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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 perme?ability 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.