Identification Of PRRSV NSP9 Interacting With N Protein And Interactions Of NSP9 With Host Proteins Effect PRRSV Replication

Porcine reproductive and respiratory syndrome(PRRS) is a contagious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV), and it has caused enormous economic losses to the pork industry. Non-structural protein 9(NSP9) is the RNA-dependent RNA polymerase(RdRp) and palys an important role in the virus transcription and replication. N protein is the most abundant viral protein expressed in infected cells. One of the most important functions of N protein is to assemble with the viral RNA to form viral nucleocapsid. Our studies have shown that NSP9 interacts with N protein in PRRSV type 2 strain NVSL97-7895, and the two proteins bind to each other through direct protein-protein recognition. However, the domain involved in the interaction is not clear and whether the interaction exists in other PRRSV strains is unknown. In this study, we identified the domains of NSP9 interacting with N protein and verified the interaction between NSP9 and N protein in other PRRSV strains. The main methods and results are listed as below:(1)We tested the interaction of truncated NSP9 with N protein, and found that the key region was in the 453-646 amino acid residues of NSP9.(2)We investigated the interactions between NSP9 and N proteins of PRRSV type 1 strain Olot/91 and type 2strainTA-12 using yeast two-hybrid assay. The result showed that NSP9 interacted with N protein of Olot/91 strain, but negative result was obtained for TA-12 strain.(3) We expressed and purified PRRSV TA-12 NSP9 and N protein in prokaryotic expression system.The protein-protein interaction between NSP9 and N protein was confirmed by Pull-down experiment.(4) Adding three different inhibitors(respectively inhibiting the host proteins HSP90?Na/K-ATPase and calmodulin which interact with NSP9) to PRRSV infected Marc-145 cells, we showed that the inhibitors significantly decreased virus titers and viral mRNA levels.In conclusion, the key domain of NSP9 interacting with N protein was identified and the existence of the interation was confirmed in different PRRSV strains in this study. Our study laid the foundation for further researches on the function of NSP9 and N protein, PRRSV–host interactions, and the screening of drug targets against PRRSV.