Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Vaccinia virus hijacks EGFR signalling to enhance virus spread through rapid and directed infected cell motility

Abstract

Cell motility is essential for viral dissemination1. Vaccinia virus (VACV), a close relative of smallpox virus, is thought to exploit cell motility as a means to enhance the spread of infection1. A single viral protein, F11L, contributes to this by blocking RhoA signalling to facilitate cell retraction2. However, F11L alone is not sufficient for VACV-induced cell motility, indicating that additional viral factors must be involved. Here, we show that the VACV epidermal growth factor homologue, VGF, promotes infected cell motility and the spread of viral infection. We found that VGF secreted from early infected cells is cleaved by ADAM10, after which it acts largely in a paracrine manner to direct cell motility at the leading edge of infection. Real-time tracking of cells infected in the presence of EGFR, MAPK, FAK and ADAM10 inhibitors or with VGF-deleted and F11-deleted viruses revealed defects in radial velocity and directional migration efficiency, leading to impaired cell-to-cell spread of infection. Furthermore, intravital imaging showed that virus spread and lesion formation are attenuated in the absence of VGF. Our results demonstrate how poxviruses hijack epidermal growth factor receptor-induced cell motility to promote rapid and efficient spread of infection in vitro and in vivo.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: VGF is required for VACV-induced cell motility and virus spread.
Fig. 2: VGF activates cell motility through EGFR–MAPK–FAK signalling.
Fig. 3: ADAM10-mediated VGF release triggers cell motility in a paracrine manner.
Fig. 4: VGF is required for lesion formation in vivo.

Similar content being viewed by others

Data availability

All data and reagents will be made available upon reasonable request.

References

  1. Sanderson, C. M., Way, M. & Smith, G. L. Virus-induced cell motility. J. Virol. 72, 1235–1243 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Valderrama, F., Cordeiro, J. V., Schleich, S., Frischknecht, F. & Way, M. Vaccinia virus-induced cell motility requires F11L-mediated inhibition of RhoA signaling. Science 311, 377–381 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Ridley, A. J. Rho GTPase signalling in cell migration. Curr. Opin. Cell Biol. 36, 103–112 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Friedl, P. & Wolf, K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat. Rev. Cancer 3, 362–374 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Reig, G., Pulgar, E. & Concha, M. L. Cell migration: from tissue culture to embryos. Development 141, 1999–2013 (2014).

    Article  CAS  PubMed  Google Scholar 

  6. Cheng, F. et al. KSHV-initiated notch activation leads to membrane-type-1 matrix metalloproteinase-dependent lymphatic endothelial-to-mesenchymal transition. Cell Host Microbe 10, 577–590 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. Abaitua, F., Zia, F. R., Hollinshead, M. & O’Hare, P. Polarized cell migration during cell-to-cell transmission of herpes simplex virus in human skin keratinocytes. J. Virol. 87, 7921–7932 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Dawson, C. W., Laverick, L., Morris, M. A., Tramoutanis, G. & Young, L. S. Epstein–Barr virus-encoded LMP1 regulates epithelial cell motility and invasion via the ERK–MAPK pathway. J. Virol. 82, 3654–3664 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kivi, N., Greco, D., Auvinen, P. & Auvinen, E. Genes involved in cell adhesion, cell motility and mitogenic signaling are altered due to HPV 16 E5 protein expression. Oncogene 27, 2532–2541 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Irwin, C. R. & Evans, D. H. Modulation of the myxoma virus plaque phenotype by vaccinia virus protein F11. J. Virol. 86, 7167–7179 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cordeiro, J. V. et al. F11-mediated inhibition of RhoA signalling enhances the spread of vaccinia virus in vitro and in vivo in an intranasal mouse model of infection. PLoS ONE 4, e8506 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Handa, Y., Durkin, C. H., Dodding, M. P. & Way, M. Vaccinia virus F11 promotes viral spread by acting as a PDZ-containing scaffolding protein to bind myosin-9A and inhibit RhoA signaling. Cell Host Microbe 14, 51–62 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. McFadden, G. & Moyer, R. in Cytokine Reference (eds Oppenheim, J. J. & Feldmann, M.) 841–846 (Academic Press, San Diego, 2000).

  14. Chang, W., Lim, J. G., Hellstrom, I. & Gentry, L. E. Characterization of vaccinia virus growth factor biosynthetic pathway with an antipeptide antiserum. J. Virol. 62, 1080–1083 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. King, C. S., Cooper, J. A., Moss, B. & Twardzik, D. R. Vaccinia virus growth factor stimulates tyrosine protein kinase activity of A431 cell epidermal growth factor receptors. Mol. Cell. Biol. 6, 332–336 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Twardzik, D. R., Brown, J. P., Ranchalis, J. E., Todaro, G. J. & Moss, B. Vaccinia virus-infected cells release a novel polypeptide functionally related to transforming and epidermal growth factors. Proc. Natl Acad. Sci. USA 82, 5300–5304 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Stroobant, P. et al. Purification and characterization of vaccinia virus growth factor. Cell 42, 383–393 (1985).

    Article  CAS  PubMed  Google Scholar 

  18. Buller, R. M., Chakrabarti, S., Cooper, J. A., Twardzik, D. R. & Moss, B. Deletion of the vaccinia virus growth factor gene reduces virus virulence. J. Virol. 62, 866–874 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Lai, A. C. & Pogo, B. G. Attenuated deletion mutants of vaccinia virus lacking the vaccinia growth factor are defective in replication in vivo. Microb. Pathog. 6, 219–226 (1989).

    Article  CAS  PubMed  Google Scholar 

  20. Buller, R. M., Chakrabarti, S., Moss, B. & Fredrickson, T. Cell proliferative response to vaccinia virus is mediated by VGF. Virology 164, 182–192 (1988).

    Article  CAS  PubMed  Google Scholar 

  21. Hickman, H. D. et al. Direct priming of antiviral CD8+ T cells in the peripheral interfollicular region of lymph nodes. Nat. Immunol. 9, 155–165 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Bidgood, S. R. & Mercer, J. Cloak and dagger: alternative immune evasion and modulation strategies of poxviruses. Viruses 7, 4800–4825 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Postigo, A., Martin, M. C., Dodding, M. P. & Way, M. Vaccinia-induced epidermal growth factor receptor–MEK signalling and the anti-apoptotic protein F1L synergize to suppress cell death during infection. Cell. Microbiol. 11, 1208–1218 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Andrade, A. A. et al. The vaccinia virus-stimulated mitogen-activated protein kinase (MAPK) pathway is required for virus multiplication. Biochem. J. 381, 437–446 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Tzahar, E. et al. Pathogenic poxviruses reveal viral strategies to exploit the ErbB signaling network. EMBO J. 17, 5948–5963 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kim, H. S. et al. Tyrosine phosphorylation of phospholipase C-γ1 by vaccinia virus growth factor. Virology 214, 21–28 (1995).

    Article  CAS  PubMed  Google Scholar 

  27. Bonjardim, C. A. Viral exploitation of the MEK/ERK pathway—a tale of vaccinia virus and other viruses. Virology 507, 267–275 (2017).

    Article  CAS  PubMed  Google Scholar 

  28. Huang, C., Jacobson, K. & Schaller, M. D. MAP kinases and cell migration. J. Cell Sci. 117, 4619–4628 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Langhammer, S., Koban, R., Yue, C. & Ellerbrok, H. Inhibition of poxvirus spreading by the anti-tumor drug gefitinib (Iressa). Antiviral Res. 89, 64–70 (2011).

    Article  CAS  PubMed  Google Scholar 

  30. Schweneker, M. et al. The vaccinia virus O1 protein is required for sustained activation of extracellular signal-regulated kinase 1/2 and promotes viral virulence. J. Virol. 86, 2323–2336 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Singh, A. B. & Harris, R. C. Autocrine, paracrine and juxtacrine signaling by EGFR ligands. Cell. Signal. 17, 1183–1193 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Blobel, C. P. ADAMs: key components in EGFR signalling and development. Nat. Rev. Mol. Cell Biol. 6, 32–43 (2005).

    Article  CAS  PubMed  Google Scholar 

  33. Ludwig, A. et al. Metalloproteinase inhibitors for the disintegrin-like metalloproteinases ADAM10 and ADAM17 that differentially block constitutive and phorbol ester-inducible shedding of cell surface molecules. Comb. Chem. High Throughput Screen. 8, 161–171 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Cudmore, S., Cossart, P., Griffiths, G. & Way, M. Actin-based motility of vaccinia virus. Nature 378, 636–638 (1995).

    Article  CAS  PubMed  Google Scholar 

  35. Doceul, V., Hollinshead, M., van der Linden, L. & Smith, G. L. Repulsion of superinfecting virions: a mechanism for rapid virus spread. Science 327, 873–876 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yakimovich, A. et al. Infection: a generic framework for computational simulation of virus transmission between cells. mSphere 1, e00078-15 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Yang, M. & Huang, C. Z. Mitogen-activated protein kinase signaling pathway and invasion and metastasis of gastric cancer. World J. Gastroenterol. 21, 11673–11679 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Payne, L. G. Significance of extracellular enveloped virus in the in vitro and in vivo dissemination of vaccinia. J. Gen. Virol. 50, 89–100 (1980).

    Article  CAS  PubMed  Google Scholar 

  39. Yang, H. et al. Antiviral chemotherapy facilitates control of poxvirus infections through inhibition of cellular signal transduction. J. Clin. Invest. 115, 379–387 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Aldaz-Carroll, L. et al. Epitope-mapping studies define two major neutralization sites on the vaccinia virus extracellular enveloped virus glycoprotein B5R. J. Virol. 79, 6260–6271 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Mercer, J. & Helenius, A. Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells. Science 320, 531–535 (2008).

    Article  CAS  PubMed  Google Scholar 

  42. Tinevez, J. Y. et al. TrackMate: an open and extensible platform for single-particle tracking. Methods 115, 80–90 (2017).

    Article  CAS  PubMed  Google Scholar 

  43. Yakimovich, A. et al. Plaque2.0—a high-throughput analysis framework to score virus–cell transmission and clonal cell expansion. PLoS ONE 10, e0138760 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Breiman, L. Random forests. Mach. Learn. 45, 5–32 (2001).

    Article  Google Scholar 

  45. Arganda-Carreras, I. et al. Trainable Weka Segmentation: a machine learning tool for microscopy pixel classification. Bioinformatics 33, 2424–2426 (2017).

    Article  CAS  PubMed  Google Scholar 

  46. Efron, B. Bootstrap methods: another look at the jackknife. Ann. Stat. 7, 1–26 (1979).

    Article  Google Scholar 

  47. Ferreira, N., Klosowski, J. T., Scheidegger, C. E. & Silva, C. T. Vector field k-means: clustering trajectories by fitting multiple vector fields. Comput. Graph. Forum 32, 201–210 (2013).

    Article  Google Scholar 

  48. Cush, S. S. et al. Locally produced IL-10 limits cutaneous vaccinia virus spread. PLoS Pathog. 12, e1005493 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Pieri, L., Sassoli, C., Romagnoli, P. & Domenici, L. Use of periodate-lysine-paraformaldehyde for the fixation of multiple antigens in human skin biopsies. Eur. J. Histochem. 46, 365–375 (2002).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank K. Gonciarz and I. F. Sbalzarini for helpful discussions. We thank B. Siegenthaler and J. Crouse for initial work on the project. We thank all members of the Mercer lab for helpful discussions and comments on the manuscript. We thank A. Vaughan, V. Jäggin, T. Lopes, T. Lummen, T. Horn and D. Loeffler for technical support. We thank B. Moss (NIAID) for the vSC20 virus. This research was supported by core funding to the MRC Laboratory for Molecular Cell Biology, University College London (MC_UU12018/7) (J.M.), the European Research Council (649101–UbiProPox) (J.M.), ETH Zurich (G.F. and D.J.M.) and the Division of Intramural Research of the NIAID (G.V.R. and H.D.H.). C.B. is funded by the MRC Laboratory for Molecular Cell Biology PhD programme.

Author information

Authors and Affiliations

Authors

Contributions

C.B., S.K. and J.M. conceived the project. C.B., A.Y., S.K. and J.M. designed the experiments. G.V.R. and H.D.H. designed, performed and analysed the intravital imaging. G.F. and D.J.M. designed and performed the cell sorting and single-cell imaging. C.B., A.Y. and J.M. analysed the data. C.B., A.Y., H.D.H., G.F. and J.M. wrote the manuscript. All authors discussed the experiments, read and approved the manuscript.

Corresponding author

Correspondence to Jason Mercer.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–5, Supplementary Table 1.

Reporting Summary

Supplementary Video 1

VACV WR and mutant plaque formation.

Supplementary Video 2

VACV plaque formation in the presence of EGFR, MEK or FAK inhibitors.

Supplementary Video 3

VACV plaque formation adjacent to wounds.

Supplementary Video 4

VACV plaque formation in the presence of ADAM10 inhibitor.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Beerli, C., Yakimovich, A., Kilcher, S. et al. Vaccinia virus hijacks EGFR signalling to enhance virus spread through rapid and directed infected cell motility. Nat Microbiol 4, 216–225 (2019). https://doi.org/10.1038/s41564-018-0288-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-018-0288-2

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing