Wang Y, Zhang X
Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
J Virol. 2000 Nov;74(22):10571-80. doi: 10.1128/jvi.74.22.10571-10580.2000.
While the 5' cis-acting sequence of mouse hepatitis virus (MHV) for genomic RNA replication has been determined in several defective interfering (DI) RNA systems, it remains elusive for subgenomic RNA transcription. Previous studies have shown that the leader RNA in the DI genome significantly enhances the efficiency of DI subgenomic mRNA transcription, indicating that the leader RNA is a cis-acting sequence for mRNA transcription. To further characterize the cis-acting sequence, we made a series of deletion mutants, all but one of which have an additional deletion of the cis-acting signal for replication in the 5' untranslated region. This deletion effectively eliminated the replication of the DI-chloramphenicol acetyltransferase (CAT)-reporter, as demonstrated by the sensitive reverse transcription (RT)-PCR. The ability of these replication-minus mutants to transcribe subgenomic mRNAs was then assessed using the DI RNA-CAT reporter system. Results from both CAT activity and mRNA transcripts detected by RT-PCR showed that a 5'-proximal sequence of 35 nucleotides (nt) at nt 25 to 59 is a cis-acting sequence required for subgenomic RNA transcription, while the consensus repeat sequence of the leader RNA does not have such effect. Analyses of the secondary structure indicate that this 35-nt sequence forms two stem-loops conserved among MHVs. Deletion of this sequence abrogated transcriptional activity and disrupted the predicted stem-loops and overall RNA secondary structure at the 5' untranslated region, suggesting that the secondary structure formed by this 35-nt sequence may facilitate the downstream consensus sequence accessible for the discontinuous RNA transcription. This may provide a mechanism by which the 5' cis-acting sequence regulates subgenomic RNA transcription. The 5'-most 24 nt are not essential for transcription, while the 9 nt immediately downstream of the leader enhances RNA transcription. The sequence between nt 86 and 135 had little effect on transcription. This study thus defines the cis-acting transcription signal at the 5' end of the DI genome.
虽然在几种缺陷干扰（DI）RNA系统中已经确定了小鼠肝炎病毒（MHV）基因组RNA复制的5'顺式作用序列，但亚基因组RNA转录的顺式作用序列仍不清楚。先前的研究表明，DI基因组中的前导RNA显著提高了DI亚基因组mRNA转录的效率，这表明前导RNA是mRNA转录的顺式作用序列。为了进一步表征顺式作用序列，我们构建了一系列缺失突变体，除了一个之外，所有突变体在5'非翻译区都额外缺失了复制的顺式作用信号。如灵敏的逆转录（RT）-PCR所示，这种缺失有效地消除了DI-氯霉素乙酰转移酶（CAT）报告基因的复制。然后使用DI RNA-CAT报告系统评估这些无复制能力突变体转录亚基因组mRNA的能力。CAT活性和RT-PCR检测到的mRNA转录本的结果均表明，第25至59位核苷酸处5'近端的35个核苷酸（nt）序列是亚基因组RNA转录所需的顺式作用序列，而前导RNA的共有重复序列没有这种作用。二级结构分析表明，这个35-nt序列形成了两个在MHV中保守的茎环。删除该序列消除了转录活性，并破坏了5'非翻译区预测的茎环和整体RNA二级结构，这表明由这个35-nt序列形成的二级结构可能有助于下游共有序列可用于不连续RNA转录。这可能提供了一种5'顺式作用序列调节亚基因组RNA转录的机制。最5'端的24 nt对转录不是必需的，而前导序列下游紧邻的9 nt增强了RNA转录。第86至135位核苷酸之间的序列对转录影响很小。因此，本研究确定了DI基因组5'端的顺式作用转录信号。
