THE POLYADENYLATION SIGNAL OF INFLUENZA-VIRUS RNA INVOLVES A STRETCH OF URIDINES FOLLOWED BY THE RNA DUPLEX OF THE PANHANDLE STRUCTURE
THE POLYADENYLATION SIGNAL OF INFLUENZA-VIRUS RNA INVOLVES A STRETCH OF URIDINES FOLLOWED BY THE RNA DUPLEX OF THE PANHANDLE STRUCTURE
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DOI：
10.1128/jvi.65.6.2861-2867.1991
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发表时间：
1991-06-01
影响因子：
5.4
通讯作者：
PALESE, P
中科院分区：
文献类型：
--
作者：
LUO, GX;LUYTJES, W;PALESE, P
转染体外重建的RNA-蛋白质复合物的细胞并感染流感病毒作为辅助后，适当的RNA被转录和扩增，蛋白质被表达。该系统使我们能够研究病毒 RNA 转录所涉及的信号。为了进行分析，我们使用了质粒衍生的 RNA，其中含有氯霉素乙酰转移酶 (CAT) 报告基因，两侧是甲型/WSN/33 流感病毒 NS RNA 片段的非编码序列。然后将突变引入 5' 和 3' 末端，并对所得 RNA 进行研究以确定其体外转录及其在 RNA-蛋白质转染系统中的 CAT 表达活性。结果表明，负链 RNA 5' 端的一段不间断的尿苷对于 mRNA 的合成至关重要。此外，由 5' 和 3' 末端核苷酸产生的双链 RNA“panhandle”结构似乎是聚腺苷酸化所必需的，因为这些碱基对的开放减少了 mRNA 合成并消除了突变 RNA 的 CAT 活性表达。最后，研究表明这种双链 RNA 结构要求不是序列特异性的，因为合成的 GC 夹可以取代病毒编码的 RNA 双链体。数据表明，病毒RNA聚合酶通过滑动（口吃）机制添加poly(A)，当它碰到靠近尿苷片段的双链RNA屏障时，就会发生这种机制。
Appropriate RNAs are transcribed and amplified and proteins are expressed after transfection into cells of in vitro-reconstituted RNA-protein complexes and infection with influenza virus as the helper. This system permits us to study the signals involved in transcription of virus RNAs. For the analysis we used a plasmid-derived RNA containing the reporter gene for chloramphenicol acetyltransferase (CAT) flanked by the noncoding sequences of the NS RNA segment of influenza A/WSN/33 virus. Mutations were then introduced into both the 5' and 3' ends, and the resulting RNAs were studied to determine their transcription in vitro and their CAT expression activity in the RNA-protein transfection system. The results reveal that a stretch of uninterrupted uridines at the 5' end of the negative-strand RNA is essential for mRNA synthesis. Also, a double-stranded RNA "panhandle" structure generated by the 5'- and 3' -terminal nucleotides appears to be required for polyadenylation, since opening up of these base pairs diminished mRNA synthesis and eliminated expression of CAT activity by the mutant RNAs. Finally, it was shown that this double-stranded RNA structural requirement is not sequence specific, since a synthetic GC clamp can replace the virus-coded RNA duplex. The data suggest that the viral RNA polymerase adds poly(A) by a slippage (stuttering) mechanism which occurs when it hits the double-stranded RNA barrier next to the stretch of uridines.