title: Genetic manipulation of a transcription-regulating sequence of porcine reproductive and respiratory syndrome virus reveals key nucleotides determining its activity.
abstract: The factors that determine the transcription-regulating sequence (TRS) activity of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unclear. In this study, the effect of mutagenesis of conserved C nucleotides at positions 5 and 6 in the leader TRS (TRS-L) and/or canonical body TRS7 (TRS-B7) on the synthesis of subgenomic (sg) mRNA and virus infectivity was investigated in the context of a type 2 PRRSV infectious cDNA clone. The results showed that a double C mutation in the leader TRS completely abolished sg mRNAs synthesis and virus infectivity, but a single C mutation did not. A single C or double C mutation in TRS-B7.1 or/and TRS-B7.2 impaired or abolished the corresponding sg mRNA synthesis. Introduction of identical mutations in the leader and body TRSs partially restored sg mRNA7.1 and/or sg mRNA7.2 transcription, indicating that the base-pairing interaction between sense TRS-L and cTRS-B is a crucial factor influencing sg mRNA synthesis. Analysis of the mRNA leader-body junctions of mutants provided evidence for a mechanism of discontinuous minus-strand transcription. This study also showed that mutational inactivation of TRS-B7.1 or TRS-B7.2 did not affect the production of infectious progeny virus, and the sg mRNA formed from each of them could express N protein. However, TRS-B7.1 plays more important roles than TRS-B7.2 in maintaining the growth characteristic of type 2 PRRSV. These results provide more insight into the molecular mechanism of genome expression and subgenomic mRNA transcription of PRRSV.
authors.affiliation: Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Shanghai, 200241, China.
authors.forename: Haihong
authors.initials: H
authors.lastname: Zheng
authors.forename: Keyu
authors.initials: K
authors.lastname: Zhang
authors.forename: Xing-Quan
authors.initials: XQ
authors.lastname: Zhu
authors.forename: Changlong
authors.initials: C
authors.lastname: Liu
authors.forename: Jiaqi
authors.initials: J
authors.lastname: Lu
authors.forename: Fei
authors.initials: F
authors.lastname: Gao
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhou
authors.forename: Hao
authors.initials: H
authors.lastname: Zheng
authors.forename: Tao
authors.initials: T
authors.lastname: Lin
authors.forename: Liwei
authors.initials: L
authors.lastname: Li
authors.forename: Guangzhi
authors.initials: G
authors.lastname: Tong
authors.forename: Zuzhang
authors.initials: Z
authors.lastname: Wei
authors.forename: Shishan
authors.initials: S
authors.lastname: Yuan
mesh_terms: D000818:Animals; D001483:Base Sequence; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D008969:Molecular Sequence Data; D019318:Porcine Reproductive and Respiratory Syndrome / Q000821:virology*; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics* / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid; D013552:Swine; D014158:Transcription, Genetic