The Effect Of GP5 Expression In Marc-145 Cells To Porcine Reproductive And Respiratory Syndrome Virus Infection

Porcine reproductive and respiratory syndrome(PRRS) is caused by PRRS virus(PRRSV), a member of the Arteriviridae family,order Nidoviridales. This enveloped virus contains a 15-kb positive-strand RNA genome that encodes a replicase polyprotein(open reading frames [ORFs] 1a and 1b) and six structural proteins(ORFs 2 to 7). The products of ORFs 2 to 4 are minor membrane-associated glycoproteins(GP2, GP3,and GP4, respectively). The products of ORFs 5 to 7 are the three major structural proteins(GP5, N, and M proteins, respectively). The major neutralizing epitope of porcine reproductive and respiratory syndrome virus(PRRSV) is mainly located on virus glycoprotein 5(GP5). Immunization with exogenous GP5 or exposure to native GP5 by means of DNA immunization can provide some degree of immune protection to PRRSV infection in pigs. However, during PRRSV infection in pigs, the production of neutralization antibodies induced by GP5 is delayed or suppressed. This suggests that other PRRSV proteins interfere with the function of GP5 in inducing host responses during virus infection. Here, to exclude the impacts of the other PRRSV proteins and determine the role of GP5 in the replication of PRRSV in vitro, the following results are confirmed.1. To determine the role of GP5 in the replication of PRRSV in vitro, a Marc-145 cell line stably expressing GP5(Marc-145-GP5Flag) was constructed. IFA and Western blot results show that GP5 Flag was expressed only in Marc-145-GP5 Flag cells.2. Cell proliferation and cell apoptosis measurements indicated that the expression of GP5 in Marc-145 cells did not disturb the cells’ viability, and did not induce cell apoptosis.3. Following infection with different PRRSV strains SD16, VR2332, JXA1, PRRSV replication in Marc-145-GP5 Flag cells was inhibited significantly, but not attachment to cells.4. Type I interferon assay results showed that beta interferon(IFN-β) in the Marc-145-GP5 Flag cells were increased at mRNA and protein levels. When siRNA was introduced into the cells to knock down IFN-β mRNA, PRRSV infectivity of these cells was recovered.Based on the above results, to determine the role of GP5 in the replication of PRRSV in vitro, a Marc-145 cell line stably expressing GP5(Marc-145-GP5Flag) was constructed. These data suggest that early GP5 expression is not favorable for further infection by PRRSV, because it not only stimulates production of neutralization antibodies in pigs, but also induces IFN-β production in host cells.