Analysis Of The Poly(A) Cis-Acting Element For Replication Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the family Arteriviridae in the order Nidovirales. PRRSV genome is a positive-sense , single-stranded RNA of～15 kb in length, with a cap structure at its 5' end and a poly(A) tail at its 3' end. The poly(A) tail of positive-sense single-stranded RNA virus plays very important roles in viral replication cycle. Currently, the poly(A) tail of PRRSV genome to viral infectivity, and the functional and forming mechanisms are not clear. Based on our PRRSV infectious clone of North American strain, we constructed series of PRRSV cDNA clones with partial deletion of poly (A) tail and mutating the possible polyadenylation signals, then investigated the function of poly (A) tail in viral replication and transcription.The concise research contents are presented as follows.1. In this study, based on the North American PRRSV full-length infectious cDNA clone pCBC2, through PCR-based mutagenesis, a series of PRRSV cDNA clones with partial deletion of poly(A) tail were obtained. Then the RNAs via in vitro transcription were transfected into MARC-145 cells for further cytopathic effect observation. The viral RNAs in the supernatant of those with cytopathic effect were further extracted, and the poly (A) tail lengths in progeny virus genomes were identified by G-Tailing method. The results showed that the poly(A) tail affects the infectivity of PRRSV and the replication of genomic RNA. The minimum essential length is 10; the truncated poly (A) tail in the infectious mutants can be repaired during infection process.2. Based on the North American PRRSV full-length infectious cDNA clone pCBC2, through PCR-based mutagenesis, the PRRSV cDNA clones pC1G,pC5G,pC10G,pC15G which was substituted "A" to "G" in the position 1,5,10,15 of the poly(A) tail were obtained. Then the RNAs via in vitro transcription were transfected into MARC-145 cells for further cytopathic effect observation. The viral RNAs in the supernatant of those with cytopathic effect were further extracted, and the poly (A) tail in progeny virus genomes were identified by G-Tailing method. Then the total RNA were extracted when the virus infected the MARC-145 cells for 24 hours, and the minus-strand RNA initiation site was identified by 5' RACE. The results indicated that the pC5G,pC10G and pC15G RNA of in vitro transcription could induce cytopathic effect, the above progeny virus genome couldn't be detected the substituted "G" in the poly(A) tail, and the 5' terminus of minus-strand RNA had a poly(U) tract in different length.3. This study intend to identify the possible polyadenylation signals in PRRSV genemes. Based on the North American PRRSV full-length infectious cDNA clone pCBC2, and the secondary structure of 3'UTR was predicted. In the premises of makeing the secondary structure not be changed, through PCR-based mutagenesis, a series of PRRSV cDNA clones with the substituted nucleotides in the primary sequence were obtained. Then the RNAs via in vitro transcription were transfected into MARC-145 cells for further cytopathic effect observation. The viral RNAs in the supernatant of those with cytopathic effect were further extracted, and the mutants were identified by RT-PCR. The results showed some substituted nucleotides (mabe including the polyadenylation signals) in the primary sequence influenced the infectivity of PRRSV.