Adaptive mutations at lysine residues of PRRSV-2 nsp12 enable evasion of host proteasomal degradation to promote subgenomic RNA synthesis.

Ubiquitin signaling in viral infections can either enhance immune responses and degrade viral proteins or be exploited by viruses to target host antiviral factors. Porcine reproductive and respiratory syndrome virus (PRRSV) poses a threat to the swine industry, with its non-structural proteins (nsps) critical for virulence and replication. Here, we investigated the role of ubiquitination in regulating the stability of PRRSV-2 nsps and found that nsp12 was specifically targeted for degradation via the ubiquitin-proteasome system. Mechanistically, nsp12 underwent K48- and K63-linked polyubiquitination at lysine residues 89, 91, 127, and 130. RNF114 served as an E3 ubiquitin ligase for nsp12, and its enzymatic activity was essential for both nsp12 degradation and viral replication. Notably, evolutionary analyses revealed that PRRSV-2 nsp12 acquired lysine-to-arginine substitutions at positions 89, 127, and 130 during viral adaptation, which facilitated the synthesis of subgenomic RNA. Consistently, recombinant PRRSV-2 carrying the K91/127/130R mutations within nsp12 exhibited enhanced replication, while a revertant strain with the R89K mutation in nsp12 showed attenuated infectivity. Mass spectrometry analysis further identified significant enrichment of ubiquitination-related modifications among nsp12-interacting proteins. These findings provide valuable insights for anti-PRRSV-2 drug design and highlight the challenge posed by adaptive mutations in viral proteins to the swine industry. IMPORTANCE: Ubiquitin-mediated proteasomal degradation is a central antiviral mechanism; however, how porcine reproductive and respiratory syndrome virus (PRRSV) counteracts this host defense has remained unclear. Here, we demonstrate that PRRSV-2 has evolved a strategy to escape this restriction. We show that while the viral non-structural protein 12 (nsp12) is normally targeted for degradation by ubiquitination, adaptive mutations that eliminate key ubiquitination sites allow the virus to evade proteasomal clearance. This escape not only stabilizes nsp12 but also promotes the synthesis of viral subgenomic RNA, thereby enhancing viral replication. Our findings reveal a novel immune evasion mechanism in which a virus acquires specific mutations to subvert ubiquitin-dependent host defenses, highlighting a critical challenge for the development of effective vaccines and antivirals against PRRSV-2.