Dissection Of Secondary Structure And Function Of 5' Un-translated Region For Porcine Reproductive And Respiratory Syndrome Virus

In this study, based on reverse genetic system, we focus on the porcine reproductive and respiratory syndrome virus (PRRSV) 5′UTR and intend to dissect the functions for mechanisms about genomic replication and subgenomic transcription. PRRSV are identified as two genotypes, type I (European type, EU) and type II (North American type, NA). In order to define the key cis-acting elements for that regulate the discrete steps of the viral life cycle, based on the PRRSV infectious clones of two genotypes, we change the primary sequences and secondary structure about 5′UTR by standard site-directed PCR mutagenesis. The detailed contents are discussed as follows:We analyze 128 stains of the 5′UTR in PRRSV two genotypes by DNASTAR 7.1, meantime, we predict the secondary structure by software Mfold,RNAstructure 5.0 and Vienna RNAfold. The results show that: firstly, the sequence length of 5′UTR differs in two types (220 and 190nt, respectively) and the sequences between them show only 33% sequence homology. There is a much conserved region in the 5′UTR including the known transcription-regulating sequence. Secondly, although the primary sequences between two genotypes of PRRSV are distinguishing, the secondary structures show kindly similarity. It's shown that there is an independent high-ordered structure ---- Leader TRS Hairpin (LTH) in the 5′UTR, which can function independently in viral discontinuous transcription.Higher-order structures in the 5′un-translated region (UTR) of positive-strand RNA viruses are known in many cases to make function in genome replication, transcription, and translation. Here we describe evidence supporting that a conserved stem-loop structure in the 5′UTR of porcine reproductive and respiratory syndrome virus (PRRSV) is necessary for viral subgenomic mRNA synthesis. This stem-loop structure shows phylogenetic conservation among arteriviruses and appears to have a homolog in some coronaviruses. In this study, based on the PRRSV type II infectious full-length cDNA clone, we delimit that the stem-loop 2, named N-SL2 in PRRSV type II 5′UTR, can be functionally substituted by the counterpart in different genotype of PRRSV. Moreover, we find that the functional region in N-SL2 is the stem instead of the ternary-loop through mutagenesis. To maintain the stem integrity of N-SL2 is crucial for subgenomic mRNA synthesis, meantime, virus can regain infectivity through restore the structure of N-SL2. These results suggest that stem-loop 2 in PRRSV 5′UTR is a key structural element for viral RNA synthesis.The objective of this study is to analyze the secondary structure and function of PRRSV 5′UTR. Based on the PRRSV type 2 full-length infectious cDNA clone pAPRRS, a series of mutants which can modify a conserved stem structure, named N-SL5 in PRRSV type II 5′UTR, are obtained through PCR-based mutagenesis. It is found that the secondary structure of the conserved stem loop in PRRSV 5′UTR isn't essential for virus rescue. Nonetheless, the primary sequence of the stem structure is functional for viral replication. In addition, it is demonstrated that a key nucleotide in PRRSV 5′UTR directly regulate the viral replication. All results demonstrate that the essential sequences in PRRSV 5′UTR are functional in viral genomic replication. It is known that the 5′UTR plays a crucial role in the life cycle of positive-strand RNA viruses. In this study, based on the PRRSV type 2 full-length infectious cDNA clone pAPRRS, a series of mutants which can modify a conserved hairpin structure are obtained through PCR-based mutagenesis, in order to analysis the secondary structure and functions of an independent functional entity in N-SL5 of PRRSV 5′UTR. The results show that the secondary structure of the conserved hairpin structure in N-SL5 of PRRSV 5′UTR is non-essential for virus replication, and that the loop of the structure can only tolerate two nucleotides (nt) mutation. Meanwhile, it is found that the conserved stem of the hairpin structure is non-necessary for virus rescue. All results demonstrate that an independent functional entity is found in PRRSV 5′UTR.In conclusion, the results that we got from this study can lay foundations in development of the marker vaccine and further study on the structural and functional analysis of PRRSV 5′UTR.