Shmulevitz Maya, Yameen Zareen, Dawe Sandra, Shou Jingyun, O'Hara David, Holmes Ian, Duncan Roy
Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.
J Virol. 2002 Jan;76(2):609-18. doi: 10.1128/jvi.76.2.609-618.2002.
Previous studies of the avian reovirus strain S1133 (ARV-S1133) S1 genome segment revealed that the open reading frame (ORF) encoding the final sigmaC viral cell attachment protein initiates over 600 nucleotides distal from the 5' end of the S1 mRNA and is preceded by two predicted small nonoverlapping ORFs. To more clearly define the translational properties of this unusual polycistronic RNA, we pursued a comparative analysis of the S1 genome segment of the related Nelson Bay reovirus (NBV). Sequence analysis indicated that the 3'-proximal ORF present on the NBV S1 genome segment also encodes a final sigmaC homolog, as evidenced by the presence of an extended N-terminal heptad repeat characteristic of the coiled-coil region common to the cell attachment proteins of reoviruses. Most importantly, the NBV S1 genome segment contains two conserved ORFs upstream of the final sigmaC coding region that are extended relative to the predicted ORFs of ARV-S1133 and are arranged in a sequential, partially overlapping fashion. Sequence analysis of the S1 genome segments of two additional strains of ARV indicated a similar overlapping tricistronic gene arrangement as predicted for the NBV S1 genome segment. Expression analysis of the ARV S1 genome segment indicated that all three ORFs are functional in vitro and in virus-infected cells. In addition to the previously described p10 and final sigmaC gene products, the S1 genome segment encodes from the central ORF a 17-kDa basic protein (p17) of no known function. Optimizing the translation start site of the ARV p10 ORF lead to an approximately 15-fold increase in p10 expression with little or no effect on translation of the downstream final sigmaC ORF. These results suggest that translation initiation complexes can bypass over 600 nucleotides and two functional overlapping upstream ORFs in order to access the distal final sigmaC start site.
先前对禽呼肠孤病毒S1133株（ARV - S1133）S1基因组片段的研究表明，编码最终σC病毒细胞附着蛋白的开放阅读框（ORF）在S1 mRNA 5'端下游600多个核苷酸处起始，且之前有两个预测的小的不重叠ORF。为了更清楚地定义这种不寻常的多顺反子RNA的翻译特性，我们对相关的纳尔逊湾呼肠孤病毒（NBV）的S1基因组片段进行了比较分析。序列分析表明，NBV S1基因组片段上3'近端的ORF也编码一个最终的σC同源物，呼肠孤病毒细胞附着蛋白共有的卷曲螺旋区域特有的N端延伸七肽重复序列的存在证明了这一点。最重要的是，NBV S1基因组片段在最终σC编码区上游包含两个保守的ORF，相对于ARV - S1133预测的ORF有所延伸，并以连续、部分重叠的方式排列。另外两株ARV的S1基因组片段序列分析表明，其基因排列与预测的NBV S1基因组片段相似，为重叠的三顺反子。ARV S1基因组片段的表达分析表明，所有三个ORF在体外和病毒感染细胞中均具有功能。除了先前描述的p10和最终的σC基因产物外，S1基因组片段从中央ORF编码一种功能未知的17 kDa碱性蛋白（p17）。优化ARV p10 ORF的翻译起始位点导致p10表达增加约15倍，对下游最终σC ORF的翻译几乎没有影响。这些结果表明，翻译起始复合物可以绕过600多个核苷酸和两个功能性重叠的上游ORF，以便进入远端的最终σC起始位点。
