Zúñiga Sonia, Sola Isabel, Alonso Sara, Enjuanes Luis
Centro Nacional de Biotecnología, CSIC, Department of Molecular and Cell Biology, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
J Virol. 2004 Jan;78(2):980-94. doi: 10.1128/jvi.78.2.980-994.2004.
Coronavirus transcription leads to the synthesis of a nested set of mRNAs with a leader sequence derived from the 5' end of the genome. The mRNAs are produced by a discontinuous transcription in which the leader is linked to the mRNA coding sequences. This process is regulated by transcription-regulating sequences (TRSs) preceding each mRNA, including a highly conserved core sequence (CS) with high identity to sequences present in the virus genome and at the 3' end of the leader (TRS-L). The role of TRSs was analyzed by reverse genetics using a full-length infectious coronavirus cDNA and site-directed mutagenesis of the CS. The canonical CS-B was nonessential for the generation of subgenomic mRNAs (sgmRNAs), but its presence led to transcription levels at least 10(3)-fold higher than those in its absence. The data obtained are compatible with a transcription mechanism including three steps: (i) formation of 5'-3' complexes in the genomic RNA, (ii) base-pairing scanning of the nascent negative RNA strand by the TRS-L, and (iii) template switching during synthesis of the negative strand to complete the negative sgRNA. This template switch takes place after copying the CS sequence and was predicted in silico based on high base-pairing score between the nascent negative RNA strand and the TRS-L and minimum DeltaG.
冠状病毒转录会导致合成一组嵌套的mRNA，这些mRNA带有源自基因组5'端的前导序列。这些mRNA是通过一种不连续转录产生的，在前导序列与mRNA编码序列相连。这个过程受每个mRNA之前的转录调控序列（TRS）调节，包括一个高度保守的核心序列（CS），它与病毒基因组中以及前导序列（TRS-L）3'端存在的序列具有高度同源性。通过使用全长感染性冠状病毒cDNA和对核心序列进行定点诱变的反向遗传学方法分析了TRS的作用。典型的CS-B对于亚基因组mRNA（sgmRNA）的产生并非必需，但它的存在会使转录水平比不存在时至少高10³倍。获得的数据与一种包括三个步骤的转录机制相符：（i）在基因组RNA中形成5'-3'复合物；（ii）TRS-L对新生负链RNA进行碱基配对扫描；（iii）在负链合成过程中进行模板切换以完成负链sgRNA。这种模板切换在复制CS序列后发生，并且基于新生负链RNA与TRS-L之间的高碱基配对得分和最小ΔG在计算机上进行了预测。
