Dissection Of Structure And Function Of3’UTR Terminal Sequence Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is an RNA virus causing respiratory and reproductive syndrome (PRRS) which is characterized by reproductive failure in breeding stock and respiratory problems in piglets, and causing enormous loss for swine industry. PRRS has been epidemic in the global swine population, and then resulted in enormously economical cost for the swine industry. The genome of PRRSV is a positive single-stranded RNA, about15kb in length, and consists of a5’cap, a5’untranslated region (UTR), ten open reading frames, followed by a3’UTR and a poly (A) tail. PRRSV isolates are divided into two distinct genotypes:Type I (the European) and Type II (the North American). The3’UTR of positive-stranded RNA virus genome plays an important role for viral cycle as the virus negative strand genome replication and subgenomic mRNA transcription initiate at the3’end, yet the properties of3’UTR of PRRSV remain undefined. In this study, based on the type II PRRSV infectious clone, we focus on PRRSV3’UTR terminal conserved sequences and performed a mutagenesis analysis to define the structure and function of the cis-acting elements in the3’end. The detailed experimentations are described below.1Bio-infomatic analysis about the primary sequence and secondary structure of PRRSV3’ UTRUsing the bio-infomatic software MegAlign, RNAalifold, Mfold, the primary sequence and secondary structure of PRRSV genomic3’UTR of100strains from GenBank were analyzed. The results showed that (i) the homology of the3’UTR nucleotide sequence of the two type PRRSV is between69%to77%;(ii) there are very conserved sequences(5’-AGUCACCUAUUCAAUUAGGG UGGG GGC AACCA CCG AAUU-3’) present in the3’UTR;(iii) the3’UTR of PRRSV formed a highly conserved RNA structure, a long bulged-stem loop structure, and in the top there is a highly conserved stem loop structure, another end of the nucleotide segment containing a single chain, this structure may be essential for viral replication.2Study on the effect of3’UTR terminal sequence5’-AAUU-3’in PRRS viral replicationBased on the porcine reproductive and respiratory syndrome virus (PRRSV) infectious full-length cDNA clone pAPRRS, serial site mutations of5’-AAUU-3’were engineered into the original pAPRRS by PCR-based mutagenesis. These plasmid DNAs were transfected to BHK-21cells, the protein translation and RNAs synthesis were analyzed by indirect immunofluorescence (IFA) and reverse transcription PCR (RT-PCR), respectively. The supernatants of the transfected cells were collected and subsequently used to infect MARC-145cells to detect the production of progeny viruses. Viral infectivity was identified by PRRSV-specific CPE, the virological characteristics were identified by plaque assay and multistep growth curve, and the genomic RNAs of rescued virus were analyzed by RT-PCR. We found that the protein translation and RNAs synthesis was normal in the mutants pAM01C, pAM03T, pAM03Q pAM03C, pAM04G, pAM04C, however, only pAM01C, pAM04C and pAM04G could rescue virus. So it could be concluded that (i) the3’-terminal U is optimal for viral replication;(ii) wild-type sequence at the2nd and3rd position of the tetranucleotide is absolutely for viral replication;(iii) the4th position from the3’end of the genome also play a key role in virus replication. Analysis with the MFold program indicated that all mutants retained an RNA structure identical to the wild type.3Dissection of the function of terminal sequences5’-AACCA-3’in PRRSV3’UTRThe PRRSV3’UTR with a stem loop confirmed by conserved sequences5’-AACCA-3’is predicted to play a key role for viral replication and could be a poly(A) tailing signal for virus. In order to identify the function of these sequences and their secondary structure, serial mutations were engineered into the terminal sequences5’-AACCA-3’in the original infectious full length cDNA clone pAPRRS3’UTR to change their primary and secondary structures. These plasmid DNAs were transfected into BHK-21 cells. The results showed that the stem-loop structure of5’-AACCA-3’was not crucial for viral infectivity, while the primary structure of5’-AACCA-3’played a key role in the process of viral replication. Each of the single point mutants had the similar replication with the WT; in the serial deletion mutants, single base deletion had no effect on viral infectivity; dinucleotide deletion reversed the last dinucleotide CA also had no effect, however, trinucleotide and tetranucleotide deletion mutants had no viability. These studies suggested that the primary sequence conservation of5’-AACCA-3’rather than its surrounding secondary structure is vital for PRRS viral subgenomic RNA synthesis and viral infectivity. And these conserved sequences may be a tailing signal of poly (A) in3’-end region.In conclusion, the results that we got from this study can lay foundations in development of the genetic engineering vaccine and further study on the structural and functional analysis of PRRS V3’UTR.