Abstract
Host factors that are involved in modulating cellular vesicular trafficking of virus progeny could be potential antiviral drug targets. ADP-ribosylation factors (ARFs) are GTPases that regulate intracellular vesicular transport upon GTP binding. Here we demonstrate that genetic depletion of ARF4 suppresses viral infection by multiple pathogenic RNA viruses including Zika virus (ZIKV), influenza A virus (IAV) and SARS-CoV-2. Viral infection leads to ARF4 activation and virus production is rescued upon complementation with active ARF4, but not with inactive mutants. Mechanistically, ARF4 deletion disrupts translocation of virus progeny into the Golgi complex and redirects them for lysosomal degradation, thereby blocking virus release. More importantly, peptides targeting ARF4 show therapeutic efficacy against ZIKV and IAV challenge in mice by inhibiting ARF4 activation. Our findings highlight the role of ARF4 during viral infection and its potential as a broad-spectrum antiviral target for further development.
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Data availability
All data supporting the findings of this study are provided in the source data within this paper. For RNA-seq analysis, clean reads were aligned to the green monkey genome (Chlorocebus_sabeus 1.1). Raw RNA-seq files for analysis in this study were uploaded to the NCBI Sequence Read Archive and are publicly available under Bioproject PRJNA1152728. Previously published data, including structures deposited in the PDB and UniProt databases, were utilized for modelling molecular docking. This includes inactive human ARF4 (PDB ID: 1Z6X)46, yeast ARF1 (PDB ID: 2KSQ), the Arf1–Brag2 complex (PDB ID: 4C0A) and the AlphaFold-predicted structure of human GBF1 (UniProt ID: Q92538). Source data are provided with this paper.
Code availability
This paper does not include original code.
Change history
23 June 2025
A Correction to this paper has been published: https://doi.org/10.1038/s41564-025-02060-1
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Acknowledgements
We thank L. Lu (Fudan University, Shanghai, China), P.-G. Wang (Capital Medical University, Beijing, China) and R. Bruzzone (HKU-Pasteur Research Pole, Hong Kong SAR, China) for help in discussion; and V. Malhorta (Center for Genomic Regulation, Barcelona, Spain) for the gift of the soluble horseradish peroxidase construct. We would like to acknowledge Li Ka Shing Translational Omics Platform (LKSTOP) for equipment support. This study was supported in part by the National Key Research and Development Project of China (2022YFC2303700), the National Natural Science Foundation of China (82172271), the Start-up Fund (7100119) from Li Ka Shing Institute of Health Sciences and State Key Laboratory of Pathogen and Biosecurity (SKLPBS2019) to M.-Y.L. P.P.-H.C. was supported by the University Grants Committee’s Collaborative Research Fund (C6036-21G) and General Research Fund (16301319). Y.-Q.D. was supported by the Key-Area Research and Development Program of Guangdong Province (2022B1111020002). Work in the T.T.-Y.L. lab is supported by grants from InnoHK, an initiative of the Innovation and Technology Commission, the Government of the Hong Kong Special Administrative Region. Work in the S.S. lab is supported by the Wellcome Trust (220776/Z/20/Z and 223107/Z/21/Z to S.S. and 225010/Z/22/Z to V.G.S.). C.-F.Q. was supported by the National Science Fund for Distinguished Young Scholars (81925025) and the Innovation Fund for Medical Sciences (2019-I2M-5-049) from the Chinese Academy of Medical Sciences. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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C.-F.Q., S.S. and M.-Y.L. conceived the study and wrote the paper. M.-Y.L., K.D., X.-H.C., L.Y.-L.S., Z.-R.G., T.S.N., V.G.S., Q.-W.T., S.W.v.L., H.-H.W., Y.L., T.T.-Y.L., M.-X.S., N.-N.Z., Y.Z., T.-S.C., F.Y. and Y.-Q.D. conducted and analysed the experiments. P.P.-H.C. and Z.-R.G. performed transcriptome profiles analysis. All authors reviewed and approved the paper.
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C.-F.Q. and M.-Y.L. have filed a patent (no. 202311658259.0, China, 2023) related to the finding reported in this paper.
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Extended data
Extended Data Fig. 1 ZIKV infection is inhibited in ARF4 deleted HeLa cells.
a) CL from WT and ARF4−/− HeLa cells were collected to verify the deletion efficiency by WB with the anti-ARF4 antibody. GAPDH was used as loading control. b) ARF4−/− and WT HeLa cells were challenged with ZIKV at an MOI of 0.1. Culture medium was collected daily until cytopathic effects were observed in WT cells which appeared at 3 dpi. Viral titres were determined by plaque assay on Vero cells and expressed as PFU/ml. Results are shown as means ± SD from three independent experiments. The p-value were determined versus WT by two-way ANOVA with multiple comparisons. The p-value was labelled accordingly hereafter if p < 0.05.
Extended Data Fig. 2 Endogenous ARF4 is re-distributed upon ZIKV infection though ZIKV structural proteins do not bind to endogenous ARF4.
a) CL from ZIKV infected Vero cells (MOI = 5, 48hrs) were collected to perform immunoprecipitation (IP) assay using anti-ARF4 antibody to pull down endogenous ARF4. Final IP eluates were subject to WB using antibodies against viral E, prM and capsid proteins, as well as the host ARF4 protein respectively. GAPDH was used as loading control. b) Vero cells were mock or ZIKV infected with MOI of 10 and fixed at 36 hpi. Viral E protein was co-stained with endogenous ARF4 using their specific antibodies. Scale bar=10 μM.
Extended Data Fig. 3 ARF4 is not necessary for bulk protein secretion via the constitutive pathway.
a) a) CL were collected from ZIKV NS1 transfected cells (48 hrs post transfection) to perform GST pull down using GST fused VHS-GAT bait. Immunoblot was used to detect ARF4 activation using a specific antibody in CL and eluates (input and pull-down samples, respectively). Empty vector-transfected and ZIKV- infected samples served as a negative and positive controls, respectively. NS1 levels in CL and pull down elutes were detected on a separate immunoblot. b) ARF4 activity was calculated as the percentage of activated ARF4 in eluates relative to total ARF4 in CL. c-d) ARF4 activation was detected as described above using ssHRP stably expressed Vero cells. GAPDH was used as loading control. e-g) NS1 and ssHRP in SN and CL were either detected by anti-NS1 antibody or quantified by using a Microbeta luminometer. GAPDH was used as loading control. Secretion of NS1 or ssHRP was calculated as the percentage of total amount (SN + CL). The data are representative of three independent experiments and shown as mean ± SD. The p-value were determined versus ZIKV infected samples by two-way ANOVA with multiple comparisons.
Extended Data Fig. 4 ARF4 deletion interrupts ZIKV prM cleavage.
a) Vero WT and ARF4−/− cells were infected by ZIKV at an MOI of 10. CL were collected at indicated timepoints post infection to performed WB using antibodies against both prM and cleaved M proteins. GAPDH here is used as loading control. b) The percentage of M to total expressing (prM+M) was calculated as an index of ZIKV prM cleavage. Data are representative of at least three independent experiments and shown as mean ± SD. The p-value were determined versus ZIKV infected samples by two-way ANOVA with multiple comparisons.
Extended Data Fig. 5 ARF4 regulates sorting of newly formed viral particles during intracellular transport.
a,b, ZIKV infected Vero WT and ARF4−/− cells were fixed (MOI = 10, 24 hrs) to perform TEM observation to check viral release in intracellular sub-cellular organelles (a) and from cell surface membrane (b). Virions-containing vesicles are indicated by blue stars. Dispersed unpacked virions are indicated by red arrows. Sub-cellular organelles-early endosome (LL), late endosome (LE) or multivesicular body (MVB)-like compartments, as well as membrane protrusions are highlighted by yellow. Scale bar=500 nm or 200 nm in upper right panel and its scaled panel.
Extended Data Fig. 6 The CC50 and IC50 curves of ARF4 targeting peptides (ARF4TPs).
The CC50 (a) and IC50 (b) of ARF4TPs in Vero cells were measured as described in methods. Data are representative of at least three independent experiments and shown as mean only (a) or Mean ± SEM (b).
Extended Data Fig. 7 ARF4TP-4 is safe for mice.
a) Schematic diagram of safety experiment in mice. b) Body weight of PBS (n = 3) or ARF4TP-4 treated (n = 5) mice were measured daily till 12 days after last injection. Date are means ± SEM. c-d) The ALT (c) and creatinine (d) in the sera collected at indicated day from PBS (n = 5) or ARF4TP-4 (n = 5) injection mice were calculated by using the ALT assay kit and creatinine kit (NJJCBIO). Each coloured plot in above panels represents a randomly picked mouse. All error bars reflect ± SEM. e) Tissues were collected and fixed by 4% PFA for HE staining at 12 days post the 3rd time ARF4TP-4 injection. Scale bar, 100 μM.
Extended Data Fig. 8 ARF4 deletion relieves IAV induced the body weight lost and histopathological changes in lung.
a) Body weight was daily measured after IAV challenge in both WT and in ARF4-/+ mice (n = 8) for 14 days or till the body weight reduction was up to 25%. For group mean plots in b), weight loss is shown as mean ± SEM. c-d) H&E staining and histopathological score of IAV infected lung sections collected from WT and ARF4−/+ mice at 6 dpi. Each colored plot in above panels represents a randomly picked mouse. Scale bar, 100 μM.
Extended Data Fig. 9 ARF4TP-4 obviously prevents the body weight lost and histopathological changes after IAV challenge.
ARF4TP-4 treatment and IAV challenge were performed as described in Fig. 6h. a) Body weight daily monitor was performed in both PBS and ARF4TP-4 treated mice (n = 8) for 14 days post IAV inoculation or till the body weight reduction was up to 25%. For group mean plots in b), weight loss is shown as mean ± SEM). c, d) H&E staining and histopathological score of IAV infected lung sections collected from PBS and ARF4TP-4 treated mice at 6 dpi. Each coloured plot in above panels represents a randomly picked mouse. Scale bar, 100 μM.
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Li, MY., Deng, K., Cheng, XH. et al. ARF4-mediated intracellular transport as a broad-spectrum antiviral target. Nat Microbiol 10, 710–723 (2025). https://doi.org/10.1038/s41564-025-01940-w
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DOI: https://doi.org/10.1038/s41564-025-01940-w
