The E3 ubiquitin ligase IDOL suppresses PRRSV replication by targeting viral Nsp3 for SQSTM1-mediated selective autophagic degradation

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating pathogens affecting the global swine industry. Autophagy plays an important role in both host defense and PRRSV infection. However, the mechanisms by which the host exploits the autophagy pathway to antagonize PRRSV infection remain largely unknown. Here, we perform transcriptomic analysis of porcine alveolar macrophages isolated from PRRSV-infected piglets and reveal a significant upregulation of the mRNA levels for the E3 ubiquitin ligase IDOL. These findings are further validated by in vitro experiments. Functional studies reveal that IDOL acts as a host restriction factor for PRRSV: ectopic expression of IDOL markedly suppresses viral replication, while knockdown of endogenous IDOL enhances it. Mechanistically, IDOL catalyzes K63-linked polyubiquitination of the viral nonstructural protein 3 (Nsp3) at lysine 101 (K101) and recruits the selective autophagy receptor SQSTM1, thereby targeting Nsp3 for degradation via the autophagy-lysosomal pathway and effectively suppressing viral replication. Notably, the ubiquitin-associated and LC3-interacting region domains of SQSTM1 are essential for this autophagic clearance of Nsp3. Furthermore, using a reverse genetics approach, we generate an Nsp3 K101R mutant virus and confirm that this specific mutation enables the virus to evade IDOL-mediated Nsp3 degradation, thereby restoring its replication competence. In summary, we uncover an autophagy-dependent antiviral mechanism, establish IDOL as an innate immune regulator with a non-canonical function, and define a viral immune evasion strategy, positioning IDOL as a promising therapeutic target for PRRSV control. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) represents a major threat to global swine production, inflicting substantial economic damage and endangering food security. Here, we identify the E3 ubiquitin ligase IDOL as a critical host restriction factor that combats PRRSV through a previously unrecognized autophagy-mediated mechanism. We demonstrate that PRRSV infection robustly upregulates the transcription factor ZNF460, which directly drives IDOL expression. During PRRSV infection, IDOL drives the autophagic degradation of Nsp3 by catalyzing its K63-linked ubiquitination at lysine 101 and engaging the selective autophagy receptor SQSTM1, triggering a potent antiviral response. Using reverse genetics, we generate an Nsp3 K101R mutant virus and demonstrate that this mutation enables the virus to escape IDOL-mediated degradation and restore replication competence, providing genetic validation of this antiviral axis. Collectively, our findings establish an autophagy-dependent antiviral mechanism, define IDOL as an intrinsic immune regulator with a previously unrecognized function, and elucidate a precise viral immune evasion strategy, thereby positioning IDOL as a promising therapeutic target for PRRSV intervention.