EHD4 facilitates Zika virus infection by regulating extracellular vesicle-associated viral release and cell attachment

Yue Zhang; Xiu-Li Yan; Kao Deng; Meng-Jiao He; Hui Wang; Zi-Jin Cui; Yu-Yan Li; Lei Cao; Wenlong Ma; Byung-Ho Kang; Hoi Ki Leung; Peter Pak-Hang Cheung; Cheng-Feng Qin; Ming-Yuan Li; Xiao-Feng Li

doi:10.1016/j.virs.2026.04.009

October 2026

Citation: Yue Zhang, Xiu-Li Yan, Kao Deng, Meng-Jiao He, Hui Wang, Zi-Jin Cui, Yu-Yan Li, Lei Cao, Wenlong Ma, Byung-Ho Kang, Hoi Ki Leung, Peter Pak-Hang Cheung, Cheng-Feng Qin, Ming-Yuan Li, Xiao-Feng Li. EHD4 facilitates Zika virus infection by regulating extracellular vesicle-associated viral release and cell attachment .VIROLOGICA SINICA, 2026, 41(5) : 1091-1105. http://dx.doi.org/10.1016/j.virs.2026.04.009

EHD4 facilitates Zika virus infection by regulating extracellular vesicle-associated viral release and cell attachment

Yue Zhang ^a,b ,
Xiu-Li Yan ^b ,
Kao Deng ^b,c ,
Meng-Jiao He ^b ,
Hui Wang ^b ,
Zi-Jin Cui ^b ,
Yu-Yan Li ^b ,
Lei Cao ^e ,
Wenlong Ma ^f ,
Byung-Ho Kang ^f ,
Hoi Ki Leung ^f ,
Peter Pak-Hang Cheung ^c ,
Cheng-Feng Qin ^{b
,,} ,
Ming-Yuan Li ^{c,d,b
,,} ,
Xiao-Feng Li ^{a,b
,,}

a. School of Basic Medical Sciences, Anhui Medical University, Hefei, 230032, China;
b. State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China;
c. Department of Chemical Pathology and Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong, Hong Kong SAR, China;
d. Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, 518063, China;
e. National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China;
f. State Key Laboratory of Agrobiotechnology, AoE Centre for Plant Vacuole Biology and Biotechnology, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China

Corresponding author: Cheng-Feng Qin, qincf@bmi.ac.cn
Ming-Yuan Li, mingyuanli@cuhk.edu.hk
Xiao-Feng Li, xiaofeng_li_bj@163.com
Received Date: 31 March 2026
Accepted Date: 14 September 2026
Available online: 19 September 2026

Abstract

Extracellular vesicles (EVs) and other extracellular membrane-associated particles can contribute to viral dissemination; however, the host factors that regulate the composition and infectivity of EV-associated viral material remain incompletely understood. Here, comparative proteomic profiling identified Eps15 homology domain-containing protein 4 (EHD4) as a candidate host factor enriched in EV/virion-containing preparations from Zika virus (ZIKV)-infected Vero cells. Genetic ablation of EHD4 in HeLa cells impaired the propagation of ZIKV, Japanese encephalitis virus, and dengue virus serotype 2, whereas EHD4 re-expression restored viral propagation. In ZIKV-infected EHD4-knockout (KO) cells, a greater proportion of extracellular viral RNA was recovered in EV-enriched fractions than in wild-type (WT) cells. However, viral material recovered from KO-derived EV-enriched fractions exhibited reduced cell-binding activity and lower infectivity. Partial disruption of membrane integrity attenuated the difference in infectivity between WT- and KO-derived preparations, suggesting that membrane-associated components contribute to the functional properties of the associated viral material. ZIKV replicon assays showed no major effect of EHD4 on viral RNA replication. Immunofluorescence showed partial colocalization of EHD4 with CD63-positive compartments and ZIKV envelope protein. Transmission electron microscopy revealed that EHD4 loss did not affect multivesicular body (MVB) formation, but virus-like particles were readily observed in MVB lumens of WT cells and rarely in KO cells, suggesting that EHD4 promotes viral sorting into MVB luminal vesicles. Quantitative proteomic analysis further identified reduced abundance of a subset of membrane-associated proteins in KO-derived EV-enriched fractions. Collectively, these findings identify EHD4 as a host factor that influences the extracellular partitioning, composition, and infectivity of flaviviral material recovered in EV-enriched fractions. The molecular mechanisms underlying these effects remain to be defined.
- Zika virus (ZIKV)
- , Extracellular vesicle
- , EHD4

Electronic Supplementary Material

10.1016j.virs.2026.04.009-ESM.docx
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