人肠道病毒D68型5′ UTR间隔区域对下游基因表达的影响

Effect of EV-D68 virus 5′ UTR spacer region on downstream gene expression

摘要:

【背景】人肠道病毒D68型(EV-D68)属于小RNA病毒科肠道病毒属人肠道病毒D组，在2014年8月至2015年1月间，该病毒引起的感染在北美显著增多，我国也出现流行。相对于原始的Fermon毒株，流行株5′ UTR区域几乎都存在一两处缺失，在起始密码子ATG前还存在两处ataaca重复序列，这两处位点的功能尚未见报道。【目的】探讨流行株5′ UTR区域的缺失对下游基因表达的影响，以及ataaca重复序列的功能。【方法】通过序列比对分析现阶段流行株与原始毒株Fermon株5′ UTR的差异以及保守区域，利用分子生物学方法缺失上述区域后，利用双荧光素酶报告系统分析5′ UTR中上述区域对下游报告基因的影响。【结果】序列比对分析发现，目前流行的EV-D68毒株在对应于Fermon株基因组5′ UTR的685?707区域发生了23个碱基的缺失，而部分毒株还在718?729区域发生了第二处缺失。荧光素酶结果显示，仅第一处缺失可以极大地提高下游基因的表达量，同时具有两处缺失则与野生型几乎相当，而仅缺失第二段序列则降低了下游基因的表达量。此外，还发现第一处缺失中的序列可能对下游基因表达起到了抑制作用，而靠近起始密码子的ataaca序列作用尚不明确。【结论】目前流行的EV-D68毒株在5′ UTR区域大多出现了一两处缺失，第一处缺失极大地增强了下游报告基因的表达，而第二处缺失具有相反的功能，这一现象可能与起始密码子前的两处ataaca重复序列有关。

Abstract:

[Background] EV-D68 belongs to the enterovirus D group of the Enterovirus genus of the small RNA virus family. Between August 2014 and January 2015, the infection caused by the virus increased significantly in North America, and also appeared in China. Compared with the original Fermon strain, the epidemic strain has almost one or two deletions in the 5′ UTR region, and there are two repeated ataaca sequences before the translation start codon. [Objective] We explored the effect of the deletion of 5′ UTR region of epidemic strains on downstream gene expression and the function of ataaca repeat sequence. [Methods] Sequence comparison was used to analyze the differences and conserved regions of 5′ UTR between the current epidemic strains and the original Fermon strain. The above regions were deleted by using molecular clone methods and then the dual-luciferase reporter system was used to analyze the effect on downstream luciferase reports genes. [Results] Sequence alignment analysis found that the current epidemic EV-D68 strain has a 23 base deletion in the region corresponding to the 685?707 of the 5′ UTR of the Fermon strain genome, while some strains have an additional deletion in the 718?729 regions. The luciferase assay showed that only the first deletion can greatly increase the expression of downstream genes, while the two deletions occur at the same time are almost equivalent to the wild type, while the deletion of the second sequence only reduces the expression of downstream genes slightly. In addition, we also found that the ataaca sequence in the first deletion may have a suppressive effect on downstream gene expression, and the role of the ataaca sequence near the start codon is not yet clear. [Conclusion] At present, most of the epidemic EV-D68 strains have one or two deletions in the 5′ UTR region. The first deletion greatly enhances the expression of downstream reporter genes, while the second deletion has the opposite function. The phenomenon may be related to the repeated ataaca sequences before the start codon.