Study On The Glycosylation Of GP3 Protein Of Porcine Reproductive And Respiratory Syndrome Virus Strain HuN4-F112

Porcine Reproductive and Respiratory Syndrome(PRRS) is still epidemic in most of the world,since first out break in North America in 1987. It can cause mild to severe reproductive failure in sows and respiratory problems in piglets. In 2006, a high fever syndrome, of which causative agent was later identified as highly pathogenic PRRSV (HP-PRRSV), was emerged in China, leading to serious reproductive obstacles and high mortality in all-age-pigs.GP3 is a secondary structural protein of PRRSV. It participates in viral formation with GP2a and GP4 Protein in the form of heterotrimer.GP3 is associated with protective immunity of PRRSV. It is the most heavily glycoslated envelop protein in PRRSV.Glycosylation is the post-translational modification process. Previous studies showed that glycosylation plays an important role not only in viral recognition, invasion, replication and immune escape of the host, but also closly related with the virulence and antigenicity of viruses.Howerer, it is still not clear enough about how the glycosylations of GP3 protein functions in PRRSV infection.In this study, we analysed the amino acid sequence of GP3 protein of a HP-PRRSV derived vaccine strain HuN4 -F112 with software,7 potential N-linked glycosylation sites were found. On the basis of infectious clone of HuN4-F112, N29,N42,N50,N13land N195 were chose to deglycosylation in single site or multiple sites by the way of site-directed mutagenesis PCR.After transcription and transfection in vitro and passaged on Marc-145 cells, viruses were successfully rescued from molecular cDNA clones with mutations of glycosylation site GP3-N29Q, GP3-N42Q, GP3-N50Q, GP3-N131Q, GP3-N29/50Q, GP3-N29/195Q. Meanwhile, it was failed to rescue viruses witn multiple mutations of glycosylation sites N29/42, N29/131, N29/50/131, N29/42/50/131/195.Sequencing results showed that the rescued viruses all contain the designed mutations in the glycosylation sites.Data from the multi-step growth curve of the rescued viruses indicated that the mutations of glycosylation sites N29, N42, N50, and N131 in GP3 protein didn't affect the viral progeny production, but these viruses grew at 10-fold lower titers than their parental PRRSV, especially the N50 mutation, whose titer was 6 log lower than its parental virus. Mutations with sites N29/42, N29/131, N29/50131, N29/42/50/131/195 did not result in infectious progeny production, sequence analysis found that they were the same as HuN4-F1112 except the designed mutation sites. Our results demonstrated that deglycosylation of sites N29, N42, N50, N131 could affect virus infection to the cells in varying degree, and deglycosylation of multiple sites resulting in failure of viral infection or replication. The outcome of the study may lay a basis for further study on the relationship between glycosylation and immunogenicity of PRRSV.