SELECTED HOST-CELL CAPPED RNA FRAGMENTS PRIME INFLUENZA VIRAL-RNA TRANSCRIPTION INVIVO
SELECTED HOST-CELL CAPPED RNA FRAGMENTS PRIME INFLUENZA VIRAL-RNA TRANSCRIPTION INVIVO
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DOI：
10.1093/nar/9.17.4423
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发表时间：
1981-01-01
影响因子：
14.9
通讯作者：
KRUG, RM
中科院分区：
文献类型：
--
作者：
BEATON, AR;KRUG, RM
流感病毒 RNA 体外转录是由病毒核酸内切酶从加帽 RNA 上切割下来的加帽 RNA 片段引发的。本研究的目的是确定在体外研究中观察到的病毒核酸内切酶和转录酶的特异性是否也在受感染的细胞中观察到。通过使用含有病毒粒子 RNA (vRNA) 和体内病毒 mRNA 的 cDNA 拷贝的双链 DNA，将流感 WSN 病毒的 NS（非结构）基因克隆到 pBR322 中。我们确定了所克隆的特定 NS 病毒 mRNA 分子的 5' 末端序列，以及在两种不同细胞系中合成的整个体内 NS 病毒 mRNA 群体的 5' 末端序列。对于后者的测定，我们使用来自克隆 DNA 的限制性片段进行逆转录酶催化的总体内病毒 mRNA 的延伸。结果表明，体内和体外病毒RNA转录在两个重要方面是相似的：（i）转录起始不是由vRNA 3'末端U引导的A残基，而是由vRNA 3'倒数第二个C引导的G残基开始； (ii)优先使用含有3'端A残基的加帽RNA片段作为引物，从而在病毒mRNA中产生与vRNA的3'端UC互补的AG序列。实际上，对于体内转录，A 末端加帽片段的子集，即包含 3' 倒数第二个 C 残基的 tfibse，是优选的引物。后者的特异性在之前的体外研究中尚未观察到。
Influenza viral RNA transcriptionin vitrois primed by capped RNA fragments cleaved from capped RNAs by a viral endonuclease. The present study was undertaken to determine whether the specificities of the viral endonuclease and transcriptase observed inin vitrostudies are also observed in the infected cell. The NS (nonstructural) gene of influenza WSN virus was cloned in pBR322 by using a double-stranded DNA containing a cDNA copy of both virion RNA (vRNA) andin vivoviral mRNA. We determined the 5′ terminal sequence of the particular NS viral mRNA molecule which was cloned and also the 5′ terminal sequences of the entire population ofin vivoNS viral mRNAs synthesized in two different cell lines. For the latter determination we used a restriction fragment from the cloned DNA for the reverse transcriptase-catalyzed extension of totalin vivoviral mRNA. The results indicate thatin vivoandin vitroviral RNA transcription are similar in two important respects: (i) transcription initiates not with an A residue directed by the 3′ terminal U of the vRNA, but with a G residue directed by the 3′ penultimate C of the vRNA; and (ii) capped RNA fragments containing a 3′ terminal A residue are preferentially used as primers, thereby generating an AG sequence in the viral mRNA complementary to the 3′ terminal UC of the vRNA. Actually, forin vivotranscription, a subset of A-terminated capped fragments, namely tfibse containing a 3′ penultimate C residue, are the preferred primers. The latter specificity had not been observed in previousin vitrostudies.