The Interference Study Of The Porcine Reproductive And Respiratory Syndrome Virus Nsp10 Gene And Identification The Phosphorylation Sites Of N Protein

Porcine respiratory and reproductive syndrome virus(PRRSV)was first isolated in the U.S.A in 1987 and,then,became prevalent worldwide.In 1996,PRRSV was first reported in China.Since then,it has spread rapidly throughout the country.In 2006,highly pathogenic(HP)-PRRSV characterized by a high fever and high mortality emerged in China and caused great economic losses.In 2014,the Nsp9 and Nsp10 were reported to be responsible for the virulence of the highly pathogenic porcine respiratory and reproductive syndrome virus(PRRSV).Thus,a phylogenetic analysis based on the Nsp9 and Nsp10 genes of PRRSV is of great significance.In this study,94 strains were collected from NCBI(National Center of Biotechnology Information).The sequence analysis showed that the minimum homology value of Nsp9 gene and amino acid were 86.0% and 94.4%,repectively.The minimum homology value of Nsp10 gene and amino acid were 84.2% and 93.0%,respectively.Compared with the GP5,they are more conservative.The SNAP analysis found that the N-terminus of the Nsp10,and the C-terminus of Nsp9 and Nsp10 showed high amino acid substitution rate.The amino acid analysis of Nsp9 and Nsp10 showed that the five amino acid may have relationship with the PRRSV virulence,including residues 384,519 and 544 of Nsp9 as well as residue 408 and 410 of Nsp10.It provides a theoretical basis for the study of Nsp9 and Nsp10 functions.According to the Nsp10 gene and the designning method of siRNA,three siRNAs were selected,namly siRNA-Nsp10-696,siRNA-Nsp10-875 and siRNA-Nsp10-1095.Three plasmids were constructed and transfected into Marc-145 cells followed by PRRSV infection.The IFA result showed all siRNAs could inhibit the PRRSV cytopathic effect(CPE).The western-blotting and growth curve showed the siRNAs could inhibit the PRRSV replication ability and protein expression.The real-time PCR results showed the inhibitory effect of siRNA-Nsp10-696,siRNA-Nsp10-875 and siRNA-Nsp10-1095 reached 99.24%,99.90% and 99.25% in Marc-145.The research indicated that the Nsp10 were an effective potential target for antiviral drug.The nucleocapsid(N)protein is the most abundant protein of PRRSV.It could interacte with Nsp9,Nsp10 and viral RNA.However,the phosphorylation sites of the N protein are still unclear.The function of phosphorylation modification still remains unkown.In this study,the phosphorylation sites of N protein and the function of phosphorylation of N protein were analysed.The recombinant N protein were expressed by baculovirus expression system,and purified by ion exchange chromatography and Ni chromatography.Two phosphorylation sites were found by liquid chromatography tandem mass spectrometry(LC-MS/MS).We mutated the phosphorylation sites(Ser105Ala and Ser120Ala)by reversing genetics platform and three mutated viruses were rescued,namely A105,A120 and A105-120.The IFA results suggested that the mutations did not affect the virus rescue.The growth curve showed that compared with wild-type virus,the titers of the mutated viruses at 48 hpi were significantly decreased.It is indicated that the mutations impaired the viral replication ability.The real-time PCR results were consistent with the multistep growth kinetics,the Nsp9 and N mRNA level were decreased since the mutations.However,the amount of the N expression did not change.The results indicated that the mutations may impaired the N protein function.For investigating whether the phosphorylation affected the N protein function,we constructed four plasmids,namely pCA-XH-GD,pCA-A105,pCA-A120 and pCA-A105-120.The plasmids transfected into BHK-21,the laser confocal results showed the mutations did not affect the N protein distribution in cells.The laser confocal results of the infected Marc-145 showed the same result.It is indicated that the mutations did affect the nuclear localization signal and export signal.Since the N protein could regulate the IL-10,the pCA-XH-GD,pCA-A105,pCA-A120 and pCA-A105-120 were transfected into the PAMs,respectively.The real-time PCR results showed that mutations at residues 105 and 120 were associated with down-regulation of the IL-10 mRNA level.Howerver,the relationship between the IL-10 mRNA and viral replication ability as well as whether the phosphorylation modification could impair the other functions of N protein need to be further investigated.In conclusion,we have designed three the siRNAs that could inhibit the PRRSV replication.Our results also found a novel phosphorylation site of the N protein and proved that the phosphorylation of N may play an important role in viral growth and N protein function.These studies could help us to understand the PRRSV life cycle and function of the phosphorylation modification of N protein.