Inhibition Of Porcine Reproductive And Respiratory Syndrome Virus Replication By Short Hairpin Targetting Non-Structural Protein12in Marc-145Cells

Porcine reproductive and respiratory syndrome virus (PRRSV) which is single-stranded positive-sense RNA virus is a member of the family Arteriviridae in the order Nidovirales. The～15kb PRRSV genome contains nine overlapping open reading frames (ORFs). ORF1a and lb comprise about80%of the genome. The upstream ORF1a encodes a single polyprotein la (ppla), which is predicted to be cleaved to form nine non-structural proteins:NSP1through8. The downstream ORF1b is expressed as a fusion protein with pp1a by a mechanism involving ribosomal frameshift. The ORF1ab gene product is referred to as polyprotein lab (pp1ab). Proteolytic cleavage of the polyprotein generates the polypeptides Nsp9through12. The remaining ORFs (2a,2b and3-7) code for viral structural glycoprotein GP2a, GP2b,GP3,GP4,GP5, unglycosylated2b/E and membrane (M) proteins and a nucleocapsid protein (N).RNA interference (RNAi) is a naturally occurring cellular mechanism of gene suppression which is induced small interfering RNA (siRNA) molecules homologous to some region of the target gene. The applications of RNAi can be mediated through two types of molecules; the chemically synthesized double-stranded small interfering RNA (siRNA) and vector based short hairpin RNA (shRNA). Optimized shRNA constructs allow for high potency and sustainable effects using low copy numbers resulting in less off-target effects, and it induces long lasting RNA silencing in mammalian cells, while the effect of short interfering RNA (siRNA) is generally transient in transfected animal cells. In the recent years, RNAi has become widely used as an experimental tool to study viral and cellular gene function, to identify genes that regulate virus infection and replication, and it represents a new feasible approach to develop effective treatments for viral infections.In this study, we designed two shRNA targeting different regions of NSP12gene to study the function of NSP12gene. The results showed that both of the shRNA were validated to inhibit PRRSV replication in Marc-145cells. Additionally, we demonstrated that shRNA targetting NSP12can inhibit PRRSV subgenome replication.