SYNTHETIC ANALOGS OF ROTAVIRUS GENOMIC RNA EXPRESSING A FOREIGN MARKER GENE

轮状病毒是人类病毒性胃肠炎的重要原因， 动物轮状病毒基因组由11个单顺反子片段组成 双链（ds）RNA编码六种结构（VP 1，VP 2，VP 3， VP 4，VP 6和VP 7）和五种非结构蛋白（NSP 1，NSP 2，NSP 3， NSP4、NSP5）。 基因组片段的正链是无法区分的 来自病毒mRNA，含有5 '末端甲基化帽结构， 缺乏3 '-poly（A）尾。 除了七到十个的短序列 在5 '-和3'-末端的核苷酸，11个基因组片段共享 没有可检测的同源性。保守的末端序列已被证明 包含在基因组复制中重要的顺式作用信号。 相反，保守末端和5 '-和 3 '-非编码序列（NCS）在轮状病毒基因表达调控中的作用 不知道。以前的研究已经确定，虽然11个基因组 片段以等摩尔浓度存在于受感染的细胞中， 片段的转录导致非等摩尔的 病毒mRNA水平。11种病毒蛋白的相对水平 在感染细胞中产生的抗体并不反映病毒的相对水平， mRNA的存在表明病毒中的顺式作用信号 mRNA对于调节轮状病毒基因表达是重要的。目标 本项目的目的是鉴定5 '-和 3 '-NCS影响病毒蛋白的表达。初步研究 研究了5 '-和3'-NCS对翻译的贡献， 轮状病毒mRNA的合成类似物。这些类似物由基因 对于细菌氯霉素乙酰转移酶（CAT），其侧翼为5 '- 和3 '-NCS，并测定轮状病毒基因1、2、3、4、6或9的 兔网织红细胞的翻译效率。结果显示： (i)携带基因6的同源5 '-和3'-NCS的类似物诱导了 最高水平的CAT表达，（ii）RNA片段6的3 '-NCS可以 增强其他轮状病毒mRNA类似物的翻译效率 无论5 '-NCS如何，（iii）翻译增强元件是 定位于从该区域上游约80个核苷酸的区域。 RNA片段6的3 '-NCS的3'-末端。我们最近的研究比较了 野生型基因6 mRNA与基因6 mRNA翻译效率 在3 '-NCS中含有缺失。结果证实了这一结果 获得轮状病毒-CAT类似物，并建立一个重要的 顺式作用元件位于轮状病毒基因6 mRNA的3 ′-NCS， 其功能是上调基因6产物VP 6的表达， 轮状病毒粒子的主要蛋白质成分。

Rotaviruses are an important cause of viral gastroenteritis in humans and animals. The rotavirus genome consists of eleven monocistronic segments of double-stranded (ds)RNA which encode six structural (VP1, VP2, VP3, VP4, VP6 and VP7) and five nonstructural proteins (NSP1, NSP2, NSP3, NSP4, NSP5). The plus-strand of the genome segments is indistinguishable from viral mRNA, containing a 5'-terminal methylated cap structure but lacking a 3'-poly(A) tail. Except for short sequences of seven to ten nucleotides at the 5'- and 3'-termini, the eleven genome segments share no detectable homology. The conserved terminal sequences have been shown to contain cis-acting signals that are important in genome replication. In contrast, the role of the conserved termini and the 5'- and 3'-noncoding sequences (NCS) in regulating rotavirus gene expression is not known. Previous studies have established that while the eleven genome segments exist in equimolar concentrations in the infected cell, transcription of the segments leads to the synthesis of non-equimolar levels of viral mRNA. The relative levels of the eleven viral proteins made in the infected cell do not reflect the relative levels of viral mRNA that are present indicating that cis-acting signals in the viral mRNAs are important for regulating rotavirus gene expression. The goal of this project is to identify those cis-acting signals in the 5'- and 3'-NCS that affect expression of the viral proteins. Initial studies examined the contribution of the 5'- and 3'-NCS on translation using synthetic analogs of rotavirus mRNAs. These analogs consisted of the gene for bacterial chloramphenicol acetyltransferase (CAT) flanked by the 5'- and 3'-NCS of the rotavirus genes 1, 2, 3, 4, 6 or 9 and were assayed for translational efficiency in rabbit reticulocytes. The results revealed: (i) analogs carrying homologous 5'- and 3'-NCS of gene 6 induced the highest levels of CAT expression, (ii) the 3'-NCS of RNA segment 6 could enhance the translation efficiency of other rotavirus mRNA analogs regardless of the 5'-NCS, (iii) the translation enhancement element was localized to a region approximately 80 nucleotides upstream from the 3'-end of the 3'-NCS of RNA segment 6. Our recent studies have compared the translational efficiency of wildtype gene 6 mRNA with gene 6 mRNA containing deletions in the 3'-NCS. The results confirmed the results obtained with the rotavirus-CAT analogs and establish that an important cis-acting element resides in the 3'-NCS of rotavirus gene 6 mRNA which functions to up regulate the expression of the gene 6 product, VP6, a major protein component of the rotavirus virion.