Molecular Defining Cis-acting Elements Of 3′-untranslated Region For Porcine Reproductive And Respiratory Syndrome Virus Replication

Porcine reproductive and respiratory syndrome virus (PRRSV) is causative agent of porcine reproductive and respiratory syndrome, which is characterized by reproductive failure in breeding stock and respiratory tract illness in young pigs. 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, about 15 kb in length, and encodes at least nine open reading frames (ORFs). In addition, the PRRSV genome contains a 5′untranslated region (5′UTR), which carries a cap at its 3′end, and a 3′untranslated region (3'UTR) which the poly(A) tail is attached. Little is known about PRRSV genomic RNA replication, subgenomic mRNA transcription, protein translation, virus particle assembly and viral pathogenesis etc. The virus genome 3′UTR and poly (A) tail play important role in viral replication because of the virus negative strand genome replication and subgenomic mRNA transcription initiating at the 3 'end. Using the North American type PRRSV full-length infectious cDNA clone, we performed a mutagenesis analysis to define the structure and function of the 3′UTR and the poly(A) of the PRRS viral genome in viral replication.1. In silico analysis of the primary sequence and secondary structure of genomic 3′UTR of PRRSVUsing the bioinformatics software Clustal W, RNAalifold, Mfold, the primary sequence and secondary structure of PRRSV genomic 3′UTR of 128 strains from GenBank were analyzed. The results shown that (i) The type I PRRSV 3′UTR is 37 nt shorter than that of type II; (ii) The homology of the 3 'UTR nucleotide sequence of the two type PRRSV is between 69.3% to 77.2%; (iii) There are very conserved sequence (5′-AGUCACCUAUUCAAUUAGGG UGGG GGC AACCA CCG AAUU-3') present in the 3′UTR; (iv) The 3′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 necessary for viral replication.2. Study on the effect of the mutagenesis of 3′UTR in PRRS viral replicationIn this study, using PCR site-directed mutagenesis, the nucleotide mutation including replacement,insertion,deletion were introduced to the PRRSV 3′UTR. Then 11 mutants were successfully constructed. MACR-145 cells were transfected with full-length PRRSV cDNA clones containing desired mutations. The expression of viral proteins was monitored by an indirect immunofluorescence assay (IFA) using antibody recognizing the structural protein N. The result shown that (i) the conserved sequence of PRRSV 3′UTR is required for the PRRS viral replication; (ii) The mutant with replacement or deletion in the conserved sequence loses their ability to replicate; these conserved sequences may be recognized by viral or cellular protein. While replacement in the non-conserved sequence found that these non-conserved region is not essential for viral replication. However, deletion within the non-conserved sequence showed that the virus could not be rescued. In addition, insertion in the 3′terminal sequence is not tolerated.3. Function of the 3′terminal sequence(5′-AAUU-3′) of the PRRSV genome in viral replicationThe 3′untranslated region (3′UTR) plays an important role in positive single-stranded RNA virus replication and subgenomic RNA transcription, but the cis-acting elements in 3′UTR of the porcine reproductive and respiratory syndrome virus (PRRSV) have remained largely undefined. Using an infectious full-length cDNA clone of PRRSV, we performed mutagenesis analysis to define the function of the last 4 nts (5′-AAUU-3′) of the 3′UTR of the PRRSV genome in viral replication. We engineered point mutations into the pAPRRS cDNA clone to replace the wild-type 5′-AAUU-3′with other potential nucleotide. 12 mutants were constructed. MARC-145 cells were transfected with the full-length infectious cDNA clone and mutant PRRSV cDNA clones. The virus could be rescued from the mutant pAM04T,pAM04G,pAM04C and pAM01C. To examine the role of the conserved sequence (5′-AAUU-3′) of PRRSV genome in viral replication the production of viral proteins was monitored by an indirect immunofluorescence assay (IFA) using monoclonal antibody recognizing protein nsp2 and N, after 72h post-transfection. The result shown that 3′-terminal sequence significantly affect viral replication, including (i) the terminal 3′U is optimal for PRRSV replication. Replacement of the wild-type 3′U with a purine A or G resulted in a substantial abolishment in RNA replication. In contrast, replacement with a pyrimidine C resulted in a replication level similar to that of the wild-type, The conserved 3′penultimate U and 3′third A are absolutely essential for viral replication, (iii) The nucleotide sequences of the 4th (A) position are not essential for viral replication. However, deletion of the wild-type 3′4th nucleotide entirely abolished the viral replication. To further characterize the possible effect of 3′UTR mutation on virological properties, viral plaque assay was conducted. There were no morphology and size differences of the viral plaques between and among mutant virus and that of the parental vAPRRS except for the vAM01C.4. An improved full-length cDNA clone with HDV ribozymeUsing the three-step PCR, we engineered an HdvRz followed by the bovine growth hormone polyadenylation sequence (BGH) at the 3′end of our original cDNA clone (pAPRRS), resulting in pAPRRS-HB. Transfection of MARC-145 cells with either pAPRRS or pAPRRS-HB revealed IFA-positive cells, indicating that both infectious clones are replication competent. Moreover, more IFA-positive cells were observed in transfections with the HdvRz-containing clone than in transfections with the non-HdvRz-containing clone, suggesting that the HdvRz at the 3′end of the replicon improves replication efficiency. The rescue efficacy of PRRSV with this system was approximately 10-fold higher than the traditional DNA-launched system without the engineered ribozyme elements, as determined by the virus titer of the recovery virus level in transfected MARC-145 cells. 5. Study on the effect of the different length of the poly(A) tail in PRRS viral replicationIn this study, based on the North American PRRSV full-length infectious cDNA clone pAPRRS-HdvRz, through PCR-based mutagenesis, a series of PRRSV cDNA mutant with the length of 0, 1, 2, 3, 4, 5, 8, 10 of poly(A) tail were obtained. MACR-145 cells were transfected with full-length PRRSV cDNA clones containing desired mutations. The expression of viral proteins was monitored by an indirect immunofluorescence assay (IFA) using monoclonal antibody recognizing protein nsp2 and N 72 hours after transfection. The result showed that the virus could not be rescued from the mutant pAMA0, pAMA1, pAMA2, pAMA3 while the pAMA4, pAMA5, pAMA8, pAMA10 could rescued the virus, indicating the minimal length of the poly(A) is 4 As for viral replication. And the poly (A) involved in gene translation and subgenomic RNA transcription. The TCID50 assay result showed that the titer level raised with the length of poly(A) increasing while there is no different with the parental virus when the length of poly(A) is 10.