Role Of Porcine Reproductive And Respiratory Syndrome Virus Nucleocapsid Protein In Viral Transcription

Since reported in the 1990 s,porcine reproductive and respiratory syndrome(PRRS)has caused dramatic economic losses to the swine industry worldwide.The etiology of PRRS,PRRSV shows high level of genetic variety and could lead to persistant infection and immunosupression of the infected pigs.The universal use of PRRS vaccines played an important role in preventing and controlling this disease.However,several highly pathogenic PRRSV virants emerged,which was thought to be result of immune pressure.In order to uncover the mysterious veil of PRRSV,a number of studies have been made focusing on viral pathogenicity and immunity mechanisms,but the correlation of the charatericstic transcription and replication mechanism to viral pathogenicity remains obscure.Thus,there is a need to explore the potential transcriptional factors and machanism that regulating the balance of viral genomic RNA and subgenomic RNA production.In order to establish PRRSV N protein Western blot analysis,heat shock expression vetor pCold-I was used for the prokaryotic expression of PRRSV N protein.The purified N protein was used as immunogen and immunized to BALB/c mice and the serum was collected after 4 times immunization.The newly prepared polyclonal antibody showed specific immunogenicity to mammalian expressed or anthentic PRRSV N proteins when tested by Western blot analysis and indirect immunoflorecence assay,which layed a solid foundation of this study.The phosphorylation of the nucleocapsid protein is a common feature of the Nidoviruses.In order to establish a reliable method for the identification of the phosphorylation site of PRRSV N protein,Phos-tag analysis was used in this study.The result showed that phosphorylated mammalian expressed N-HA or viral N protein could be separated into two specific bands under Phos-tag SDS-PAGE electrophoresis.Phosphorylation of the immunoprecipitaed N-HA protein was removed by treatment of calf intestinal alkaline phosphatase,indicating only one amino acid site was phosphorylated in PRRSV N protein.Subsequently,a series of mutant N protein expression vectors were constructed by overlap PCR.Moreover,other N gene containing all the serine-alanie substitution was synthesized and clone into p CAGGS vetor as a negative contral.The result showed that Ser120-Ala mutation was responsible for the disappear of the upper band,indicating the Ser120 was the phosphorylation site of PRRSV N protein.In order to further investigate the role of N protein phosphorylation to viral replication.Reverse genetic manipulation was used to mutant N protein phosphorylation.The rescued virus N protein showed only one band compared to its parental virus or the wild type contral,indicating the successful blocking of PRRSV N protein phosphorylation.N protein phosphorylation showed no influence on viral infectivity,plaque morphology or protein subcellular locaolization.However,the mutant virus showed impaired growth ability and the viral titter between 24-72 hours post infection was about 10 fold lower than that of parental virus.Quantantive PCR revealed that the relative aboundance of the mutant viral genome RNA and long subgenomic RNAs was significantly lower at the early step of infection,whichmay be the reason of impaired growth ability.All this results demonstrated that N protein phosphorylation is not essential for virus infectivity,and this post translational modification may participated in regulating viral transcription.Except for phosphorylation,N protein could also interact with many cellular proteins.In this study,we found that PRRSV N prtein could interact with cellular DEAD box RNA helicase 5(DDX5),which is a member of the DexD/H-box family,and colocalized in the cytoplasma.The interaction domain was proved to be N-N terminal interaction without the mediation of RNA.To further analysis the potential role of DDX5 in viral replication,siRNA interference was performed to downregulate the expression level of DDX5.The result showed that PRRSV replication was promoted when cellular DDX5 expression level was down-regulated.On the contrary,the transcription level of viral genomic RNA and long subgenomic RNAs were significantly increased when DDX5 expression was silenced.This result indicated a negative regulation role of DDX5 during viral continous transcription.