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01.12.2018 | Research | Ausgabe 1/2018 Open Access

Virology Journal 1/2018

Enhanced human enterovirus 71 infection by endocytosis inhibitors reveals multiple entry pathways by enterovirus causing hand-foot-and-mouth diseases

Virology Journal > Ausgabe 1/2018
Meichun Yuan, Jingjing Yan, Jingna Xun, Chong Chen, Yuling Zhang, Min Wang, Wenqi Chu, Zhigang Song, Yunwen Hu, Shuye Zhang, Xiaoyan Zhang
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Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12985-017-0913-3) contains supplementary material, which is available to authorized users.



Human 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.


Viral 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.


CPZ 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.


CPZ 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.
Additional file 1: Figure S1. Pretreatment of EV71 by CPZ had no effect on subsequent infection in A549 cells. EV71 was pretreated by DMSO or CPZ (20 μM), and then infected A549 cells (MOI=5). The “0h” indicated the infection in the presence or absence of CPZ (20 μM) without CPZ pretreatment. 12 h later, virus was removed and VP-1 expression was examined at 24 h. Means of three experiments are shown. *, p<0.05. Figure S2. Characterization of the CPZ effect. HepG2 and A549 cells were infected with EV71 (MOI=5) for 12 h, and then CPZ (20 μM) or DMSO was added at indicated time points. 24 hpi, cells were subjected to VP-1 staining. CPZ, filled square; DMSO, filled circle. Figure S3. CPZ enhanced UV-inactivated EV71 uptake in A549 cells. a. A549 cells were incubated with EV71 or UV-inactive EV71 at an MOI of 50 at 4 °C for 2 h, and then shifted to 37 °C and treated by CPZ (20 μM) or DMSO. 6 hpi, cells were stained with VP-1 antibody (Red, VP-1; DAPI, nuclei). Scale bar, 100 μm. b. Frequency of VP-1 foci in infected A549 cell was compared using paired Student’s t test. *, p<0.05. Figure S4. The knockdown efficiency of CME in A549 and RD cells. a-b. The mRNA levels of targeted genes were measured by qPCR after 48 h transfection (normalized to 18s mRNA). c. The protein levels were detected by western blot at 96 h post-transfection. GAPDH was used as an internal control. The bar plots were summarized from three independent experiments. Figure S5. The effect of CPZ on EV71 infection in A549 cells. The full length blots of Fig. 1b. Figure S6. CPZ facilitates the entry step of EV71 infection. Part of the Fig. 4c image was enlarged to show the viral foci in A549 cells more clearly. (DOC 2327 kb)
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