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Chikungunya virus cell-to-cell transmission is mediated by intercellular extensions in vitro and in vivo

Nature Microbiology volume 8, pages 1653–1667 (2023)Cite this article

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Abstract

Chikungunya virus (CHIKV) has recently emerged to cause millions of human infections worldwide. Infection can induce the formation of long intercellular extensions that project from infected cells and form stable non-continuous membrane bridges with neighbouring cells. The mechanistic role of these intercellular extensions in CHIKV infection was unclear. Here we developed a co-culture system and flow cytometry methods to quantitatively evaluate transmission of CHIKV from infected to uninfected cells in the presence of neutralizing antibody. Endocytosis and endosomal acidification were critical for virus cell-to-cell transmission, while the CHIKV receptor MXRA8 was not. By using distinct antibodies to block formation of extensions and by evaluation of transmission in HeLa cells that did not form extensions, we showed that intercellular extensions mediate CHIKV cell-to-cell transmission. In vivo, pre-treatment of mice with a neutralizing antibody blocked infection by direct virus inoculation, while adoptive transfer of infected cells produced antibody-resistant host infection. Together our data suggest a model in which the contact sites of intercellular extensions on target cells shield CHIKV from neutralizing antibodies and promote efficient intercellular virus transmission both in vitro and in vivo.

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Fig. 1: Cell-to-cell transmission promotes CHIKV infection in cell culture.
Fig. 2: Role of MXRA8 and the endocytic pathway in cell-to-cell transmission.
Fig. 3: Tetherin expression inhibits CHIKV cell-to-cell transmission.
Fig. 4: Properties of CHIKV infection in HeLa cells.
Fig. 5: Inhibition of ILE blocks cell-to-cell transmission.
Fig. 6: CHIKV cell-to-cell transmission in mice.

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All data supporting the findings of this study are available within the paper, its Extended data or Source data files. Representative microscopy images are included in the main or extended data figures. Additional microscopy image files are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank all the members of our laboratory for their helpful discussions, C. Martin for comments on the paper, and L. Kim and A. Fayed for technical support. We thank the Einstein Analytical Imaging Facility and the Flow Cytometry Core Facility for use of their instruments, and A. Briceno for training on the SP5 confocal microscope and J. Zhang for training on the BD LSR-II analyser. We thank E. Frolova (University of Alabama) for providing the CHIKV-GFP (181/25) infectious clone, Z. Bornholdt (Mapp Biopharmaceutial) for providing mAb chCHK-152, and F. Rey (Institut Pasteur) for helpful discussions on CHIKV antibodies. This work was supported by National Institutes of Health grants to M.K. from NIGMS (R01GM057454), to T.E.M. from NIAID (R01AI141436), to J.R.L. from NIAID (R01AI125462), to M.S.D. from NIAID (R01AI143673) and by NCI Cancer Center Support Grant P30CA013330. The development of mAb E10-18 was funded by the Investissement d’Avenir programme, Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (ANR-10-LABX-62-IBEID), the ‘Investissements d’Avenir’ programme (ANR-10-IHUB-0002, ANR-15-CE15-00029 ZIKAHOST and the INCEPTION programme ANR-16-CONV-0005), Institut Pasteur, Inserm. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Author notes

  1. Deceased: Thérèse Couderc.

Authors and Affiliations

  1. Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA

    Peiqi Yin, Judy J. Wan & Margaret Kielian

  2. Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO, USA

    Bennett J. Davenport, Brian C. Ware & Thomas E. Morrison

  3. Department of Medicine, Washington University School of Medicine, Saint Louis, MO, USA

    Arthur S. Kim & Michael S. Diamond

  4. Department of Pathology & Immunology, Washington University School of Medicine, Saint Louis, MO, USA

    Arthur S. Kim & Michael S. Diamond

  5. Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO, USA

    Michael S. Diamond

  6. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA

    Karen Tong & Jonathan R. Lai

  7. Institut Pasteur, Inserm U1117, Biology of Infection Unit, Université de Paris, Paris, France

    Thérèse Couderc & Marc Lecuit

  8. Department of Infectious Diseases and Tropical Medicine, APHP, Institut Imagine, Necker-Enfants Malades University Hospital, Paris, France

    Marc Lecuit

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Contributions

P.Y. and M.K. conceived the project; P.Y. performed the experimental work with B.J.D. performing the mouse experiments; J.J.W. provided important experimental background on ILE, antibody blocking of ILE and ELISA, M.K. and T.E.M. supervised the research; A.K., M.S.D., B.C.W., K.T., T.C., M.L. and J.R. L. developed and contributed key reagents; P.Y. and M.K. wrote and edited the paper. All authors reviewed and revised the paper and agreed to the published version of the paper. T.C., now deceased, reviewed and approved an earlier version of this paper.

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Correspondence to Margaret Kielian.

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Competing interests

P.Y., B.J.D., J.J.W., A.S.K., B.C.W., K.T., T. C., M. L., T.E.M. and M.K. report no competing interests. J.R.L. is a paid consultant for Celdara Medical, LLC. M.S.D. is a consultant for Inbios, Vir Biotechnology, Senda Biosciences, Ocugen, Moderna and Immunome. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Vir Biotechnology, Moderna, Generate Biomedicine and Emergent BioSolutions.

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Extended data

Extended Data Fig. 1 Growth properties of CHIKV and CHIKV-GFP in MEF cells.

a, Schematic representation of CHIKV 181/25 and CHIKV 181/25 GFP infectious clone constructs. b, Growth kinetics of CHIKV and CHIKV-GFP in MEF cells. MEF cells were inoculated with CHIKV or CHIKV-GFP (MOI = 10) for 2 h at 37 °C, then washed to remove unbound virus. Virus production at the indicated times was quantitated by ICA. The graphs represent the means and range for 2 independent experiments. c, Schematic of experiments to test CHIKV cell-to-cell transmission. After infection of producer cells, dye-labeled target cells are added, co-culture is performed under various conditions, and virus in the medium and infection of target cells are quantitated. d, Representative gating scheme to identify uninfected and CHIKV-GFP-infected producer and target cells.

Source data

Extended Data Fig. 2 Infection of MEF or U-2 OS target cells in the presence of mAb DEN-4G2 or chCHK-152.

a, MEF producer cells were inoculated with CHIKV-GFP (MOI = 10) for 2 h, washed, and then co-cultured for 12 h with MEF target cells in the presence of the indicated concentrations of mAb chCHK-152 or DEN-4G2, a control mAb against an unrelated virus. The media were collected, and cells were analyzed by flow cytometry. Data are representative of 3 independent experiments. b, Quantitation of target cell infection in samples prepared as in Extended Data 2a. The bar graph represents the mean ± S.D. of 3 independent experiments (shown as points). Statistical significance was calculated by unpaired two-tailed t-tests. c, U-2 OS producer cells were infected with CHIKV-GFP (MOI = 10), washed, and incubated at 37 °C for 2 h. U-2 OS target cells stained with CMRA dye were then plated onto the producer cells in the presence of the indicated concentrations of chCHK-152 and co-cultured for 12 h. Cells were then fixed and analyzed by flow cytometry. d, Quantitation of target cell infection in samples from Extended Data 2c. The bar graph represents the mean ± S.D. of 3 independent experiments (shown as points). Statistical significance was calculated by unpaired two-tailed t-tests.

Source data

Extended Data Fig. 3 Effect of Bafilomycin or Dyngo-4a on ILE formation.

a, Examples of ILE induced by CHIKV-GFP infection in U-2 OS treated with Bafilomycin A1 or Dyngo-4a. U-2 OS cells were infected with CHIKV-GFP (MOI = 0.5) for 1 h, treated with Bafilomycin A1 (100 nM), Dyngo-4a (60 μM) or DMSO vehicle for 1 h, then cultured for 9 h. ILE visualized as in Fig. 1b. White arrowheads indicate ILE. Bar = 20 μm. b, ILE were quantitated as described in Fig. 1c. Data shown represent the mean of 2 independent experiments, with points showing the results from each experiment.

Source data

Extended Data Fig. 4 Characterization of inducible Rab5 U-2 OS cell lines.

a, Clonal U-2 OS cell lines inducibly expressing GFP-tagged Rab5-WT or Rab5-DN were cultured in the presence or absence of doxycycline (dox) for 16 h and analyzed by flow cytometry. b, The indicated U-2 OS cell lines were incubated in the presence or absence of doxycycline for 16 h and infected with SFV at an MOI of 1 for 12 h. Cells were stained with antibody to detect E2/E1 and quantitated by flow cytometry. The bar graph represents the mean ± S.D of 3 independent experiments (shown as points). Statistical significance was calculated using unpaired two-tailed multiple t-tests. c, Clonal U-2 OS cell lines were cultured in the presence of doxycycline (dox) for 16 h and infected with CHIKV for 11 h. ILE were visualized as in Fig. 1b. White arrowheads indicate ILE. Bar = 20 μm. d, Cells were treated and infected as in panel c, and ILE in infected cells were quantitated as in Fig. 1c. Data shown represent the mean of 2 independent experiments (shown as points).

Source data

Extended Data Fig. 5 ILE formation in U-2 OS-tetherin cell lines.

Examples of ILE induced by CHIKV-GFP infection in U-2 OS expressing WT-tetherin and L-tetherin vs. the parental cells. The indicated U-2 OS cells were incubated with or without doxycycline for 16 h and infected with CHIKV-GFP (MOI = 0.5) for 11 h. Cells were permeabilized and stained with antibodies against the E2/E1 proteins (red, pseudo color) and tubulin (green, pseudo color). The GFP reporter channel is not shown. White arrowheads indicate ILE. Bar = 20 μm.

Extended Data Fig. 6 MAb E10-18 does not inhibit free virus infection of target cells.

MEF producer cells were infected with CHIKV-GFP (MOI = 10) for 2 h, washed, and then co-cultured for 12 h with MEF target cells in the presence of the indicated concentrations of mAbs chCHK-152 or E10-18. Infection of target cells was determined by flow cytometry. The bar graph represents the mean ± S.D. of 3 independent experiments (shown as points). Statistical significance was calculated by unpaired two-tailed t-tests.

Source data

Extended Data Fig. 7 E1-K61T-G64S escapes chCHK-166 inhibition of ILE formation.

Examples of ILE from experiment in Fig. 5h. MEF cells were infected with CHIKV-GFP or CHKV-GFP E1-K61T-G64S for 2 h, then cultured in presence or absence of 20 μg/mL mAb chCHK-166 for 9 h. ILE were visualized as in Fig. 1b. White arrowheads indicate ILE. Bar = 20 μm.

Extended Data Fig. 8 CHK-152 N297Q pre-treatment can block CHIKV infection in ipsilateral ankle.

a, WT C57BL/6 mice were treated with 100 μg of chCHK-152 N297Q i.p. (n = 2) or with an equal volume of PBS (n = 1) as a negative control. At 6 h following antibody or PBS treatment, mice were inoculated with 103 PFU CHIKV-Venus s.c. in the left footpad. At 24 h post-virus inoculation, single cell suspensions were prepared from collagenase/DNase digested ipsilateral ankle tissue, incubated with fixable viability dye (Vi-421), and stained with CD45 BUV395. Figure created with Biorender.com. b, Representative dot plots showing the gating scheme to identify host cells that are productively infected with CHIKV-Venus. c, Flow plots from PBS and chCHK-152 N297Q pretreated mice. All plots are gated on viable singlet CD45− cells. d, Frequency of Venus+ cells in the CD45− fraction of ipsilateral ankle tissue.

Extended Data Fig. 9 FACS gating strategies for Fig. 6.

a, Representative gating scheme to identify donor Violet+ MEF cells, and endogenous host Violet- CD45− cells isolated from the ipsilateral ankle. b, In all MEF adoptive transfer experiments, a group of control mice was PBS-treated and adoptively transferred with 106 mock-infected Cell-Trace Violet loaded MEF cells. Flow plots show that the presence of Violet+ MEF cells does not interfere with detection of Venus+ cells.

Extended Data Fig. 10 ILE formation in primary joint cells infected with CHIKV ex vivo.

Examples of ILE quantitated in Fig. 6f. Ankle tissue from C57BL/6 mice was collected, subjected to collagenase/Dnase digestion, and cultured for 24 h. The cells were infected with WT CHIKV strain AF15561 or CHIKV strain 181/25 for 16 h, fixed and stained with antibodies to E1/E2(red) and tubulin (green). White arrowheads indicate ILE. Bar = 20 μm.

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Yin, P., Davenport, B.J., Wan, J.J. et al. Chikungunya virus cell-to-cell transmission is mediated by intercellular extensions in vitro and in vivo. Nat Microbiol 8, 1653–1667 (2023). https://doi.org/10.1038/s41564-023-01449-0

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