Host E3 ligase RNF2 restricts African swine fever virus replication through targeting viral pA104R for its K5 site ubiquitination degradation

The African swine fever virus (ASFV) -encoded late structural protein pA104R is a putative histone-like protein, which is also a DNA-binding related protein required for ASFV DNA replication, transcription, and genome packaging. However, the molecular mechanism underlying pA104R-host protein interactions remain unknown. To identify proteins potentially interacting with ASFV-pA104R, a primary porcine alveolar macrophage (PAM) cDNA yeast two-hybrid library was constructed, and the pig E3 ubiquitin ligase RING-finger protein 2 (RNF2) was identified, which specifically negatively regulates the proliferation of ASFV. Mechanistically, RNF2 inhibits ASFV replication by promoting the proteasomal degradation of ASFV-pA104R through K48-linked ubiquitination at pA104R lysine 5 (K5). Further studies showed that the K5R mutation impairs the interaction between pA104R and RNF2 and antagonizes for pA104R degradation by RNF2. An ASFV mutant carrying a pA104R point mutation (ASFV CN/SC/2019 pA104R-K5R) was generated based on the ASFV CN/SC/2019 (wild-type) strain. Furthermore, our findings indicate that ASFV CN/SC/2019 pA104R-K5R enhances viral replication and virulence, potentially by increasing viral transcription and/or modulating the host immune response. Accordingly, compared with the parental strain, ASFV CN/SC/2019 pA104R-K5R was more pathogenic and severe lesions in swine. Collectively, our study identifies an intrinsic antiviral protein RNF2 that mediates ASFV CN/SC/2019 pA104R-K5 site ubiquitination emerges as a potential determinant of viral replication and pathogenicity.