The Molecular Mechanisms Of TRIM28 In Regulation Of PRRSV-induced Autophagy

Porcine reproductive respiratory syndrome virus(PRRSV)infection leads to porcine reproductive and respiratory syndrome(PRRS)that is one of the most important viral infectious diseases to swine industry worldwide.The target cells of PRRSV are porcine alveolar macrophages and dendritic cells,causing animal immunosuppression and promoting co-infection with other pathogens.Understanding of the pathogenesis of PRRS is critical for the development of novel vaccines and antiviral drugs.Although RPRSV has been extensively studied for many years,the mechanisms of virus and host interaction are not completely understood.Recent study suggested that PRRSV infection could induce autophagy to promote its replication.However,the molecular mechanisms of PRRSVmediated autophagy are still unclear.TRIM28,also known as KAP1,is a nuclear protein belonging to member of the TRIM family with multiple biological functions including transcriptional inhibition and E3 ligase activity.It was shown that TRIM28 was involved in the regulation of virus replication and autophagy.However,it remains unknown whether TRIM28 plays role in the process of PRRSV replication and PPRSV-induced autophagy.We found that PRRSV infection induced autophagy and significantly increased the protein level of TRIM28 in cytoplasm without altering TRIM28 mRNA expression in Marc145 cells.Further examination confirmed that PRRSV infection induced TRIM28 translocation from nucleus to cytoplasm.Screening nonstructural proteins of PRRSV showed that Nsp4 colocalized and interact with cytoplasmic TRIM28 leading to TRIM28 translocation from nucleus to the cytoplasm.Further Co-IP assay proved that the cytoplasmic translocation of TRIM28 during PRRSV infection is dependent on chromosome region maintenance 1(CRM1),a key export receptor for the export of proteins out of the nucleus.To examine the roles of TRIM28 during PRRSV infection,specific siRNA oligoes targeting TRIM28 gene were designed to knockdown of TRIM28 expression.The results showed that knockdown of TRIM28 clearly suppressed autophagy formation induced by PRRSV infection,suggesting that TRIM28 is required for PRRSV-mediated autophagy.Mechanistically,TRIM28 interacted with Vps34,an early autophagy regulatory molecule.and the interaction between TRIM28 and Vps34 was significantly enhanced in response to PRRSV infection.Moreover,pretreatment of Leptomycin B(LMB),an inhibitor of CRM1,decreased the interaction between TRIM28 and Vps34,indicating that nuclear export of TRIM28 is crucial for TRIM28-Vps34 interaction.In addition,TRIM28 expression increased of Vps34 SUMOylation and reinforced Vps34-Beclin1 complex formation while knockdown TRIM28 reduced the SUMOylation of Vps34 as well as formation of Vps34-Beclin1 complexes.Indeed,TRIM28 mutants lacking of nuclear location signal significantly strengthened the SUMOylation of Vps34,further confirming that nuclear export of TRIM28 is pivotal for PRRSV-induced autophagy.In summary,we found that PRRSV Nsp4 was important for nuclear export of TRIM28 in a CRM1-dependent manner during PRRSV infection.Cytoplasmic TRIM28 then functioned as an SUMO E3 ligase of Vps34 to enhance Vp34 SUMOylation,promoting the formation of Vps34-Beclin1 complex to initiate autophagy.Hence,our study reveals a novel mechanism of PRRSV-mediated autophagy and provides valuable information for further understanding the pathogenesis of PRRS,which might contribute to development novel antiviral drugs.