EHD4 facilitates Zika virus infection by regulating extracellular vesicle-associated viral release and cell attachment
6 affiliations
- 1School of Basic Medical Sciences, Anhui Medical University, Hefei, 230032, China
- 2State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China
- 3Department of Chemical Pathology and Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong, Hong Kong SAR, China
- 4National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
- 5State 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
- 6Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, 518063, China
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.
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BibTeX
@article{zhang2026ehd4,
title = {{EHD4 facilitates Zika virus infection by regulating extracellular vesicle-associated viral release and cell attachment}},
author = {Yue Zhang and Xiu-Li Yan and Kao Deng and Meng-Jiao He and Hui Wang and Zi-Jin Cui and Yu-Yan Li and Lei Cao and Wenlong Ma and Byung-Ho Kang and Hoi Ki Leung and Peter Pak-Hang Cheung and Cheng-Feng Qin and Ming-Yuan Li and Xiao-Feng Li},
journal = {Virologica Sinica},
year = {2026},
month = sep,
doi = {10.1016/j.virs.2026.04.009},
url = {https://doi.org/10.1016/j.virs.2026.04.009},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - EHD4 facilitates Zika virus infection by regulating extracellular vesicle-associated viral release and cell attachment
AU - Yue Zhang
AU - Xiu-Li Yan
AU - Kao Deng
AU - Meng-Jiao He
AU - Hui Wang
AU - Zi-Jin Cui
AU - Yu-Yan Li
AU - Lei Cao
AU - Wenlong Ma
AU - Byung-Ho Kang
AU - Hoi Ki Leung
AU - Peter Pak-Hang Cheung
AU - Cheng-Feng Qin
AU - Ming-Yuan Li
AU - Xiao-Feng Li
PY - 2026
DA - 2026/09/19
JO - Virologica Sinica
DO - 10.1016/j.virs.2026.04.009
UR - https://doi.org/10.1016/j.virs.2026.04.009
AB - 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.
SN - 1674-0769
SN - 1995-820X
ER -