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Mohamed Hussain, K, J Leong, M L Ng and J H Chu. The
Essential Role of Clathrin-mediated Endocytosis in the
Infectious Entry of Human Enterovirus 71. Journal of
Biological Chemistry 2011; 286(1): 309-321.
Little is currently known about the infectious entry process
of Human Enterovirus 71 (HEV71) into host cells, which may
represent potential anti-viral targeting sites. In this
study, a targeted small-interfering RNA (siRNA) screening
platform assay was established and validated to identify and
profile key cellular genes involved in processes of
endocytosis, cytoskeletal dynamics and endosomal trafficking
essential for HEV71 infection. Screen evaluation was
conducted via the expression of well-characterised
dominant-negative mutants, bioimaging studies
(double-labeled immunofluorescence assays, transmission
electron microscopy analysis), secondary siRNA-based dosage
dependency studies and drug inhibition assays. The
infectious entry of HEV71 into RD cells was shown to be
significantly inhibited by siRNAs targeting genes associated
with clathrinmediated endocytosis (CME), that include AP2A1,
ARRB1, CLTC, CLTCL1, SYNJ1, ARPC5, PAK1, ROCK1 and WASF1.
The functional role of CME was verified by the observation
of strong co-localisation between HEV71 particles and
clathrin as well as dose-dependent inhibition of HEV71
infection upon siRNA knockdown of CME-associated genes.
HEV71 entry by CME was further confirmed via inhibition by
dominant-negative EPS15 mutants and treatment of CME drug
inhibitors, with more than 80% inhibition observed at 20μM
chlorpromazine. Furthermore, HEV71 infection was shown to be
sensitive to the disruption of human genes in regulating
early to late endosomal trafficking as well as endosomal
acidic pH. The identification of clathrin-mediated
endocytosis as the entry pathway for HEV71 infection of
susceptible host cells contributes to a better understanding
of HEV71 pathogenesis and enables future development of
anti-viral strategies against HEV71 infection.
Legend for the attached figure: Ultra-structural analysis of
HEV71 infectious entry. To visualise synchronised HEV71
entry, RD cells were incubated with HEV71 at 4oC
for 1 h, after which they were warmed to 37oC
prior to processing for transmission electron microscopy
analysis. (a) At 0 min upon warming to 37oC,
HEV71 particles (indicated by red arrows) were seen attached
to the cell surface (b) At 5 min after warm-up, HEV71
particles were observed within invaginations of the plasma
membrane (clathrin pits indicated by yellow arrows) (c) At
10 min of warm-up, HEV71 particles were seen enclosed within
clathrin-coated vesicles (d) At 15-20 min upon warm-up,
vesicles containing numerous HEV71 virus particles were
observed.
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