Myristoylation of EV71 VP4 is Essential for Infectivity and Interaction with Membrane Structure
Myristoylation of EV71 VP4 is Essential for Infectivity and Interaction with Membrane Structure
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EV71 VP4 的肉豆蔻酰化对于感染性和与膜结构的相互作用至关重要
DOI：
10.1007/s12250-020-00226-1
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发表时间：
2020-05-12
影响因子：
5.5
通讯作者：
Jiang, Chunlai
中科院分区：
文献类型：
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作者：
Cao, Jiaming;Qu, Meng;Jiang, Chunlai
肠道病毒71（EV 71）VP 4在其氨基末端甘氨酸残基处与肉豆蔻酸共衍生连接。然而，这种肉豆蔻酰化在EV 71生命周期中的作用在很大程度上仍然未知。为了研究这个问题，我们开发了一种肉豆蔻酰化缺陷型病毒和报告基因（荧光素酶）假病毒，其在EV 71 VP 4上具有Gly-to-Ala突变（G2 A）。将EV 71-G2 A基因组编码质粒转染细胞后，VP 4豆蔻酰化缺失对病毒蛋白表达和病毒形态无影响，但对病毒感染性有显著影响。此外，在豆蔻酰化缺陷型报告假病毒感染的细胞中，荧光素酶活性和病毒基因组RNA与野生型病毒相比显著降低;然而，在豆蔻酰化缺陷型病毒感染的细胞中未检测到细胞病变效应和病毒衣壳蛋白。此外，尽管在第二次盲传感染中检测到豆蔻酰化缺陷病毒RNA和蛋白质，但与野生型病毒相比，它们的数量少得多。豆蔻酰化缺陷型病毒基因组RNA和负链病毒RNA的复制均被阻断，表明豆蔻酰化影响病毒基因组RNA从衣壳释放到细胞质。此外，失去豆蔻酰化的VP 4改变了分布的VP 4-绿色荧光蛋白，从膜结构部分消失。最后，脂质体渗漏测定显示EV 71肉豆蔻酰化介导模型膜的渗透性。因此，VP 4的氨基末端豆蔻酰化是EV 71感染和衣壳-膜结构相互作用的关键。本研究为EV 71感染的分子机制和抗病毒药物设计提供了新的分子靶点。
The Enterovirus 71 (EV71) VP4 is co-translationally linked to myristic acid at its amino-terminal glycine residue. However, the role of this myristoylation in the EV71 life cycle remains largely unknown. To investigate this issue, we developed a myristoylation-deficient virus and reporter (luciferase) pseudovirus with a Gly-to-Ala mutation (G2A) on EV71 VP4. When transfecting the EV71-G2A genome encoding plasmid in cells, the loss of myristoylation on VP4 did not affect the expression of viral proteins and the virus morphology, however, it did significantly influence viral infectivity. Further, in myristoylation-deficient reporter pseudovirus-infected cells, the luciferase activity and viral genome RNA decreased significantly as compared to that of wild type virus; however, cytopathic effect and viral capsid proteins were not detected in myristoylation-deficient virus-infected cells. Also, although myristoylation-deficient viral RNA and proteins were detected in the second blind passage of infection, they were much fewer in number compared to that of the wild type virus. The replication of genomic RNA and negative-strand viral RNA were both blocked in myristoylation-deficient viruses, suggesting that myristoylation affects viral genome RNA release from capsid to cytoplasm. Besides, loss of myristoylation on VP4 altered the distribution of VP4-green fluorescent protein protein, which disappeared from the membrane structure fraction. Finally, a liposome leakage assay showed that EV71 myristoylation mediates the permeability of the model membrane. Hence, the amino-terminal myristoylation of VP4 is pivotal to EV71 infection and capsid-membrane structure interaction. This study provides novel molecular mechanisms regarding EV71 infection and potential molecular targets for antiviral drug design.