Subgenomic messenger RNA amplification in coronaviruses
Subgenomic messenger RNA amplification in coronaviruses
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DOI：
10.1073/pnas.1000378107
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发表时间：
2010-07-06
影响因子：
11.1
通讯作者：
Brian, David A.
中科院分区：
文献类型：
--
作者：
Wu, Hung-Yi;Brian, David A.
冠状病毒拥有已知最大的RNA基因组，它是一种在细胞质中复制的27至32kb(+)链分子。在病毒复制过程中，形成了一个由5到8个亚基因组(Sg)mRNAs组成的3‘端共端嵌套组，这些mRNAs也是与基因组5’端共端的，因为当sgmRNA合成模板制作时，由于(-)链合成过程中基因供体信号的不连续转录，它们携带了基因组前导。一个悬而未决的问题是，迅速而丰富地出现的sgmRNAs是否进行了转录后扩增。在这里，使用RT-PCR和头尾连接的(-)链的序列分析，我们证明了在体外产生的标记sgmRNA导入病毒感染细胞后，sgmRNA和基因组一样，可以作为(-)链合成的模板。此外，当转染的sgmRNA含有内部定位的RNA依赖的RNA聚合酶模板切换供体信号时，该位点发生不连续转录，并产生一个较短的、3‘末端嵌套的含有前导的sgmRNA，其前导-小体连接和GFP基因的表达证明了这一点。因此，原则上，自然感染中较长的套式sgmRNAs都包含潜在的内部模板切换供体信号，可以增加较短的3‘-套式sgmRNAs的数量。这种行为对冠状病毒存活率的一个预测优势是增加了维持基因组的机会。通过WT sgmRNA(现在独立丰富的)和有缺陷的基因组之间的同源重组，基因组的3‘三分之一的完整性。
Coronaviruses possess the largest known RNA genome, a 27- to 32-kb (+)-strand molecule that replicates in the cytoplasm. During virus replication, a 3' coterminal nested set of five to eight subgenomic (sg) mRNAs are made that are also 5' coterminal with the genome, because they carry the genomic leader as the result of discontinuous transcription at intergenic donor signals during (-)-strand synthesis when templates for sgmRNA synthesis are made. An unanswered question is whether the sgmRNAs, which appear rapidly and abundantly, undergo posttranscriptional amplification cation. Here, using RT-PCR and sequence analyses of head-totail-ligated (-) strands, we show that after transfection of an in vitro-generated marked sgmRNA into virus-infected cells, the sgmRNA, like the genome, can function as a template for (-)-strand synthesis. Furthermore, when the transfected sgmRNA contains an internally placed RNA-dependent RNA polymerase template-switching donor signal, discontinuous transcription occurs at this site, and a shorter, 3' terminally nested leader-containing sgmRNA is made, as evidenced by its leader-body junction and by the expression of a GFP gene. Thus, in principle, the longer-nested sgmRNAs in a natural infection, all of which contain potential internal templateswitching donor signals, can function to increase the number of the shorter 3'-nested sgmRNAs. One predicted advantage of this behavior for coronavirus survivability is an increased chance of maintaining genome. tness in the 3' one-third of the genome via a homologous recombination between the (now independently abundant) WT sgmRNA and a defective genome.