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Volume 41 Issue 5
October 2026

    . doi: 10.1016/j.virs.2026.04.009
    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
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    EHD4通过调控细胞外囊泡相关病毒释放及其细胞吸附促进寨卡病毒感染

    • a. 安徽医科大学基础医学院, 中国合肥 230032。;

    • b. 病原微生物生物安全全国重点实验室, 军事科学院军事医学研究院, 中国北京 100071。;

    • c. 香港中文大学化学病理学系及李嘉诚健康科学研究所, 中国香港特别行政区。;

    • d. 香港中文大学(深圳)研究院, 中国深圳 518063。;

    • e. 中国科学院生物物理研究所大分子国家重点实验室, 中国北京 100101。;

    • f. 香港中文大学生命科学学院农业生物技术国家重点实验室、AoE 植物液泡生物学与生物技术中心, 中国香港特别行政区。

    • 细胞外囊泡（extracellular vesicles，EVs）及其他细胞外膜相关颗粒可参与病毒播散，但调控EV相关的病毒物质组成及感染性的宿主因子还未充分认识。本研究通过比较蛋白质组学分析发现，Eps15同源结构域蛋白4（EHD4）是在寨卡病毒（ZIKV）感染Vero细胞来源的EV/病毒颗粒混合制备物中富集的候选宿主因子。在HeLa细胞中敲除EHD4后，寨卡病毒、日本脑炎病毒及登革病毒2型的增殖均受到抑制，而重新表达EHD4可恢复病毒增殖。在ZIKV感染的EHD4敲除（KO）细胞中，与野生型（WT）细胞相比，有更高比例的病毒RNA存在于EV富集组分。然而，来源于KO细胞EV富集组分中的病毒物质的细胞结合能力和感染性均减弱，而部分破坏EV的膜结构后，WT和KO来源制备物之间的感染性差异有所减弱，提示膜相关成分参与决定其携带的病毒物质的功能特性。ZIKV复制子实验显示，EHD4对病毒RNA复制本身并无明显影响。免疫荧光结果显示，EHD4与CD63阳性区室及ZIKV包膜蛋白存在部分共定位。透射电子显微镜也观察到EHD4缺失并不影响多泡体（multivesicular body，MVB）的形成，WT细胞的MVB腔内可较容易观察到病毒样颗粒，而在KO细胞中则较少见，提示EHD4可能促进病毒相关物质向MVB腔内囊泡的分选。进一步的定量蛋白质组学分析发现，KO来源的EV富集组分中一部分膜相关蛋白的丰度降低。综合上述结果表明，EHD4是一种可影响黄病毒相关物质在EV富集组分中的胞外分配、组成及感染性的宿主因子。其作用的具体分子机制仍有待阐明。

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

    • 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

    • 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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      1. Aguilar, R.C., Jones, T., Naslavsky, N., Caplan, S., 2020. Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission. Plos One, 15.

      2. Aitken, C.E., Lorsch, J.R., 2012. A mechanistic overview of translation initiation in eukaryotes. Nature Structural & Molecular Biology, 19, 568-576.

      3. Brito Querido, J., Diaz-Lopez, I., Ramakrishnan, V., 2023. The molecular basis of translation initiation and its regulation in eukaryotes. Nature Reviews Molecular Cell Biology, 25, 168-186.

      4. Dick, G.W., 1952. Zika virus. II. Pathogenicity and physical properties. Trans R Soc Trop Med Hyg, 46, 521-534.

      5. Duffy, M.R., Chen, T.H., Hancock, W.T., Powers, A.M., Kool, J.L., Lanciotti, R.S., Pretrick, M., Marfel, M., Holzbauer, S., Dubray, C., et al., 2009. Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med, 360, 2536-2543.

      6. Fu, Y., Xiong, S., 2023. Exosomes mediate Coxsackievirus B3 transmission and expand the viral tropism. PLoS Pathog, 19, e1011090.

      7. Gold, A.S., Feitosa-Suntheimer, F., Araujo, R.V., Hekman, R.M., Asad, S., Londono-Renteria, B., Emili, A., Colpitts, T.M., 2020. Dengue Virus Infection of Aedes aegypti Alters Extracellular Vesicle Protein Cargo to Enhance Virus Transmission. International Journal of Molecular Sciences, 21, 6609.

      8. Gottesman, M.M., Ling, V., 2005. The molecular basis of multidrug resistance in cancer: The early years of P-glycoprotein research. FEBS Letters, 580, 998-1009.

      9. Grant, B.D., Caplan, S., 2008. Mechanisms of EHD/RME-1 Protein Function in Endocytic Transport. Traffic, 9, 2043-2052.

      10. Hashimoto, Y., Porkolab, G., Hudson, N., O'Callaghan, J., Hanley, N., Delaney, C., Deli, M. A., Westenskow, P., Campbell, M. 2025. EHD4 and ASAP2 are critical negative regulators of the claudin-5-based endothelial barrier. The FEBS Journal, 293, 2067-2085.

      11. Horna-Terron, E., Pradilla-Dieste, A., Sanchez-De-Diego, C., Osada, J., 2014. TXNDC5, a Newly Discovered Disulfide Isomerase with a Key Role in Cell Physiology and Pathology. International Journal of Molecular Sciences, 15, 23501-23518.

      12. Hui, L., Qian, D., Guo Y., Yi Z., Guo T., Li A., Wu Y., 2026. Breaking barriers and beyond: Mechanisms and pathological implications of Zika virus traversal across blood-tissue interfaces. Virologica Sinica, 41, 247-260.

      13. Hurley, J.H., Hanson, P.I., 2010. Membrane budding and scission by the ESCRT machinery: it's all in the neck. Nature Reviews Molecular Cell Biology, 11, 556-566.

      14. Kalluri, R., Lebleu, V.S., 2020. The biology, function, and biomedical applications of exosomes. Science, 367, eaau6977.

      15. Kowal, J., Arras, G., Colombo, M., Jouve, M., Morath, J.P., Primdal-Bengtson, B., Dingli, F., Loew, D., Tkach, M., Thery, C., 2016. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci U S A, 113, E968-E977.

      16. Lim, S.-K., Lim, J.K., Yoon, I.-K., 2017. An Update on Zika Virus in Asia. Infection & Chemotherapy, 49, 91-100.

      17. Liu, Y., Li, Q., Shao, S., Ji, X., Gao, W., Fan, Y., Liu, M., Wang, Y., Bai, J., 2024. HSPA4 Enhances BRSV Entry via Clathrin-Mediated Endocytosis Through Regulating the PI3K-Akt Signaling Pathway and ATPase Activity of HSC70. Viruses, 16, 1784.

      18. Martinez-Rojas, P.P., Quiroz-Garcia, E., Monroy-Martinez, V., Agredano-Moreno, L.T., Jimenez-Garcia, L.F., Ruiz-Ordaz, B.H., 2020. Participation of Extracellular Vesicles from Zika-Virus-Infected Mosquito Cells in the Modification of Naive Cells’ Behavior by Mediating Cell-to-Cell Transmission of Viral Elements. Cells, 9, 123.

      19. Mccullough, J., Colf, L.A., Sundquist, W.I., 2013. Membrane Fission Reactions of the Mammalian ESCRT Pathway. Annual Review of Biochemistry, 82, 663-692.

      20. Melo, A.A., Hegde, B.G., Shah, C., Larsson, E., Isas, J.M., Kunz, S., Lundmark, R., Langen, R., Daumke, O., 2017. Structural insights into the activation mechanism of dynamin-like EHD ATPases. Proceedings of the National Academy of Sciences, 114, 5629-5634.

      21. Moi, M.L., Nguyen, T.T.T., Nguyen, C.T., Vu, T.B.H., Tun, M.M.N., Pham, T.D., Pham, N.T., Tran, T., Morita, K., Le, T.Q.M., Dang, D.A., Hasebe, F., 2017. Zika virus infection and microcephaly in Vietnam. The Lancet Infectious Diseases, 17, 805-806.

      22. Monel, B., Compton, A.A., Bruel, T., Amraoui, S., Burlaud-Gaillard, J., Roy, N., Guivel-Benhassine, F., Porrot, F., Genin, P., Meertens, L., et al., 2017. Zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells. The EMBO Journal, 36, 1653-1668.

      23. Mukhopadhyay, S., Kuhn, R.J., Rossmann, M.G., 2005. A structural perspective of the flavivirus life cycle. Nat Rev Microbiol, 3, 13-22.

      24. Naslavsky, N., Caplan, S., 2011. EHD proteins: key conductors of endocytic transport. Trends in Cell Biology, 21, 122-131.

      25. Nolte-‘T Hoen, E., Cremer, T., Gallo, R.C., Margolis, L.B., 2016. Extracellular vesicles and viruses: Are they close relatives? Proceedings of the National Academy of Sciences, 113, 9155-9161.

      26. Ostrowski, M., Carmo, N.B., Krumeich, S., Fanget, I., Raposo, G., Savina, A., Moita, C.F., Schauer, K., Hume, A.N., Freitas, R.P., et al., 2009. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nature Cell Biology, 12, 19-30.

      27. Pierson, T.C., Diamond, M.S., 2020. The continued threat of emerging flaviviruses. Nat Microbiol, 5, 796-812.

      28. Raab-Traub, N., Dittmer, D.P., 2017. Viral effects on the content and function of extracellular vesicles. Nat Rev Microbiol, 15, 559-572.

      29. F.A., Ran, P.D., Hsu, J., Wright, V., Agarwala, D.A., Scott, F., Zhang, Genome engineering using the CRISPR-Cas9 system, Nat Protocols, 8, 11, 2013, 10.1038/nprot.2013.143.

      30. Richter, J.D., Sonenberg, N., 2005. Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature, 433, 477-480.

      31. Roth, H., Magg, V., Uch, F., Mutz, P., Klein, P., Haneke, K., Lohmann, V., Bartenschlager, R., Fackler, O.T., Locker, N., Stoecklin, G., Ruggieri, A., Buchmeier, M.J., 2017. Flavivirus Infection Uncouples Translation Suppression from Cellular Stress Responses. mBio, 8, e02150-16.

      32. Safadi, D.E., Lebeau, G., Lagrave, A., Melade, J., Grondin, L., Rosanaly, S., Begue, F., Hoareau, M., Veeren, B., Roche, M., Hoarau, J.J., Meilhac, O., Mavingui, P., Despres, P., Viranaicken, W., Krejbich-Trotot, P., 2023. Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection. Viruses, 15, 364.

      33. Shives, K., Massey, A., May, N., Morrison, T., Beckham, J., 2016. 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression. Viruses, 8, 287.

      34. St. John, A.L., Ishida, K., Yagi, H., Kato, Y., Morita, E., 2023. N-linked glycosylation of flavivirus E protein contributes to viral particle formation. PLOS Pathogens, 19, e1011681.

      35. Stenmark, H., 2009. Rab GTPases as coordinators of vesicle traffic. Nature Reviews Molecular Cell Biology, 10, 513-525.

      36. Sullivan, D.C., Huminiecki, L., Moore, J.W., Boyle, J.J., Poulsom, R., Creamer, D., Barker, J., Bicknell, R., 2003. EndoPDI, a Novel Protein-disulfide Isomerase-like Protein That Is Preferentially Expressed in Endothelial Cells Acts as a Stress Survival Factor. Journal of Biological Chemistry, 278, 47079-47088.

      37. Tabata, K., Arimoto, M., Arakawa, M., Nara, A., Saito, K., Omori, H., Arai, A., Ishikawa, T., Konishi, E., Suzuki, R., Matsuura, Y., Morita, E., 2016. Unique Requirement for ESCRT Factors in Flavivirus Particle Formation on the Endoplasmic Reticulum. Cell Reports, 16, 2339-2347.

      38. Vader, P., Mol, E.A., Pasterkamp, G., Schiffelers, R.M., 2016. Extracellular vesicles for drug delivery. Advanced Drug Delivery Reviews, 106, 148-156.

      39. Van Niel, G., D'angelo, G., Raposo, G., 2018. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol, 19, 213-228.

      40. Vanlandingham, P.A., Ceresa, B.P., 2009. Rab7 Regulates Late Endocytic Trafficking Downstream of Multivesicular Body Biogenesis and Cargo Sequestration. Journal of Biological Chemistry, 284, 12110-12124.

      41. Vora, A., Zhou, W., Londono-Renteria, B., Woodson, M., Sherman, M.B., Colpitts, T.M., Neelakanta, G., Sultana, H., 2018. Arthropod EVs mediate dengue virus transmission through interaction with a tetraspanin domain containing glycoprotein Tsp29Fb. Proc Natl Acad Sci U S A, 115, E6604-E6613.

      42. Votteler, J., Sundquist, Wesley i., 2013. Virus Budding and the ESCRT Pathway. Cell Host & Microbe, 14, 232-241.

      43. Wang, Y., Li, Q., Hu, D., Gao, D., Wang, W., Wu, K., Wu, J., 2021. USP38 Inhibits Zika Virus Infection by Removing Envelope Protein Ubiquitination. Viruses, 13, 2029.

      44. Weissenhorn, W., Poudevigne, E., Effantin, G., Bassereau, P., 2013. How to get out: ssRNA enveloped viruses and membrane fission. Current Opinion in Virology, 3, 159-167.

      45. Wongsurawat, T., Athipanyasilp, N., Jenjaroenpun, P., Jun, S.R., Kaewnapan, B., Wassenaar, T.M., Leelahakorn, N., Angkasekwinai, N., Kantakamalakul, W., Ussery, D.W., Sutthent, R., Nookaew, I., Horthongkham, N., 2018. Case of Microcephaly after Congenital Infection with Asian Lineage Zika Virus, Thailand. Emerg Infect Dis, 24, 1758-1761.

      46. Yang, Y., Hong, Y., Cho, E., Kim, G.B., Kim, I.S., 2018. Extracellular vesicles as a platform for membrane-associated therapeutic protein delivery. Journal of Extracellular Vesicles, 7, 1440131.

      47. York, S.B., Sun, L., Cone, A.S., Duke, L.C., Cheerathodi, M.R., Meckes, D.G., Goodrum, F., 2021. Zika Virus Hijacks Extracellular Vesicle Tetraspanin Pathways for Cell-to-Cell Transmission. mSphere, 6, 101128msphere0019221.

      48. Zhao, F., Xu, Y., Liu, N., Lv, D., Chen, Y., Liu, Z., Jin, X., Xiao, M., Lavillette, D., Zhong, J., Bartenschlager, R., Long, G., 2023. Extracellular vesicles from Zika virus-infected cells display viral E protein that binds ZIKV-neutralizing antibodies to prevent infection enhancement. EMBO J, 42, e112096.

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

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

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