Dissection Of Cis-Acting Elements In5’Untranslated Region Of Porcine Reproductive And Respiratory Syndrome Virus

PRRSV is the pathogen of Porcine Reproductive and Respiratory Syndrome (PRRS), which characterized by reproductive failure in breeding stock and respiratory problems in piglets. Now, it is the tremendous threat for the swine husbandry worldwide. Recently, significant economic losses were caused because of PRRS. PRRSV was divided into two distinct genotypes:Type I (the European type) and Type Ⅱ (the North American type). It should be unearthed that PRRSV genome replication, subgenomic mRNA transcription, protein translation, viral particle assembly and pathogenisis. At present, a large number of researchs demonstrated that5’ UTR of PRRSV was very important for viral replication. It was largely unknown that the possible cis-acting element and its regulatory mechanism. In this study, based on the type II PRRSV full-length infectious clone pAPRRS, we constructed a range of5’terminal sequences deleted mutants and UTR of two genotypes substituted mutants in PRRSV5’UTR. RNA/DNA transfection, virus rescue and virologic analysis was conducted for dissecting the putative cis-acting element and its structure and function, which could provide basis for interpreting PRRSV pathogenesis. The detailed experimental content was described as follows.1.Cis-acting structural element in5’UTR is essential for infectivity of Porcine Reproductive and Respiratory Syndrome VirusIt is believed that the genomic5’untranslated region (UTR) of Arterivirus plays crucial roles in viral genomic replication, subgenomic mRNA transcription and protein translation, yet the structure and function still remain largely unknown. In this study, we conducted serial nucleotide truncation, ranging from1to190nucleotides, to the5’UTR of the porcine reproductive and respiratory syndrome virus (PRRSV) infectious full-length cDNA clone pAPRRS. In vitro synthetic RNAs were transfected into MARC-145cells for further genetic and virologic analysis. Our results demonstrated that the first three nucleotides of PRRSV5’UTR were dispensable for virus viability, which however was repaired with foreign sequences. In order to assess if the primary sequence or structural element play more important regulatory roles, the CMV promoter-driven5’UTR truncation mutant cDNA clones were directly transfected into the BHK-21cell lines. We found that PRRSV tolerated the first16nucleotides sequence alteration of5’UTR without losing virus viability. However, these revertant viruses contained a range of non-templated with unknown origin exogenous nucleotides in the repaired5’end. Further analyses revealed that the5’proximal stem-loop1(SL1) in the highly structured5’UTR was invariably required for virus infectivity. Taken together, we conclude that authentic5’-proximal primary sequence is nonessential, but the resultant structural elements are probably indispensable for PRRSV infectivity.2. A cis-acting structural element related with viral infection was existed in Porcine Reproductive and Respiratory syndrome Virus5’UTRIn this study, in order to dissect the structure and function of PRRSV5’UTR, based on porcine reproductive and respiratory syndrome virus (type II, attenuated strain) full-length cDNA clone pAPRRS, a range of5’terminal sequences deleted mutants in PRRSV5’UTR were constructed. The effect of these alteration in viral infection, genomic replication, sg mRNA transcription and protein translation were analyzed via reverse genetic manipulation. A serial of unknown origin AU-rich exogenous sequences were detected in5’proximal region of the rescued virus. Because the transcriptional initiation sites for T7and CMV promoter were different, we introduced a GC-rich sequence in the mutant site in order to investigate virus rescue was template dependent or independent. The results showed that the repairment mechanism was template independent. Further secondary structure analysis showed that the SL1of PRRS5’UTR was indispensable for virus infectivity.3.5’ UTR of type I could be fully functional in type II genomic backboneThe5’untranslated region (UTR) of the genomic RNA is believed to be vital for the replication and transcription of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), yet its functional mechanism remains unclear. We got a chimeric clone, pAPLV5via substituting type I5’UTR into the backbone of type II attenuated strain infectious full-length cDNA clone, pAPRRS. Through twice passage after directly transfecting into MARC-145cells, the chimeric virus vAPLV5was rescued, chimeric virus was rescued and developed similar cytopathic effect. Remarkably, comparing their biological characteristics, the chimeric virus displayed the similar virological properties with the type II parental virus, including plaque morphology and growth kinetics. Northern blot analysis also showed that the chimeric virus maintained the same subgenomic mRNA profile. These results demonstrated that5’UTR of PRRSV type I can functionally replace that of the type II, and RNA secondary structure of the5’UTR plays a critical role for the replication of PRRSV.4. The functional dissection on heterologous5’untranslated Region for Infectivity of Porcine Reproductive and Respiratory syndrome VirusIn order to deeply analyze the type I5’ UTR how to play function in type II genomic backbone, after full-length sequencing, we found there were several nucleotide mutations in chimeric virus genome, mainly distributed in Nsp2and Nsp9. We introduced four mutant sites into the chimeric clone, pAPLV5. CPE appeared immediately when the four mutants were transfected into MARC-145cells. Viral titers were higher than parental chimeric virus and the plaque size was smaller than vAPLV5. Semi-quantitative RT-PCR was conducted for negative-strand genomic RNAs and sg mRNAs analysis. The results showed that the RNA synthesis level of the four mutant viruses were higher than vAPLV5. Taken together, the regulatory function was tightly related with RdRp, heterologous5’UTR with revertant RdRp played joint role in virus rescuing processes.It is the first report that the type I5’ UTR could play regulatory function in type II genomic backbone and fully functional. In addition, we did related research to explore the origin of5’UTR5’terminal exogenous sequences, results showed that the insertion sequences which used for repair was template independent. Taken together, in this study, we dissected the primary sequence and possible cis-acting element in PRRSV5’UTR. The results that we got could lay foundations in development of the marker vaccine and further study on the structural and functional analysis of PRRSV5’UTR.