Hema M, Kao C Cheng
Department of Biophysics and Biochemistry, Texas A&M University, College Station, Texas 77843, USA.
J Virol. 2004 Feb;78(3):1169-80. doi: 10.1128/jvi.78.3.1169-1180.2004.
Bromoviral templates for plus-strand RNA synthesis are rich in A or U nucleotides in comparison to templates for minus-strand RNA synthesis. Previous studies demonstrated that plus-strand RNA synthesis by the brome mosaic virus (BMV) RNA replicase is more efficient if the template contains an A/U-rich template sequence near the initiation site (K. Sivakumaran and C. C. Kao, J. Virol. 73:6415-6423, 1999). These observations led us to examine the effects of nucleotide changes near the template's initiation site on the accumulation of BMV RNA3 genomic minus-strand, genomic plus-strand, and subgenomic RNAs in barley protoplasts transfected with wild-type and mutant BMV transcripts. Mutations in the template for minus-strand synthesis had only modest effects on BMV replication in barley protoplasts. Mutants with changes to the +3, +5, and +7 template nucleotides accumulated minus-strand RNA at levels similar to the the wild-type level. However, mutations at positions adjacent to the initiation cytidylate in the templates for genomic and subgenomic plus-strand RNA synthesis significantly decreased RNA accumulation. For example, changes at the third template nucleotide for plus-strand RNA3 synthesis resulted in RNA accumulation at between 18 and 24% of the wild-type level, and mutations in the third template nucleotide for subgenomic RNA4 resulted in accumulations at between 7 and 14% of the wild-type level. The effects of the mutations generally decreased as the mutations occurred further from the initiation nucleotide. These findings demonstrate that there are different requirements of the template sequence near the initiation nucleotide for BMV RNA accumulation in plant cells.
与负链RNA合成的模板相比，用于正链RNA合成的雀麦花叶病毒模板富含A或U核苷酸。先前的研究表明，如果模板在起始位点附近包含富含A/U的模板序列，那么雀麦花叶病毒（BMV）RNA复制酶进行的正链RNA合成效率更高（K. Sivakumaran和C. C. Kao，《病毒学杂志》73:6415 - 6423，1999年）。这些观察结果促使我们研究模板起始位点附近的核苷酸变化对用野生型和突变型BMV转录本转染的大麦原生质体中BMV RNA3基因组负链、基因组正链和亚基因组RNA积累的影响。负链合成模板中的突变对大麦原生质体中BMV的复制只有适度影响。模板核苷酸+3、+5和+7发生变化的突变体积累的负链RNA水平与野生型水平相似。然而，基因组和亚基因组正链RNA合成模板中起始胞苷酸相邻位置的突变显著降低了RNA积累。例如，正链RNA3合成的第三个模板核苷酸发生变化导致RNA积累量为野生型水平的18%至24%，亚基因组RNA4的第三个模板核苷酸发生突变导致积累量为野生型水平的7%至14%。随着突变距离起始核苷酸越来越远，突变的影响通常会降低。这些发现表明，植物细胞中BMV RNA积累对起始核苷酸附近的模板序列有不同要求。
