Molecular Mechanisms Of The Interactions Of PRRSV Nsp2with DDX3X And Nsp9with DDX5

Porcine reproductive and respiratory syndrome virus (PRRSV) has been considered the most significant pathogen severely impacting the swine industry worldwide. The nonstructural proteins (Nsps) of PRRSV play important roles in viral replication, immunomodulation and pathogenesis. To investigate the interactions of Nsps and host cellular proteins and their roles in viral replication regulation and pathogenicity is of significance for elucidating the pathogenesis of PRRSV. In the present study, we confirmed the interactions of Nsp9with host cellular protein DDX5and Nsp2with DDX3X, and analyzed the molecular mechanisms and biological significances of the interactions, in order to provide scientific basis for the pathogenesis of PRRSV.By Co-IP assay, DDX5was shown to interact with viral Nsp9in the co-transfected HEK293cells with the DDX5-and Nsp9-expressing plasmids, and the interaction between endogenous DDX5and Nsp9was also confirmed in MARC-145cells infected with the Nsp9-expressing lentiviruses. Then, the interacting domains between DDX5and Nsp9were determined to be the DEXDc and HELICc domains in DDX5and the RdRp domain in Nsp9, respectively. Moreover, in HEK293cells, MARC-145cells and PAM cell lines co-transfected with the DDX5-and Nsp9-expressing plasmids, Nsp9was shown to co-localize with DDX5in the cytoplasm with a perinuclear pattern, and meanwhile in PRRSV-infected MARC-145cells and PAMs, endogenous DDX5was also found to co-localize with Nsp9. Finally, silencing the DDX5gene in MARC-145cells significantly impacted the replication of PRRSV, and while the over-expression of DDX5could slightly enhance viral replication. These findings indicate that DDX5positively regulates the replication of PRRSV via its interaction with viral Nsp9in vih-o, suggesting that the interaction of Nsp9and DDX5promotes PRRSV replication, providing a novel clue for exploring possible mechanisms associated with PRRSV replication.The interaction and binding regions of DDX3X and Nsp2were identified by Co-IP. The results showed that the N-terminus of Nsp2interacted with DDX3X, whereas the interaction was not mediated by cellular DNase or RNase. The confocal microscopy analysis showed that Nsp2co-localized with DDX3X in the cytoplasm in HEK293cells, MARC-145cells and PAM cell lines co-transfected with the DDX3X-and Nsp2-expressing plasmids, and meanwhile in PRRSV-infected MARC-145cells and PAMs, endogenous DDX3X was also found to co-localize with Nsp2. The interaction of DDX3X with exogenous MAVS was demonstrated by Co-IP, and the binding of the HELICc residues of DDX3X to the CARD domain of MAVS was further determined. DDX3X was shown to co-localize with MAVS in the cytoplasm by confocal microscopy analysis. Luciferase reporter assay was performed to detect the effect of DDX3X on IFN-β responsive promoter. The results showed that DDX3X could activate and promote MAVS to activate the IFN-(3promoter in a dose dependent manner. Nsp2could inhibit IFN-β and IRF3promoter, and affect the phosphorylation and relocalization of1RF3. Further analysis indicated that Nsp2could inhibit MAVS-mediated IFN-β promoter activity and IRF3phosphorylation following PRRSV infection in MARC-145cells or in HEK293cells transfected with Nsp2-expressing plasmid. Co-IP and Western blotting assays showed that the expression level of DDX3X and the interaction of DDX3X with MAVS were both influenced in the condition of PRRSV infection and Nsp2-expression. Meanwhile, the expression of DDX3X was not benifical for PRRSV replication by DDX3X-expressing lentivirus or siRNA assay. These results suggested that the interaction of Nsp2and DDX3X sequestered the interaction between DDX3X and MAVS and affected MAVS-induced IRF3phosphorylation and promoter activity. This interaction then inhibited IFN-β promoter activity and blocked the production of IFN-β, which could promote the PRRSV replication.As a whole, our study revealed the molecular mechanisms affecting viral replication by the interactions of PRRSV Nsp9with DDX5and Nsp2with DDX3X, providing a novel insight and way for further understanding PRRSV replication regulation and pathogenesis.