FAM134B Restricts African Swine Fever Virus Capsid Assembly via Reticulophagy and Its Antiviral Activity is Antagonized by the Viral Virulence-Associated Factor pMGF300-2R.

African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating viral disease in domestic pigs and wild boar. ASFV is a large DNA virus whose capsid assembly takes place within perinuclear viral factories (VFs). Accumulating evidence has demonstrated that reticulophagy confers antiviral activity against multiple RNA viruses. Nevertheless, the roles of reticulophagy in DNA virus replication and viral immunoevasion mechanisms remain largely uncharacterized. Here, we revealed that FAM134B, a reticulophagy receptor, markedly inhibits the maturation of ASFV in VFs. Mechanistically, FAM134B interacts with the major viral capsid proteins p72 and pA137R, facilitating their delivery to lysosomes for reticulophagy-mediated degradation and thus blocking ASFV capsid assembly. Additionally, we found that ASFV exploits its virulence-associated factor pMGF300-2R to target FAM134B and enhance viral replication. Further investigations revealed that pMGF300-2R binds to FAM134B and triggers its autophagic degradation via the C-terminal LC3-interacting region (LIR) motif. Importantly, the MGF300-2R-deleted ASFV mutant exhibited significantly reduced replication and virulence compared with wild-type ASFV in pigs, which was attributed to increased FAM134B expression. Collectively, this study establishes the first mechanistic link between reticulophagy and DNA virus replication and underscores the critical role of FAM134B-mediated reticulophagy in host defense against ASFV infection.