INVESTIGATION OF THE CONTROL OF CORONAVIRUS SUBGENOMIC MESSENGER-RNA TRANSCRIPTION BY USING T7-GENERATED NEGATIVE-SENSE RNA TRANSCRIPTS
INVESTIGATION OF THE CONTROL OF CORONAVIRUS SUBGENOMIC MESSENGER-RNA TRANSCRIPTION BY USING T7-GENERATED NEGATIVE-SENSE RNA TRANSCRIPTS
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DOI：
10.1128/jvi.69.10.6219-6227.1995
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发表时间：
1995-10-01
影响因子：
5.4
通讯作者：
BRITTON, P
中科院分区：
文献类型：
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作者：
HISCOX, JA;MAWDITT, KL;BRITTON, P
冠状病毒传染性胃肠炎病毒（TGEV）的亚基因组mRNA不是以等摩尔量产生的。我们已经开发了一个报告基因系统来研究这种差异亚基因组mRNA合成的控制。通过TGEV聚合酶的mRNA的转录从含有T7启动子的DNA原位产生的反义RNA模板获得。产生了一系列基因盒;这些基因盒包含转录相关序列（塔斯）（也称为基因间序列和启动子）下游的报告基因氯霉素乙酰转移酶（CAT）基因，据信参与TGEV亚基因组mRNA 6和7的合成。设计基因盒，使得在3'末端具有与TGEV塔斯互补的序列或修饰形式的CAT基因的负义RNA拷贝将通过T7 RNA聚合酶原位合成。使用这个系统，我们已经证明，CAT表达的mRNA来源于T7产生的负义RNA转录只在TGEV感染的细胞，只有从具有TGEV负义TAS的转录。CAT mRNA的分析显示在5'端存在TGEV前导RNA序列，这与所有冠状病毒mRNA具有对应于基因组RNA的5'端的5'前导序列的观察结果一致。我们的结果表明，CAT mRNA是从原位合成的负义RNA模板转录的，而不需要TGEV基因组5'或3'序列上的T7产生的负义转录本（3 '-TAS-CAT-5'）。TGEV塔斯的修饰表明：（i）前导RNA 3'端和TGEV反义TAS之间潜在碱基配对的程度不是转录的亚基因组mRNA量的唯一决定因素;（ii）其他因素，包括TAS侧翼的核苷酸，参与TGEV亚基因组mRNA转录的调节。
The subgenomic mRNAs of the coronavirus transmissible gastroenteritis virus (TGEV) are not produced in equimolar amounts. We have developed a reporter gene system to investigate the control of this differential subgenomic mRNA synthesis. Transcription of mRNAs by the TGEV polymerase was obtained from negative-sense RNA templates generated in situ from DNA containing a T7 promoter. A series of gene cassettes was produced; these cassettes comprised the reporter chloramphenicol acetyltransferase (CAT) gene downstream of transcription-associated sequences (TASs) (also referred to as intergenic sequences and promoters) believed to be involved in the synthesis of TGEV subgenomic mRNAs 6 and 7. The gene cassettes were designed so that negative-sense RNA copies of the CAT gene with sequences complementary to the TGEV TASs, or modified versions, at the 3' end would be synthesized in situ by T7 RNA polymerase. Using this system, we have demonstrated that CAT was expressed from mRNAs derived from the T7-generated negative-sense RNA transcripts only in TGEV-infected cells and only from transcripts possessing a TGEV negative-sense TAS. Analysis of the CAT mRNAs showed the presence of the TGEV leader RNA sequence at the 5' end, in keeping with observations that all coronavirus mRNAs have a 5' leader sequence corresponding to the 5' end of the genomic RNA. Our results indicated that the CAT mRNAs were transcribed from the in situ-synthesized negative-sense RNA templates without the requirement of TGEV genomic 5' or 3' sequences on the T7-generated negative-sense transcripts (3'-TAS-CAT-5'). Modification of the TGEV TASs indicated (i) that the degree of potential base pairing between the 3' end of the leader RNA and the TGEV negative-sense TAS was not the sole determinant of the amount of subgenomic mRNA transcribed and (ii) that other factors, including nucleotides flanking the TAS, are involved in the regulation of transcription of TGEV subgenomic mRNAs.