Functional analysis of the influenza A virus cRNA promoter and construction of an ambisense transcription system
Functional analysis of the influenza A virus cRNA promoter and construction of an ambisense transcription system
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甲型流感病毒 cRNA 启动子的功能分析和伏义转录系统的构建
DOI：
10.1006/viro.2001.1107
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发表时间：
2001-10-25
期刊：
影响因子：
3.7
通讯作者：
Hobom, G
中科院分区：
文献类型：
--
作者：
Azzeh, M;Flick, R;Hobom, G
虽然已知甲型流感病毒RNA作为病毒mRNA和互补cRNA合成的模板，但迄今为止观察到后者仅作为复制中的中间体起作用并产生子代vRNA分子。这里显示，cRNA启动子也能够启动病毒mRNA的合成，类似于遵循引物募集的夺帽模式的vRNA促进的转录。检测cRNA促进的转录需要报告基因编码序列的倒位加上病毒多聚腺苷酸化信号的重新定位。通过RNA聚合酶I反向遗传学构建cRNA启动子变体使我们能够在报告基因读出系统中确定RNA聚合酶相关的碱基配对构象。它再次被证明符合“开瓶器”模型，与相应的vRNA构象相似，但在其结合相互作用方面略有不同。对从流感vRNA和cRNA模板分子以任一方向启动的两个转录反应的观察，使我们能够构建双顺反子、双义RNA分子，用于从病毒RNA的单个片段同时表达两种蛋白质。(C)北京：科学出版社.
While influenza A viral RNA is known to act as a template for the synthesis of both viral mRNA and complementary cRNA, the latter has been observed so far only to function as an intermediate in replication and give rise to progeny vRNA molecules. Here it is shown that the cRNA promoter is also capable of initiating viral mRNA synthesis, similar to vRNA-promoted transcription adhering to the cap-snatching mode of primer recruitment. Detection of cRNA promoted transcription required an inversion of the reporter gene coding sequence plus relocation of the viral polyadenylation signal. Construction of cRNA promoter variants through RNA polymerase I reverse genetics allowed us to determine the RNA polymerase-associated, base-paired conformation in a reporter gene read-out system. It again turned out to adhere to the "corkscrew" model, similar, but slightly different in its binding interactions from the corresponding vRNA conformation, The observation of two transcription reactions, initiated in either direction from influenza vRNA and cRNA template molecules, allowed us to construct bicistronic, ambisense RNA molecules for simultaneous expression of two proteins from a single segment of viral RNA. (C) 2001 Academic Press.