AN IN VITRO SYSTEM FOR THE LEADER-PRIMED TRANSCRIPTION OF CORONAVIRUS MRNAS
AN IN VITRO SYSTEM FOR THE LEADER-PRIMED TRANSCRIPTION OF CORONAVIRUS MRNAS
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DOI：
10.1002/j.1460-2075.1990.tb07641.x
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发表时间：
1990-12-01
期刊：
影响因子：
11.4
通讯作者：
LAI, MMC
中科院分区：
文献类型：
--
作者：
BAKER, SC;LAI, MMC
我们开发了一种体外转录系统，可以利用外源前导 RNA 进行小鼠肝炎病毒 (MHV) 的“前导引物”mRNA 转录。通过 MHV 感染细胞的溶血卵磷脂透化制备含有病毒蛋白和模板 RNA 的细胞质提取物。将与内源前导 RNA 序列不同的合成前导 RNA 添加到提取物中，并证明其掺入 MHV mRNA 中。无论添加的前导 RNA 的大小如何，外源前导 RNA 都会在同一位点连接到内源 mRNA，该位点对应于 UCUAA 五核苷酸重复区域。只有含有五核苷酸序列的前导RNA才能用于转录。五核苷酸重复区域内基因间位点和外源前导序列之间的错配在体外系统中得到纠正。因此，该体外系统建立了一种使用外源 RNA 进行反式引导的转录的新机制，并表明在冠状病毒 mRNA 合成过程中涉及特定的核糖核酸酶活性。
We have developed an in vitro transcription system which can utilize exogenous leader RNA for mouse hepatitis virus (MHV) ''leader-primed'' mRNA transcription. Cytoplasmic extracts containing viral proteins and template RNA were prepared by lysolecithin permeabilization of MHV-infected cells. Synthetic leader RNA which differed in sequence from the endogenous leader RNA was added to the extracts and demonstrated to be incorporated into MHV mRNAs. Irrespective of the size of leader RNAs added, the exogenous leader RNA was joined to the endogenous mRNA at the same site, which corresponds to a UCUAA pentanucleotide repeat region. Only leader RNAs containing the pentanucleotide sequences could be utilized for transcription. Mismatches between the intergenic site and the exogenous leader sequence within the pentanucleotide repeat region were corrected in the in vitro system. This in vitro system thus established a novel mechanism of leader-primed transcription using exogenous RNA in trans, and suggests the involvement of a specific ribonuclease activity during coronavirus mRNA synthesis.