing ubiquitination-driven antiviral immune responses and promoting viral replication. Genetic deletion of the D250R gene markedly attenuates ASFV virulence while eliciting robust immune responses in pigs. Notably, immunization with the D250R-deficient virus confers complete protection against lethal challenge with a virulent ASFV strain. Collectively, these findings reveal a previously unrecognized viral strategy to subvert host immunity and establish the D250R-deleted ASFV as a promising live-attenuated vaccine candidate for African swine fever control. This study uncovers a novel African swine fever virus (ASFV) immune-evasion mechanism targeting ubiquitin activation and identifies D250R as a key virulence factor. Loss of D250R attenuates the virus and supports its potential as a live attenuated vaccine candidate against ASF.