Todd S, Semler B L
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717, USA.
Nucleic Acids Res. 1996 Jun 1;24(11):2133-42. doi: 10.1093/nar/24.11.2133.
The specific recognition of genomic positive strand RNAS as templates for the synthesis of intermediate negative strands by the picornavirus replication machinery is presumably mediated by cis-acting sequences within the genomic RNA 3' non-coding region (NCR). A structure-infectivity analysis was conducted on the 44 nt human rhinovirus 14 (HRV14) 3' NCR to identify the primary sequence and/or secondary structure determinants required for viral replication. Using biochemical RNA secondary structure probing techniques, we have demonstrated the existence of a single stem-loop structure contained entirely within the 3' NCR, which appears to be phylogenetically conserved within the rhinovirus genus. We also report the in vivo analysis of a number of 3' NCR deletion mutations engineered into infectious cDNA clones which were designed to disrupt the stem-loop secondary structure to varying degrees. Large deletions (up to 37 nt) resulted in defective growth phenotypes, although they were not lethal. We propose that the absolute requirements for initiation of negative strand synthesis are less stringent than previously postulated, even though defined RNA secondary structure determinants may have evolved to facilitate and/or regulate the process of viral RNA replication.
微小核糖核酸病毒复制机制将基因组正链RNA特异性识别为合成中间负链的模板，大概是由基因组RNA 3'非编码区（NCR）内的顺式作用序列介导的。对44个核苷酸的人鼻病毒14（HRV14）3' NCR进行了结构-感染性分析，以确定病毒复制所需的一级序列和/或二级结构决定因素。使用生化RNA二级结构探测技术，我们证明了完全包含在3' NCR内的单个茎环结构的存在，该结构在鼻病毒属内似乎是系统发育保守的。我们还报告了对一些工程改造到感染性cDNA克隆中的3' NCR缺失突变的体内分析，这些突变旨在不同程度地破坏茎环二级结构。大的缺失（高达37个核苷酸）导致生长表型缺陷，尽管它们并非致命。我们提出，尽管特定的RNA二级结构决定因素可能已经进化以促进和/或调节病毒RNA复制过程，但负链合成起始的绝对要求比以前假设的要宽松。
