Stewart S R, Semler B L
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, CA 92697, USA.
Nucleic Acids Res. 1998 Dec 1;26(23):5318-26. doi: 10.1093/nar/26.23.5318.
In attenuated Sabin strains, point mutations within stem-loop V of the 5'-non-coding region (NCR) reduce neurovirulence and cell-specific cap-independent translation. The stem-loop V attenuation determinants lie within the highly structured internal ribosome entry site. Although stem-loop V Sabin mutations have been proposed to alter RNA secondary structure, efforts to identify such conformational changes have been unsuccessful. A previously described linker-scanning mutation (X472) modified five nucleotides adjacent to the attenuation determinant at nt 480 [for poliovirus (PV) type 1]. Transfection of X472 RNA generated only pseudo-revertants in HeLa (cervical carcinoma) or SK-N-SH (neuroblastoma) cells. Pseudo-revertants from both cell types contained nucleotide changes within the X472 linker. In addition, some neuroblastoma-isolated revertants revealed second site mutations within the pyrimidine-rich region located approximately 100 nt distal to the original lesion. Enzymatic RNA structure probing determined that the X472 linker substitution did not disrupt the overall conformation of stem-loop V but abolished base pairing adjacent to the attenuation determinant. Our analyses correlated increased base pairing proximal to the stem-loop V attenuation determinant with growth of X472 revertant RNAs (measured by northern blot analysis). Potential roles of second site mutations in the pyrimidine-rich region are discussed. In addition, our enzymatic structure probing results are shown on a consensus secondary structure model for stem-loop V of the PV 5'-NCR.
在减毒的萨宾株中，5'-非编码区（NCR）茎环V内的点突变可降低神经毒力和细胞特异性帽依赖性翻译。茎环V减毒决定簇位于高度结构化的内部核糖体进入位点内。尽管有人提出茎环V萨宾突变会改变RNA二级结构，但识别此类构象变化的努力一直未成功。先前描述的连接子扫描突变（X472）修饰了与脊髓灰质炎病毒1型（PV1）nt 480处减毒决定簇相邻的五个核苷酸。在HeLa（宫颈癌）或SK-N-SH（神经母细胞瘤）细胞中转染X472 RNA仅产生假回复突变体。来自两种细胞类型的假回复突变体在X472连接子内都含有核苷酸变化。此外，一些从神经母细胞瘤分离的回复突变体在距原始损伤约100 nt远的富含嘧啶区域内显示出第二位点突变。酶促RNA结构探测确定X472连接子取代并未破坏茎环V的整体构象，但消除了与减毒决定簇相邻的碱基配对。我们的分析将茎环V减毒决定簇近端增加的碱基配对与X472回复突变体RNA的生长（通过Northern印迹分析测量）相关联。讨论了富含嘧啶区域第二位点突变的潜在作用。此外，我们的酶促结构探测结果显示在PV 5'-NCR茎环V的共有二级结构模型上。
