Interaction Of Porcine Reproductive And Respiratory Syndrome Virus Nucleocapsid Protein With Cellular PARP-1，DHX9 And Viral RdRp And The Effects On Virus Replication

Procine reproductive and respiratory syndrome(PRRS) which caused by procine reproductive and respiratory syndrome virus(PRRSV) is a serious epidemic endangering the swine industry worldwide. The nucleocapsid(N) protein of PRRSV is the most abundant viral protein, and its major function is to encapsidate viral genomic RNA. N protein could also affect the function of a variety of cellular proteins. Analysis of host differentially expressed host proteins caused by PRRSV infection will help to understand the molecular mechanism of PRRSV pathogenesis. Meanwhile, analysis of the interaction between N protein and cellular proteins will shed light on the function of N protein in the process of viral infection. This study explored the interactions between PRRSV N protein and cellular DHX9 and viral Rd Rp, and investigated the effects of protein interactions between N protein and cellular PARP-1, DHX9 and PRRSV Rd Rp on viral proliferation. The main results are as follow:1. Thirty-nine differentially expressed proteins of procine alveolar macrophages(PAMs) in response to PRRSV infection were identified by quantitative mass spectrometry. By bioinformatic analysis, we found that the up-regulated expressions of host IL1β and IL8 could be important factors leading to the cellular pathologic changes after PRRSV infection, and PRRSV could evade host innate immunity by up-regulation or down-regulation of different cellular proteins. Further analysis of the one hundred and eight cellular partners of PRRSV N protein obtained by quantitative mass spectrometry, showed that N protein is closely related to cellular translation and RNA processing.2. Inhibiting the function of an interacting partner, PARP-1, by a small molecule inhibitor 3-AB, we found that cellular PARP-1 played an important role in viral RNA and protein syntheses as well as progeny virus production. In the 3-AB drug sensitivity test, no drug resistant mutant virus was produced after fifteen continuous passages under the slection pressure of 3-AB. These results proved that it was an effective way to identify the key cellular proteins involved in virus proliferation through the interactome of PRRSV N protein.3. By co- immunoprecipitation(coIP) assay and fluorescence microscopy observation, we confirmed that cellular DHX9, a RNA helicase, interacted with PRRSV N protein. The redistribution of endogenous cellular DHX9 from nucleus to cytoplasm in N protein overexpressed and PRRSV infected cells was observed through immunofluorescence assay. By interfering with DHX9 expression, we found that the syntheses of PRRSV genomic RNA(gRNA) and long subgenomic RNA(smRNA) were suppressed. Convers ely, over-expression of cellular DHX9 promoted the syntheses of long viral RNAs. These results indicate that cellular DHX9 in nucleus could be recruited by PRRSV N protein into cytoplasm to promote the viral long RNAs syntheses.4. PRRSV N protein was identified to interact with viral nonstructural protein 9(NSP9), the RNA-dependent RNA-polymerase(Rd Rp), in different strains. The contacting surface on NSP9 was determind at the C-terminal end 599-646 residues of the protein. Mutagenesis analyses identified the E646, E608, E611 on NSP9 and Q85 on N protein as the key amino acids involved in the protein-protein interaction. Competitive inhibition of the N-NSP9 interaction by over-expressing N protein binding fragment of NSP9 in viral infected cells, markedly inhibited the syntheses of viral total RN As and gRNA, while the inhibitory effects could be relieved by substituting the critical residues involved in the interaction. These data confirmed that N-NSP9 interaction could participate in the regulation process of viral RNA replication and transcription.In conclusion, the host proteins associating with PRRSV pathogenesis were anylyzed by high-throughput proteomic approach in this study. N protein was found to mainly interact with cellular proteins involved in RNA processing and translation. Further studies revealed that cellular PARP-1 played an important role in PRRSV biology, and DHX9 interacted with N protein to promote the viral reproduction. Most notebly, PRRSV N protein was found to interact with viral Rd Rp and benefit viral RNAs’ syntheses. These findings provided clues for the biological roles of PRRSV N interacting with cellular proteins and the molecular mechanism of viral minus-strand RNA synthesis during PPRSV infection.