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.