TRANSCRIPTION ANTITERMINATION DURING INFLUENZA VIRAL TEMPLATE RNA-SYNTHESIS REQUIRES THE NUCLEOCAPSID PROTEIN AND THE ABSENCE OF A 5' CAPPED END
TRANSCRIPTION ANTITERMINATION DURING INFLUENZA VIRAL TEMPLATE RNA-SYNTHESIS REQUIRES THE NUCLEOCAPSID PROTEIN AND THE ABSENCE OF A 5' CAPPED END
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DOI：
10.1073/pnas.83.17.6282
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发表时间：
1986-09-01
影响因子：
11.1
通讯作者：
KRUG, RM
中科院分区：
文献类型：
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作者：
BEATON, AR;KRUG, RM
复制流感病毒粒子RNA的第一步是合成这些RNA的全长转录本。这些转录本或模板RNA的合成需要：(I)非启动启动，而不是病毒mRNA合成过程中使用的封端RNA启动启动，以及(Ii)在mRNA合成过程中使用的多聚腺苷酸化位点的抗终止。为了确定模板RNA合成的机制，我们从感染细胞中制备了对模板RNA和病毒mRNAs的合成都有活性的核提取物。通过提供二核苷酸APG作为引物，我们绕过了这些提取物催化的低效非引发引发，因此，我们能够专注于抗终止步骤。当使用APG而不是封端的RNA作为引物时，发生了反终止，从而模板RNA合成，这表明5‘’封端的存在阻止了转录本3‘端的反终止。核抽提物的超速离心法得到一种含有病毒核衣壳的颗粒组分，它能合成病毒的核糖核酸，但不能合成模板RNA，还有一种含有抗终止因子的上清液。当将本身基本上没有活性的上清液在APG存在的情况下加入到颗粒中时，模板RNA的合成就会恢复。该上清液与含有针对个别病毒蛋白的抗体的蛋白A-琼脂糖孵育的耗竭实验表明，病毒核衣壳蛋白是抗终止所必需的。讨论了这些结果对控制病毒RNA复制的意义。
The first step in the replication of influenza virion RNAs is the synthesis of full-length transcripts of these RNAs. The synthesis of these transcripts, or template RNAs, requires: (i) unprimed initiation rather than the capped RNA-primed initiation used during viral mRNA synthesis, and (ii) antitermination at the polyadenylylation site used during mRNA synthesis. To determine the mechanism of template RNA synthesis, we prepared nuclear extracts from infected cells that were active in the synthesis of both template RNAs and viral mRNAs. By providing the dinucleotide ApG as primer, we circumvented the inefficient unprimed initiation catalyzed by these extracts and, as a consequence, were able to focus on the antitermination step. Antitermination, and hence template RNA synthesis, occurred when ApG but not a capped RNA was used as primer, indicating that the presence of a 5'' capped end blocked antitermination at the 3'' end of the transcript. Ultracentrifugation of the nuclear extract yielded a pellet fraction that contained viral nucleocapsids active in viral mRNA synthesis but not template RNA synthesis and a supernatant fraction that contained the antitermination factor. When the supernatant, which had essentially no activity by itself, was added to the pellet in the presence of ApG, template RNA synthesis was restored. Depletion experiments in which this supernatant was incubated with protein A-Sepharose containing antibodies to individual viral proteins demonstrated that the viral nucleocapsid protein was required for antitermination. The implications of these results for the control of viral RNA replication are discussed.