Dissection Of Function Of RdRp Porcine Erproductive And Erspiartory Syndrome Virus

In this study, we aimed at the RdRp of porcine reproductive and respiratory syndrome virus (PRRSV).PRRSV contains two genotypes, type I (European type, EU) and type II (North American type, NA), of which arerepresented by strain Lelystad Virus and strain VR-2332, respectively. However, recently PRRSV causeslarge-scale disease outbreaks and mass economic loss to the swine industry worldwide. In order to control thePRRS and find the key pathogenic factor, based on reverse genetics technique, our PRRSV infectious clones oftwo genotypes and EAV infectious clone, we define the function of RdRp, replication and transcriptionmechanism of PRRSV.1. The constructions and virological characteristics of SDD series mutants of PRRSVThe subgenomic mRNA transcription and genomic replication of PRRSV are directed by the viral replicase.The replicase is expressed in the form of two polyproteins and is subsequently processed into smallernonstructural proteins (nsps). nsp9, containing the viral replicase, has characteristic sequence motifs conservedamong the RNA-dependent RNA polymerases (RdRp) of positive-strand (PS) RNA viruses. To test whether theconserved SDD motif can tolerate other conserved motifs of RNA viruses and the influence of every residue onRdRp catalytic activity, many amino acids substitutions were introduced into it. Only one nsp9substitution, ofserine by glycine (S3050G), could rescue mutant viruses. The rescued virus was genetically stable. Alteration ofeither aspartate residue was not tolerated, destroyed the polymerase activity, and abolished virus transcription, butdid not eliminate virus replication. We also found that the SDD motif was essentially invariant for the signaturesequence of PRRSV RdRp. It could not accommodate other conserved motifs found in other RNA viralpolymerases, except the GDD motif, which is conserved in all the other PS RNA viruses. These findings indicatedthat nidoviruses are evolutionarily related to other PS RNA viruses. Our studies support the idea that the twoaspartate residues of the SDD motif are critical and essential for PRRSV transcription and represent a sequencevariant of the GDD motif in PS RNA viruses.2. Construction of genomic recombination systemRecombination mechanism resembles replication and transcription mechanism, all needs templateswitching and RdRp jumping. Also, it is reported some recombination hotspots are regulating elements ofreplication and transcription. To investigate the mechanism of PRRSV recombination, which is a way of viralvariation, and in order to investigate replication and transcription mechanism, we constructed a recombinationsystem and homologous recombination technique. Based on our reverse genetic technique and by usinghomologous recombination technique in rescuing PRRSV recombinants, we co-transfected several pairs ofreplicons to MARC-145cells. Results showed that the rescued recombinant s contained parental part sequences ofdonor and receptor. Homologous sequences shared in a pair of replicons and intermolecular homolog ous recombination occurred. The recombination sites w ere randomly distributed depending on the pairsco-transfected, including256to318nt within ORF1,13970to13992nt,14428to14494nt within ORF6and15135to15161nt within ORF7. Therefore, the homologous recombination technique not only disclosed thefeature of randomness of recombination sites, but also provided a new approach for the construction of infectiouscDNA clones of other RNA viruses.3. Construction of ORF1b chimeric of different genotypic PRRSVThrough investigating structure and function of RdRp among PRRSV I and II, EAV, based on the platformof reverse genetics technique and EVA, PRRSV infectious clones of genotype I and II, we constructed chimericpAPRRSasc-SHE1b, pAPRRSasc-EAV1b and pAPRRSasc-EAV1b234, pAPRRS-SHEnsp9p andpAPRRS-EAV2~73'. Results showed, firstly, RdRp and other nsps of one genotype, need to combine itsassociated viral genomic elements and proteins to transcript sgmRNAs of structural proteins. Secondly. in oneviral genotype, the non-conservative region of RdRp maybe can't be separated with its genes in order to functionnormally. Thirdly, in viral life cycle nsps need to function with its3'UTR, so we supposed that in one viralgenotype not only nsps must function with each other, but also RdRp needs3'UTR to function.