Mouse hepatitis virus stem-loop 4 functions as a spacer element required to drive subgenomic RNA synthesis.
Mouse hepatitis virus stem-loop 4 functions as a spacer element required to drive subgenomic RNA synthesis.
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小鼠肝炎病毒茎环 4 充当驱动亚基因组 RNA 合成所需的间隔元件。
DOI：
10.1128/jvi.05092-11
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发表时间：
2011
影响因子：
5.4
通讯作者：
Leibowitz,Julian
中科院分区：
文献类型：
--
作者：
Yang,Dong;Liu,Pinghua;Giedroc,DavidP;Leibowitz,Julian
小鼠肝炎病毒（MHV） 5 ‘非翻译区（5 ’ utr）的5 ' 140核苷酸预计包含三个二级结构，茎环1 (SL1)， SL2和SL4。SL1和SL2是亚基因组RNA合成所必需的。目前的研究重点是SL4，它包含两个碱基配对区域SL4a和SL4b。一系列的反向遗传实验表明，SL4a并不需要碱基配对。SL4b的结构、序列和8-氨基酸开放阅读框（ORF）都不是病毒复制所必需的。含有SL4a和SL4b分离缺失的病毒是可存活的。然而，SL4的缺失是致命的，携带这种缺失的基因组在指导亚基因组RNA合成方面存在缺陷。131aca133仅3 '到SL4的缺失对病毒复制有深远的影响。携带131aca133缺失的病毒在斑块大小上是异质的。我们分离了三种在5'UTR中具有第二位点突变的病毒，这种突变弥补了斑块大小的减少、生长动力学的延迟以及与131aca133缺失相关的较低滴度。在我们的模型中，第二位点突变预计会改变SL1和SL2之间或SL2和SL4之间的间距，或破坏SL4a近端部分的稳定性。用较短的与序列无关的茎环取代SL4构建的突变体是可行的。这些结果表明，MHV 5'UTR中的SL4在一定程度上作为间隔元件定位SL1、SL2和转录调控序列（TRS），这种间隔元件可能在指导亚基因组RNA合成中发挥重要作用。
The 5′ 140 nucleotides of the mouse hepatitis virus (MHV) 5′ untranslated region (5′UTR) are predicted to contain three secondary structures, stem-loop 1 (SL1), SL2, and SL4. SL1 and SL2 are required for subgenomic RNA synthesis. The current study focuses on SL4, which contains two base-paired regions, SL4a and SL4b. A series of reverse genetic experiments show that SL4a is not required to be base paired. Neither the structure, the sequence, nor the putative 8-amino-acid open reading frame (ORF) in SL4b is required for viral replication. Viruses containing separate deletions of SL4a and SL4b are viable. However, deletion of SL4 is lethal, and genomes carrying this deletion are defective in directing subgenomic RNA synthesis. Deletion of131ACA133just 3′ to SL4 has a profound impact on viral replication. Viruses carrying the131ACA133deletion were heterogeneous in plaque size. We isolated three viruses with second-site mutations in the 5′UTR which compensated for decreased plaque sizes, delayed growth kinetics, and lower titers associated with the131ACA133deletion. The second-site mutations are predicted to change either the spacing between SL1 and SL2 or that between SL2 and SL4 or to destabilize the proximal portion of SL4a in our model. A mutant constructed by replacing SL4 with a shorter sequence-unrelated stem-loop was viable. These results suggest that the proposed SL4 in the MHV 5′UTR functions in part as a spacer element that orients SL1, SL2, and the transcriptional regulatory sequence (TRS), and this spacer function may play an important role in directing subgenomic RNA synthesis.