Abstract
BackgroundHuman enterovirus 71 (EV71) was previously known to enter cells through clathrin or caveolar mediated endocytic pathways. However, we observed chlorpromazine (CPZ) or dynasore (DNS), which inhibit clathrin and dynamin mediated endocytosis, did not suppress EV71 cell entry in particular cell types. So the current knowledge of entry mechanisms by EV71 is not complete.MethodsViral infection was examined by flow cytometry or end-point dilution assays. Viral entry was monitored by immunofluorescence or pseudoviral infections. Various inhibitors were utilized for manipulating endocytic pathways. Cellular proteins were knockdown by siRNA.ResultsCPZ and DNS did not inhibit but rather enhance viral infection in A549 cells, while they inhibited infections in other cells tested. We further found CPZ did not affect EV71 binding to target cells and failed to affect viral translation and replication, but enhanced viral entry in A549 cells. Immunofluorescence microscopy further confirmed this increased entry. Using siRNA experiment, we found that the enhancement of EV71 infection by CPZ did not require the components of clathrin mediated endocytosis. Finally, CPZ also enhanced infection by Coxackivirus A16 in A549 cells.ConclusionsCPZ and DNS, previously reported as EV71 entry inhibitors, may rather lead to increased viral infection in particular cell types. CPZ and DNS increased viral entry and not other steps of viral life cycles. Therefore, our study indicated an unknown dynamin-independent entry pathway utilized by enteroviruses that cause Hand-Foot-and-Mouth Diseases.
Highlights
Human enterovirus 71 (EV71) was previously known to enter cells through clathrin or caveolar mediated endocytic pathways
We found that the infection of EV71 in A549 cells was rather enhanced when treated with increasing concentrations of CPZ (Fig. 1a)
Pretreatment of EV71 by CPZ showed on effect on subsequent infections in A549 cells (Fig. S1)
Summary
Human enterovirus 71 (EV71) was previously known to enter cells through clathrin or caveolar mediated endocytic pathways. We observed chlorpromazine (CPZ) or dynasore (DNS), which inhibit clathrin and dynamin mediated endocytosis, did not suppress EV71 cell entry in particular cell types. Hand-foot-and-mouth disease (HFMD) is a common, usually self-limiting illness affecting large numbers of infants and young children worldwide [1]. The clinical symptoms include fever, vesicular rashes on hands, feet, buttocks and the mouth. A small proportion of infected children may suffer severe complications including meningitis, acute flaccid paralysis, neurorespiratory syndrome and even fatal illness. The EV71 mature virion has a canyon around the five-fold axes, probably as the binding site for EV71 specific receptor. Several receptors have been reported, only human scavenger
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