Putative cis-acting stem-loops in the 5′ untranslated region of the severe acute respiratory syndrome coronavirus can substitute for their mouse hepatitis virus counterparts
Putative cis-acting stem-loops in the 5′ untranslated region of the severe acute respiratory syndrome coronavirus can substitute for their mouse hepatitis virus counterparts
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DOI：
10.1128/jvi.00455-06
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发表时间：
2006-11-01
影响因子：
5.4
通讯作者：
Leibowitz, Julian L.
中科院分区：
文献类型：
--
作者：
Kang, Hyojeung;Feng, Min;Leibowitz, Julian L.
建立了小鼠肝炎病毒（MHV）和严重急性呼吸综合征冠状病毒（SCoV）5 '非翻译区（5 ' UTR）5 ' 140个核苷酸的二级结构模型，预测了3种主要的螺旋茎环结构，命名为茎环1（SL 1）、SL 2和SL 4。预测SCoV 5 ' UTR含有第四个茎环，命名为SL 3，其中前导转录调控序列（TRS）折叠成发夹环。通过用相应的SCoV序列替换完整的MHV 5 ′ UTR，并通过用相应的SCoV序列分别替换MHV 5 ′ UTR推定的SL 1、推定的SL 2、TRS和推定的SL 4，构建对应于MHV/SCoV嵌合基因组的cDNA。嵌合基因组在体外转录，并在电穿孔到允许细胞后回收病毒。其中MHV 5 ' UTR SL 1、SL 2和SL 4被其SCoV对应物单独替换的基因组是活的。含有完整的SCoV 5 'UTR或预测的SCoV SL 3的嵌合体是不能存活的。含有SCoV 5 'UTR的嵌合体（其中SCoV TRS被MHV TRS替换）也是不能存活的。含有整个SCoV 5 'UTR的嵌合体不能指导任何病毒特异性RNA的合成。在MHV/5 ' UTR SCov嵌合体中用MHV TRS替换SCoV TRS允许合成负义基因组大小的RNA，但不支持正义或负义亚基因组RNA的产生7。用MHV/SCoV SL 3嵌合体获得了类似的表型。这些结果表明，除了其已知的功能，在亚基因组RNA合成的负义基因组RNA的复制的TRS的作用。
Consensus covariation-based secondary structural models for the 5 ' 140 nucleotides of the 5 ' untranslated regions (5 ' UTRs) from mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SCoV) were developed and predicted three major helical stem-loop structures, designated stem-loop 1 (SL1), SL2, and SL4. The SCoV 5 ' UTR was predicted to contain a fourth stem-loop, named SL3, in which the leader transcriptional regulatory sequence (TRS) is folded into a hairpin loop. cDNAs corresponding to MHV/SCoV chimeric genomes were constructed by replacing the complete MHV 5 ' UTR with the corresponding SCoV sequence and by separately replacing MHV 5'UTR putative SL1, putative SL2, TRS, and putative SL4 with the corresponding SCoV sequences. Chimeric genomes were transcribed in vitro, and viruses were recovered after electroporation into permissive cells. Genomes in which the MHV 5 ' UTR SL1, SL2, and SL4 were individually replaced by their SCoV counterparts were viable. Chimeras containing the complete SCoV 5'UTR or the predicted SCoV SL3 were not viable. A chimera containing the SCoV 5'UTR in which the SCoV TRS was replaced with the MHV TRS was also not viable. The chimera containing the entire SCoV 5'UTR failed to direct the synthesis of any virus-specific RNA. Replacing the SCoV TRS with the MHV TRS in the MHV/5 ' UTR SCov chimera permitted the synthesis of minus-sense genome-sized RNA but did not support the production of positive- or minus-sense subgenomic RNA7. A similar phenotype was obtained with the MHV/SCoV SL3 chimera. These results suggest a role for the TRS in the replication of minus-sense genomic RNA in addition to its known function in subgenomic RNA synthesis.