Dissection Of Structure And Function Of 3' Untranslated Region 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. PRRSV isolates are divided into two distinct genotypes: TypeⅠ(the European) and TypeⅡ(the North American). PRRSV is an enveloped, single-stranded positive-sense RNA virus. Its genome is approximately 15kb in length and consists of a 5'cap, a 5'untranslated region (UTR), ten open reading frames, followed by a 3' UTR and a poly (A) tail. It is still largely unknown about PRRSV genome replication, subgenomic transcription and protein translation. The 3'UTR of positive-stranded RNA virus genome plays a pivotal role for viral cycle, yet the properties of 3'UTR of PRRSV remain largely undefined. Using the type II PRRSV infectious clone established in the lab, we performed a mutagenesis analysis to define structure and function of the 3'UTR of PRRSV, which could provide basis for interpreting PRRSV pathogenesis and artificially regulating virus. The detailed experimentations are described below.1. The effects of the deletion proximal to the 5'end of PRRSV 3'UTR on viral replicationBased PRRSV infectious full-length cDNA clone pAPRRS, serial deletions were engineered into the original pAPRRS by PCR-based mutagenesis. These plasmid DNAs were transfected into BHK-21 cells, 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 subsequently used to infect MARC-145 cells to detect the production of progeny viruses 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. The results showed that 66 nucleotides of 5'-end sequences of the 3'UTR were dispensable for the viability of PRRSV in cultured cells, but it could influent the replication efficiency in MARC-145 cells. The 67 nucleotides deletion would decrease the transcription efficiency and lead to no viral infectivity. Furthermore, more than 67 nucleotides deletion would destroy the subgenomic mRNA synthesis and viral replication. In conclusion, our studies demonstrated that the first 66 nucleotides of the 5'proximal region of typeⅡPRRSV 3'UTR is the delimitation of maximal nonessential region for viral replication in cultured cells.2. Dissection of the function of a conserved secondary structure element in PRRSV 3'UTRThe structural elements of single positive strand RNA virus 3'UTR are believed to play a pivotal role for virus replication. The PRRSV 3'UTR with a conserved bulged stem loop structure is predicted to play a key role for virus replication. In order to identify this element, serial point mutations were engineered into the stem loop and bulge "A-GC" in the original infectious full-length cDNA clone pAPRRS 3'UTR by site-directed mutagenesis. These plasmid DNAs were transfected into BHK-21 cells. The supernatants of the transfected cells were subsequently used to infect MARC-145 cells to detect the production of progeny viruses. We have demonstrated that mutations in the stem loop sequence and loop size were tolerated, but resulted in a fitness cost of PRRSV replication in cells. The stability of the stem structure is required for RNA synthesis rather than its sequence. Once the complementary base pair in the stem is destructed, subgenomic RNA synthesis is completely disrupted. The bulge "A-GC" is an "A-VN" -like bulge that is highly functionally tolerant of base substitutions, but any structural change mutation is lethal. These studies suggested that the bulged stem loop functions as an important structural element in stimulating subgenomic RNA synthesis and viral infections in PRRSV. This secondary structure element may participate in RNA as well as protein interaction.Taken together, PRRSV 3'UTR is vital for PRRSV replication, and there maybe discrete cis-acting elements responsible for different steps of the viral life cycle. Utilizing PRRSV infectious clone pAPRRS platform, we have analyzed the structure and function of PRRSV 3'UTR, and demonstrated that:(1) the first 66 nt of the 5' proximal region of typeⅡPRRSV 3'UTR was a nonessential region for viral replication; (2) a bulged stem loop was predicted in PRRSV 3'UTR, and its structure rather than its sequence was important for subgenomic mRNA synthesis and viral infections.