Suppression Of RNA-mediated Gene Silencing By Porcine Reproductive And Respiratory Syndrome Virus

RNAi is sequence-specific gene silencing mechanism activated by thedouble-stranded RNA（dsRNA）,As a natural defense mechanism to the foreign geneor virus attack, the phenomenon of RNAi existed widly in plant,namatodes worm,drosophila and human cells. However, virus can counteract this anti-viral defense ofthe host by viral proteins and non-coding viral RNAs, RNA silencing suppressor(RSS). Currently, about20RSSs have been identified from plant, animal and humanviruses20, and most of these RSSs are interferon antagonist.Porcine reproductive and respiratory syndrome (PRRS) is an infectious diseasecharacterized by respiratory disorders or abortion in pregnant sows and Pigletsbreathing difficulties, resulting in important economic losses to the swine industry.PRRSV, a small enveloped virus including a single positive-stranded RNA genome, isa member of family Arteriviridae which, along with the Coronaviridae, are classifiedin the order Nidovirales. The genome is about15kb, containing nine open readingframes (ORFs).ORF-1encodes14non-structural proteins (nsps), In addition to thecopy, nsps have protease, polymerase function, and inhibit interferon production.ORF2-7encoed viral structural proteins. The PRRSV exist immunologicalcharacteristics of immunosuppression and persistent infection,which can caused virusimmune escape. RNAi can inhibit of viral replication and clear the virus as a innateimmune defense system. A suboptimal innate immune response and the persistentinfection were the important features of PRRSV infection, so the aims of our presentwork are to address whether PRRSV could suppress the RNA silencing, whether thePRRSV encoded RSSs which help the virus overcome the cellular antiviral RNAsilencing response.In this study, by RNAi technology, the synthetic dsRNA and shRNA or mi-RNAinterference expression plasmid were transfected into MARC-145cells. The fireflyluciferase reporter gene(Luc) is the target gene targeted by dsRNA, shRNA andmi-RNA.Luc expression was suppressed on transfected MARC-145cells by by dualfluorescence detection system. It indicated that the dsRNA, shRNA, andmi-RNA-induced gene silencing model is successfully constructed on MARC-145cells;when PRRSV and RNAi model were acted on MARC-145cells, PRRSV restoreexpression of silenced luciferase reporter gene on MARC-145cells.It showned thatPRRSV can inhibit the shRNA, dsRNA, miRNA-induced gene silencing. Whennsp1or nsp11and RNAi model were con-transfected on MARC-145cells, nsp1andnsp11, also can restore expression of suppressed luciferase. nsp1hydrolysis nsp1αand nsp1β, and nsp1inhibit the shRNA, dsRNA, miRNA-induced gene silencing isachieved by nsp1α. nsp1α and nsp11were RNA silencing suppressor; ThePCPαactivity of nsp1α and the endoribonuclease activity of nsp11are inactivated inthe mutant of Nsp1α and nsp11.The mutant of nsp1α or nsp11and RNAi model were con-transfected on MARC-145cells, however, these mutants can not inhibit theRNAi-induced gene silencing. It demonstrated that the activity of PCPα and theactivity of endoribonuclease were respectively required for nsp1α and nsp11as theRSSs.PRRSV could suppress the shRNA, dsRNA and miRNA-induced RNAsilencing on MARC-145cells. nsp1and nsp11have effect on inhibitting theRNA-induced gene silencing,and nsp1α and nsp11are RNA silencing suppressor. Theactivity of PCPα and the activity of endoribonuclease were respectively required fornsp1α and nsp11as the RSSs.