Determination Of The Interactome Of NSP7 And NSP12 From Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus With Host Cellular Proteins Using High Throughput Proteomics

Porcine reproductive and respiratory syndrome virus(PRRSV) has caused tremendous economic losses and continues to be a serious problem to the swine industry worldwide, and it is also a major threat to global food security. However, there is no satisfied vaccine and antiviral therapies approved due to the high mutation rate of this RNA virus. Although extensive research has been focused on PRRSV, the structure and function of some viral proteins like nonstructural protein 12(NSP12) and NSP7, which may play important roles in viral replication and production, still remain unknown. Some published data have been focused on the relationship between PRRSV viral proteins such as N,NSP1β,NSP2 and whole virion and the host cellular proteins,but cellular interactomes of PRRSV NSP12 and NSP7 have not been reported to the best of our knowledge. In order to better understand the function of NSPs, we determined the interaction of NSPs with cellular proteins using quantitative proteomics coupled with an immune-precipitation strategy based on the over expression of the NSPs-EGFP fusion proteins in 293 T cells.The characteristics of PRRSV NSPs were studied in both infected cells and prokaryotic expression systems. In infected Marc-145 cells, infection percentage reached the peak at 48 h.p.i. In infected PAM cells, NSP12 was detected at 12 h.p.i. and its expression reached the highest level at 24 h.p.i. in infected Marc-145 cells, which suggested the important roles of NSP12 in the early infection stage of virus growth.Nsp7 expressed in E.coli was soluble, and purified NSP7,which may be useful for its structure and function studies, was produced. In contrast, NSP12 expressed from E.coli was inclusion body. More optimization needed to be done to obtain soluble proteins.12 cellular proteins were identified having potential interaction with PRRSV NSP7. Among them, peroxiredoxin 4 was chosen in further studies, in which we found that knockdown of Peroxiredoxin 4 reduced viral production. The interactome analysis suggested that PRRSV NSPs formed multiple cellular protein interactions that may have multiple implications for both viral and host cell biology.Our data showed that 112 cellular proteins had a high probability to interact with NSP12-EGFP. The majority of those proteins were nucleic acid binding proteins or chaperones involved in RNA post-transcriptional modification, protein synthesis and cellular assembly and organization. Ingenuity Pathways Analysis(IPA) analysis identified eukaryotic initiation factor-2(e IF2) signaling pathway which involved in eukaryotic protein synthesis as the most significant pathway associated with the expression of PRRSV NSP12. Among them, cellular chaperon HSP70 was identified to interact with PRRSV NSP12 protein, and inhibition of HSP70 significantly reduced the viral m RNA synthesis and virus replication.Western blot results showed that the level of NSP12-EGFP dramatically decreased with the existence of HSP70 inhibitor compared with the control. These data suggest that NSP12 can recruit cellular proteins such as HSP70 to maintain its own stability and benefit for the virus replication.To investigate if protein degradation contributed to the reduction of NSP12-EGFP level, MG132 was used to inhibit the proteolysis by(ATP)-ubiquitin(Ub)-dependent proteasome. Results of the MG132 treatment showed that the level of NSP12-EGFP was effectively recovered by the proteasome inhibitor, suggesting that association of NSP12 with HSP70 could protect the protein from degradation.To conclude,our results shed some light on the possibility of developing potential therapeutics targeting specific cellular proteins against PRRSV infection.