REQUIREMENT OF THE 5'-END GENOMIC SEQUENCE AS AN UPSTREAM CIS-ACTING ELEMENT FOR CORONAVIRUS SUBGENOMIC MESSENGER-RNA TRANSCRIPTION
REQUIREMENT OF THE 5'-END GENOMIC SEQUENCE AS AN UPSTREAM CIS-ACTING ELEMENT FOR CORONAVIRUS SUBGENOMIC MESSENGER-RNA TRANSCRIPTION
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DOI：
10.1128/jvi.68.8.4727-4737.1994
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发表时间：
1994-08-01
影响因子：
5.4
通讯作者：
LAI, MMC
中科院分区：
文献类型：
--
作者：
LIAO, CL;LAI, MMC
我们已经开发了一个有缺陷的干扰（DI）RNA含有氯霉素乙酰转移酶报告基因，放置在基因间序列，研究小鼠肝炎病毒（MHV），原型冠状病毒的亚基因组mRNA转录。使用这个系统，我们已经确定了MHV亚基因组mRNA转录所需的序列。我们表明，这种序列的要求不同于RNA复制。除了先前鉴定的对基因间（启动子）序列的需要之外，亚基因组mRNA转录还需要来自基因组RNA 5'端的额外序列。这些上游序列包括前导RNA和前导与基因间序列之间的间隔序列，其来源于5'非翻译区和基因1的一部分。间隔区序列要求是特异性的，因为只有来自RNA基因组5'端的序列，而不是来自其他MHV基因组区域或异源序列的序列，才能从基因间序列启动亚基因组转录。这些结果强烈表明，野生型病毒亚基因组mRNA（mRNA 2至mRNA 7）和可能的对应亚基因组负义RNA不能用于mRNA扩增。此外，我们已经证明，存在于基因组5'端的部分前导序列，其缺乏前导-mRNA融合序列，仍然可以支持亚基因组mRNA转录。在这种情况下，亚基因组转录物的前导序列完全来自野生型辅助病毒，表明MPN前导RNA启动反式亚基因组mRNA转录。因此，即使前导序列不能作为mRNA合成的引物，它也可以增强亚基因组转录。这些结果表明，MHV的5 '端前导序列不仅为mRNA起始提供了反式作用引物，而且还作为亚基因组mRNA转录所需的顺式作用元件。MHV亚基因组mRNA合成的上游顺式作用元件的鉴定定义了调节RNA病毒mRNA合成的新序列要求。
We have developed a defective interfering (DI) RNA containing a chloramphenicol acetyltransferase reporter gene, placed behind an intergenic sequence, for studying subgenomic mRNA transcription of mouse hepatitis virus (MHV), a prototype coronavirus. Using this system, we have identified the sequence requirement for MHV subgenomic mRNA transcription. We show that this sequence requirement differs from that for RNA replication. In addition to the previously identified requirement for an intergenic (promoter) sequence, additional sequences from the 5' end of genomic RNA are required for subgenomic mRNA transcription. These upstream sequences include the leader RNA and a spacer sequence between the leader and intergenic sequence, which is derived from the 5' untranslated region and part of gene 1. The spacer sequence requirement is specific, since only the sequence derived from the 5' end of RNA genome, but not from other MHV genomic regions or heterologous sequences, could initiate subgenomic transcription from the intergenic sequence. These results strongly suggest that the wild-type viral subgenomic mRNAs (mRNA2 to mRNA7) and probably their counterpart subgenomic negative-sense RNAs cannot be utilized for mRNA amplification. Furthermore, we have demonstrated that a partial leader sequence present at the 5' end of genome, which lacks the leader-mRNA fusion sequence, could still support subgenomic mRNA transcription. In this case, the leader sequences of the subgenomic transcripts were derived exclusively from the wild-type helper virus, indicating that the MPN leader RNA initiates in trans subgenomic mRNA transcription. Thus, the leader sequence can enhance subgenomic transcription even when it cannot serve as a primer for mRNA synthesis. These results taken together suggest that the 5'-end leader sequence of MHV not only provides a trans-acting primer for mRNA initiation but also serves as a cis-acting element required for the transcription of subgenomic mRNAs. The identification of an upstream cis-acting element for MHV subgenomic mRNA synthesis defines a novel sequence requirement for regulating mRNA synthesis in RNA viruses.