The Effect Of GP5^Δ84-119 Stable Expression On The Replication Of Porcine Reproductive And Respiratory Syndrome Virus Anda Preliminary Study Of Its Mechanism

Porcine reproductive and respiratory syndrome（PRRS）, commonly known as blue ear disease is a contagious disease caused by porcine reproductive and respiratory syndrome virus（PRRSV）, and in pregnant sows miscarriage, stillbirth, fetal weak respiratory symptoms,mummified and other reproductive disorders and pigs of all ages, the high mortality rate of piglets, immune suppression and persistent infection as the main feature. Since PRRSV cell tropism macrophages, antibody-dependent enhancement（ADE） and persistent infection control measures and other features, pathogenic mechanism and immune mechanism of the disease is not entirely clear, for PRRS has no effective prevention and control measures at present.GP5, encoded by ORF5, is a glycosylated envelope protein and is the main structural protein of PRRSV which plays an important role in the virus assembly, invasion and induce immune response in the body. Molecular mass of GP5 is about 25 ku which with a high degree of variability, and the American type strains and European strains of GP5 were respectively composed of 200 and 201 amino acid residues. Mature GP5 is consisted of an amino-terminal signal peptide（1-31aa）, extracellular region, three transmembrane hydrophobic region and hydrophilic carboxyl terminal in cytoplasm. Three transmembrane hydrophobic regions formed two extracellular region in the viral envelope, and the glycosylation sites of the American type strains in the first extracellular amino terminal 44 and 51（in LV 46 and 53）were highly conserved among different strains. In LV strain, the glycosylation of 46asparagine（Asn） is essential for assembly and infectivity of viruses, but it does not seem to be important for the glycosylation of Asn 53. There is no relevant reports about the relationship of the second extracellular region and the viral replication. To study the role of the second extracellular domain of GP5 on PRRSV replication, a recombinant plasmid(named as pPB-GP5^Δ84-119) expressing truncated GP5 was constructed using PiggyBac Transposon System Vectors and transfected to Marc-145 cells. Marc-145 cell line stably expressing GP5^Δ84-119 was obtained by puromycin resistance screening and triple subcloning. GP5^Δ84-119 was confirmed stable expression in Marc-145 cells by RT-PCR, western blot and confocalanalysis and the cells was named as Marc-145-GP5^Δ84-119. The result of cell proliferation assay using cck-8 confirmed that the expression of GP5^Δ84-119 did not affect the proliferation of Marc-145 cells. It was found that GP5^Δ84-119 expression inhibited the replication of highly pathogenic PRRSV in Marc-145 cells through induction of IFN especially IFN-β. These findings suggested that the second extracellular domain of GP5 plays a regulatory role on PRRSV replication. Cells were treated with the PI3 K inhibitor LY294002 and ERK specific inhibitor U0126 and then inoculated with PRRSV SD16, the results showed that the changes had nothing in common between PRRSV N gene copy numbers in cells and virus titers in the supernatant in different samples which indicated thatthe regulation of PRRSV replication by GP5 was closely related with PI3 K signal pathway and MEK signal pathway.