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

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.

Key words:EV-D68, 5′ UTR, dual luciferase report system