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NPF motifs in the vaccinia virus protein A36 recruit intersectin-1 to promote Cdc42:N-WASP-mediated viral release from infected cells

Nature Microbiology volume 1, Article number: 16141 (2016) | Download Citation

Abstract

During its egress, vaccinia virus transiently recruits AP-2 and clathrin after fusion with the plasma membrane. This recruitment polarizes the viral protein A36 beneath the virus, enhancing actin polymerization and the spread of infection. We now demonstrate that three NPF motifs in the C-terminus of A36 recruit AP-2 and clathrin by interacting directly with the Epsin15 homology domains of Eps15 and intersectin-1. A36 is the first identified viral NPF motif containing protein shown to interact with endocytic machinery. Vaccinia still induces actin tails in the absence of the A36 NPF motifs. Their loss, however, reduces the cell-to-cell spread of vaccinia. This is due to a significant reduction in virus release from infected cells, as the lack of intersectin-1 recruitment leads to a loss of Cdc42 activation, impairing N-WASP-driven Arp2/3-mediated actin polymerization. Our results suggest that initial A36-mediated virus release plays a more important role than A36-driven super-repulsion in promoting the cell-to-cell spread of vaccinia.

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References

  1. 1.

    & The role of signalling and the cytoskeleton during vaccinia virus egress. Virus Res. 209, 87–99 (2015).

  2. 2.

    , , & Actin-based motility of vaccinia virus. Nature 378, 636–638 (1995).

  3. 3.

    et al. Actin based motility of vaccinia virus mimics receptor tyrosine kinase signalling. Nature 401, 926–929 (1999).

  4. 4.

    et al. Vaccinia virus utilizes microtubules for movement to the cell surface. J. Cell Biol. 154, 389–402 (2001).

  5. 5.

    & Vaccinia virus intracellular movement is associated with microtubules and independent of actin tails. J. Virol. 75, 11651–11663 (2001).

  6. 6.

    et al. Grb2 and Nck act cooperatively to promote actin-based motility of vaccinia virus. Curr. Biol. 12, 740–745 (2002).

  7. 7.

    , & SRC mediates a switch from microtubule- to actin-based motility of vaccinia virus. Science 306, 124–129 (2004).

  8. 8.

    et al. Disabling poxvirus pathogenesis by inhibition of Abl-family tyrosine kinases. Nature Med. 11, 731–739 (2005).

  9. 9.

    , , & Abl collaborates with Src family kinases to stimulate actin-based motility of vaccinia virus. Cell Microbiol. 8, 233–241 (2006).

  10. 10.

    et al. A complex of N-WASP and WIP integrates signalling cascades that lead to actin polymerization. Nature Cell Biol. 2, 441–448 (2000).

  11. 11.

    & The WH1 and EVH1 domains of WASP and Ena/VASP family members bind distinct sequence motifs. Curr. Biol. 12, 1617–1622 (2002).

  12. 12.

    , , & The rate of N-WASP exchange limits the extent of ARP2/3-complex-dependent actin-based motility. Nature 458, 87–91 (2009).

  13. 13.

    , , & WIP provides an essential link between Nck and N-WASP during Arp2/3-dependent actin polymerization. Curr. Biol. 23, 999–1006 (2013).

  14. 14.

    , & Cdc42 and the Rho GEF intersectin-1 collaborate with Nck to promote N-WASP-dependent actin polymerisation. J. Cell Sci. 127, 673–685 (2014).

  15. 15.

    et al. Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP. Nature Cell Biol. 3, 927–932 (2001).

  16. 16.

    , & Emerging roles for intersectin (ITSN) in regulating signaling and disease pathways. Int. J. Mol. Sci. 14, 7829–7852 (2013).

  17. 17.

    et al. Regulation of synaptic vesicle recycling by complex formation between intersectin 1 and the clathrin adaptor complex AP2. Proc. Natl Acad. Sci. USA 107, 4206–4211 (2010).

  18. 18.

    et al. Clathrin potentiates vaccinia-induced actin polymerization to facilitate viral spread. Cell Host Microbe 12, 346–359 (2012).

  19. 19.

    Tickets to ride: selecting cargo for clathrin-regulated internalization. Nature Rev. Mol. Cell Biol. 10, 583–596 (2009).

  20. 20.

    & EHD proteins key conductors of endocytic transport. Trends Cell Biol. 21, 122–131 (2011).

  21. 21.

    , , & Repulsion of superinfecting virions: a mechanism for rapid virus spread. Science 327, 873–876 (2010).

  22. 22.

    et al. A36-dependent actin filament nucleation promotes release of vaccinia virus. PLoS Pathogens 9, e1003239 (2013).

  23. 23.

    , , & Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP. Nature 391, 93–96 (1998).

  24. 24.

    et al. Small molecule targeting Cdc42-intersectin interaction disrupts Golgi organization and suppresses cell motility. Proc. Natl Acad. Sci. USA 110, 1261–1266 (2013).

  25. 25.

    et al. Cdc42 is not essential for filopodium formation, directed migration, cell polarization, and mitosis in fibroblastoid cells. Mol. Biol. Cell 16, 4473–4484 (2005).

  26. 26.

    et al. Vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry. Proc. Natl Acad. Sci. USA 107, 9346–9351 (2010).

  27. 27.

    , , , & Structure and Asn-Pro-Phe binding pocket of the Eps15 homology domain. Science 281, 1357–1360 (1998).

  28. 28.

    & The Eps15 homology (EH) domain. FEBS Lett. 513, 24–29 (2002).

  29. 29.

    et al. Recognition specificity of individual EH domains of mammals and yeast. EMBO J. 17, 6541–6550 (1998).

  30. 30.

    et al. Loss of cytoskeletal transport during egress critically attenuates ectromelia virus infection in vivo. J. Virol. 86, 7427–7443 (2012).

  31. 31.

    & Nck- and N-WASP-dependent actin-based motility is conserved in divergent vertebrate poxviruses. Cell Host Microbe 6, 536–550 (2009).

  32. 32.

    et al. Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains. J. Biol. Chem. 273, 31401–31407 (1998).

  33. 33.

    , , & The ear of α-adaptin interacts with the COOH-terminal domain of the Eps 15 protein. J. Biol. Chem. 271, 12111–12116 (1996).

  34. 34.

    , , , & The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15. EMBO J. 18, 1159–1171 (1999).

  35. 35.

    et al. Intersectin (ITSN) family of scaffolds function as molecular hubs in protein interaction networks. PLoS ONE 7, e36023 (2012).

  36. 36.

    et al. Intersectin adaptor proteins are associated with actin-regulating protein WIP in invadopodia. Cell Signal 27, 1499–1508 (2015).

  37. 37.

    & The non-canonical roles of clathrin and actin in pathogen internalization, egress and spread. Nature Rev. Microbiol. 11, 551–560 (2013).

  38. 38.

    & Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nature Rev. Mol. Cell Biol. 12, 517–533 (2011).

  39. 39.

    & Golgi-derived membranes that contain an acylated viral polypeptide are used for vaccinia virus envelopment. J. Virol. 55, 651–659 (1985).

  40. 40.

    , , , & Interactions between vaccinia virus IEV membrane proteins and their roles in IEV assembly and actin tail formation. J. Virol. 73, 2863–2875 (1999).

  41. 41.

    et al. A tubular EHD1-containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane. EMBO J. 21, 2557–2567 (2002)

  42. 42.

    , , , & Interactions between EHD proteins and Rab11-FIP2: a role for EHD3 in early endosomal transport. Mol. Biol. Cell 17, 163–177 (2006).

  43. 43.

    , & A role for EHD4 in the regulation of early endosomal transport. Traffic 9, 995–1018 (2008).

  44. 44.

    , & Role of phosphatidylinositol 4,5-bisphosphate in regulating EHD2 plasma membrane localization. PLoS ONE 8, e74519 (2013).

  45. 45.

    , , , & The release of vaccinia virus from infected cells requires RhoA-mDia modulation of cortical actin. Cell Host Microbe 1, 227–240 (2007).

  46. 46.

    , & F11L-mediated inhibition of RhoA-mDia signaling stimulates microtubule dynamics during vaccinia virus infection. Cell Host Microbe 1, 213–226 (2007).

  47. 47.

    et al. N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility. Nature Cell Biol. 3, 897–904 (2001).

  48. 48.

    & Near-infrared fluorescent proteins for multicolor in vivo imaging. Nature Methods 10, 751–754 (2013).

  49. 49.

    et al. Isoform diversity in the Arp2/3 complex determines actin filament dynamics. Nature Cell Biol. 18, 76–86 (2016).

  50. 50.

    , , & Open source software for quantification of cell migration, protrusions, and fluorescence intensities. J. Cell Biol. 209, 163–180 (2015).

  51. 51.

    , & Compact, synthetic, vaccinia virus early/late promoter for protein expression. Biotechniques 23, 1094–1097 (1997).

  52. 52.

    et al. Tes, a specific Mena interacting partner, breaks the rules for EVH1 binding. Mol. Cell 28, 1071–1082 (2007).

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Acknowledgements

The authors thank S. Snapper (Harvard Medical School) and C. Brakebusch (University of Copenhagen) for providing N-WASP and Cdc42 deficient cells, respectively. The authors also thank N. O'Reilly (Francis Crick Institute) for synthesizing peptides, M. Matsuda (Kyoto University) for the iRFP670 clone and S. Caplan (University of Nebraska Medical Center) for GFP-tagged EHD1–4 clones. The authors acknowledge members of the Way Laboratory and H. Walden (University of Dundee) and D. Stephens (University of Bristol) for comments on the manuscript. Research was supported by Cancer Research UK and the Francis Crick Institute.

Author information

Author notes

    • Ina Weisswange
    •  & Ashley C. Humphries

    Present addresses: Eupheria Biotech GmbH, Tatzberg 47-51, 01307 Dresden, Germany (I.W.); Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York 10029, USA (A.C.H.).

Affiliations

  1. Cellular Signalling and Cytoskeletal Function Laboratory, The Francis Crick Institute, Lincoln's Inn Fields Laboratory, 44 Lincoln's Inn Fields, London WC2A 3LY, UK

    • Xenia Snetkov
    • , Ina Weisswange
    • , Julia Pfanzelter
    • , Ashley C. Humphries
    •  & Michael Way

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Contributions

X.S. and M.W. designed the study and wrote the manuscript. X.S. performed and analysed the experiments. I.W. constructed and analysed A36 C-terminal deletion mutants. J.P. generated pLVX-Lifeact-iRFP670 HeLa cells and A.C.H. generated pE/L-GFP-intersectin-1 clones and provided valuable discussions. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Michael Way.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Figures 1-5, Supplementary Video 1-4 legends

Videos

  1. 1.

    Supplementary Video 1

    Intersectin-1 and Eps15 are simultaneously recruited to CEV inducing actin tails

  2. 2.

    Supplementary Video 2

    NPF motifs are required for viral recruitment of intersectin-1

  3. 3.

    Supplementary Video 3

    NPF motifs are required for viral recruitment of Eps15

  4. 4.

    Supplementary Video 4

    Loss of all three A36 NPF motifs results in significantly longer actin tails and a faster velocity of the virus

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DOI

https://doi.org/10.1038/nmicrobiol.2016.141