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Vaccinia virus dissemination requires p21-activated kinase 1

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Abstract

The orthopoxvirus vaccinia virus (VACV) interacts with both actin and microtubule cytoskeletons in order to generate and spread progeny virions. Here, we present evidence demonstrating the involvement of PAK1 (p21-activated kinase 1) in the dissemination of VACV. Although PAK1 activation has previously been associated with optimal VACV entry via macropinocytosis, its absence does not affect the production of intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs). Our data demonstrate that low-multiplicity infection of PAK1-/- MEFs leads to a reduction in plaque size followed by decreased production of both IMVs and EEVs, strongly suggesting that virus spread was impaired in the absence of PAK1. Confocal and scanning electron microscopy showed a substantial reduction in the amount of VACV-induced actin tails in PAK1-/- MEFs, but no significant alteration in the total amount of cell-associated enveloped virions (CEVs). Furthermore, the decreased VACV dissemination in PAK1-/- cells was correlated with the absence of phosphorylated ARPC1 (Thr21), a downstream target of PAK1 and a key regulatory subunit of the ARP2/3 complex, which is necessary for the formation of actin tails and viral spread. We conclude that PAK1, besides its role in virus entry, also plays a relevant role in VACV dissemination.

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References

  1. McFadden G (2005) Poxvirus tropism. Nat Rev Microbiol 3:201–213

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Buchkovich NJ, Yu Y, Zampieri CA, Alwine JC (2008) The TORrid affairs of viruses: effects of mammalian DNA viruses on the PI3K–Akt–mTOR signalling pathway. Nat Rev Microbiol 6:266–275

    Article  PubMed  PubMed Central  Google Scholar 

  3. Bonjardim CA, Ferreira PC, Kroon EG (2009) Interferons: signaling, antiviral and viral evasion. Immunol Lett 122:1–11

    Article  CAS  PubMed  Google Scholar 

  4. Taylor MP, Koyuncu OO, Enquist LW (2011) Subversion of the actin cytoskeleton during viral infection. Nat Rev Microbiol 9:427–439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Humphries AC, Way M (2013) The non-canonical roles of clathrin and actin in pathogen internalization, egress and spread. Nat Rev Microbiol 11:551–560

    Article  CAS  PubMed  Google Scholar 

  6. Alto NM, Orth K (2012) Subversion of cell signaling by pathogens. Cold Spring Harb Perspect Biol 4:a006114

    Article  PubMed  PubMed Central  Google Scholar 

  7. Condit RC, Moussatche N, Traktman P (2006) In a nutshell: structure and assembly of the vaccinia virion. Adv Virus Res 66:31–124

    Article  CAS  PubMed  Google Scholar 

  8. Moss B (2007) Poxviridae. In: Fields BN, Knipe DM, Howley PM (eds) Virology, 5th edn. Lippincott-Raven, Philadelfia, pp 2905–2946

    Google Scholar 

  9. Moss B (2012) Poxvirus cell entry: how many proteins does it take? Viruses 4:688–707

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Johnston JB, McFadden G (2003) Poxvirus immunomodulatory strategies: current perspectives. J Virol 77:6093–6100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Perdiguero B, Esteban M (2009) The interferon system and vaccinia virus evasion mechanisms. J Interf Cytokine Res 29:581–598

    Article  CAS  Google Scholar 

  12. Schepis A, Schramm B, de Haan CA, Locker JK (2006) Vaccinia virus-induced microtubule-dependent cellular rearrangements. Traffic 7:308–323

    Article  CAS  PubMed  Google Scholar 

  13. Schramm B, de Haan CA, Young J, Doglio L, Schleich S, Reese C et al (2006) Vaccinia-virus-induced cellular contractility facilitates the subcellular localization of the viral replication sites. Traffic 7:1352–1367

    Article  CAS  PubMed  Google Scholar 

  14. Pereira AC, Leite FG, Brasil BS, Soares-Martins JA, Torres AA, Pimenta PF et al (2012) A vaccinia virus-driven interplay between the MKK4/7–JNK1/2 pathway and cytoskeleton reorganization. J Virol 86:172–184

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Law M, Carter GC, Roberts KL, Hollinshead M, Smith GL (2006) Ligand-induced and nonfusogenic dissolution of a viral membrane. Proc Natl Acad Sci USA 103:5989–5994

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Townsley AC, Weisberg AS, Wagenaar TR, Moss B (2006) Vaccinia virus entry into cells via a low-pH-dependent endosomal pathway. J Virol 80:8899–8908

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Huang CY, Lu TY, Bair CH, Chang YS, Jwo JK, Chang W A novel cellular protein, VPEF, facilitates vaccinia virus penetration into HeLa cells through fluid phase endocytosis. J Virol 82:7988–7999

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

    Article  CAS  PubMed  Google Scholar 

  19. Rietdorf J, Ploubidou A, Reckmann I, Holmstrom A, Frischknecht F, Zettl M et al (2001) Kinesin-dependent movement on microtubules precedes actin-based motility of vaccinia virus. Nat Cell Biol 3:992–1000

    Article  CAS  PubMed  Google Scholar 

  20. Ward BM, Moss B (2001) Vaccinia virus intracellular movement is associated with microtubules and independent of actin tails. J Virol 75:11651–11663

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Frischknecht F, Moreau V, Rottger S, Gonfloni S, Reckmann I, Superti-Furga G et al (1999) Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling. Nature 401:926–929

    Article  CAS  PubMed  Google Scholar 

  22. Leite F, Way M (2015) The role of signalling and the cytoskeleton during vaccinia virus egress. Virus Res 209:87–99

    Article  CAS  PubMed  Google Scholar 

  23. Newsome TP, Scaplehorn N, Way M (2004) SRC mediates a switch from microtubule- to actin-based motility of vaccinia virus. Science 306:124–129

    Article  CAS  PubMed  Google Scholar 

  24. Ward BM (2005) The longest micron; transporting poxviruses out of the cell. Cell Microbiol 7:1531–1538

    Article  CAS  PubMed  Google Scholar 

  25. Reeves PM, Bommarius B, Lebeis S, McNulty S, Christensen J, Swimm A et al (2005) Disabling poxvirus pathogenesis by inhibition of Abl-family tyrosine kinases. Nat Med 11:731–739

    Article  CAS  PubMed  Google Scholar 

  26. Scaplehorn N, Holmstrom A, Moreau V, Frischknecht F, Reckmann I, Way M (2002) Grb2 and Nck act cooperatively to promote actin-based motility of vaccinia virus. Curr Biol 12:740–745

    Article  CAS  PubMed  Google Scholar 

  27. Donnelly SK, Weisswange I, Zettl M, Way M (2013) WIP provides an essential link between Nck and N-WASP during Arp2/3-dependent actin polymerization. Curr Biol 23:999–1006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Moreau V, Frischknecht F, Reckmann I, Vincentelli R, Rabut G, Stewart D, Way M (2000) A complex of N-WASP and WIP integrates signalling cascades that lead to actin polymerization. Nat Cell Biol 2:441–448

    Article  CAS  PubMed  Google Scholar 

  29. Weisswange I, Newsome TP, Schleich S, Way M (2009) The rate of N-WASP exchange limits the extent of ARP2/3-complex-dependent actin-based motility. Nature 458:87–91

    Article  CAS  PubMed  Google Scholar 

  30. Hunter MP, Russo A, O’Bryan JP (2013) Emerging roles for intersectin (ITSN) in regulating signaling and disease pathways. Int J Mol Sci 14:7829–7852

    Article  PubMed  PubMed Central  Google Scholar 

  31. Humphries AC, Donnelly SK, Way M (2014) Cdc42 and the Rho GEF intersectin-1 collaborate with Nck to promote N-WASP-dependent actin polymerization. J Cell Sci 127:673–685

    Article  CAS  PubMed  Google Scholar 

  32. Humphries AC, Dodding MP, Barry DJ, Collinson LM, Durkin CH, Way M (2012) Clathrin potentiates vaccinia-induced actin polymerization to facilitate viral spread. Cell Host Microbe 12:346–359

    Article  CAS  PubMed  Google Scholar 

  33. Alvarez DE, Agaisse H (2013) The formin FHOD1 and the small GTPase Rac1 promote vaccinia virus actin-based motility. J Cell Biol 202:1075–1090

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gournier H, Goley ED, Niederstrasser H, Trinh T, Welch MD (2001) Reconstitution of human Arp2/3 complex reveals critical roles of individual subunits in complex structure and activity. Mol Cell 8:1041–1052

    Article  CAS  PubMed  Google Scholar 

  35. Vadlamudi RK, Li F, Barnes CJ, Bagheri-Yarmand R, Kumar R (2004) p41-Arc subunit of human Arp2/3 complex is a p21-activated kinase-1-interacting substrate. EMBO Rep 5:154–160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Johnston JB, Barrett JW, Chang W, Chung CS, Zeng W, Masters J et al (2003) Role of the serine-threonine kinase PAK-1 in myxoma virus replication. J Virol 77:5877–5888

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Van den Broeke C, Radu M, Chernoff J, Favoreel HW (2010) An emerging role for p21-activated kinases (Paks) in viral infections. Trends Cell Biol 20:160–169

    Article  PubMed  Google Scholar 

  38. Pacheco A, Chernoff J (2010) Group I p21-activated kinases: emerging roles in immune function and viral pathogenesis. Int J Biochem Cell Biol 42:13–16

    Article  CAS  PubMed  Google Scholar 

  39. Schmidt FI, Bleck CK, Mercer J (2012) Poxvirus host cell entry. Curr Opin Virol 2:20–27

    Article  CAS  PubMed  Google Scholar 

  40. Laliberte JP, Moss B (2009) Appraising the apoptotic mimicry model and the role of phospholipids for poxvirus entry. Proc Natl Acad Sci USA 106:17517–17521

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Laliberte JP, Weisberg AS, Moss B (2011) The membrane fusion step of vaccinia virus entry is cooperatively mediated by multiple viral proteins and host cell components. PLoS Pathog 7:e1002446

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Husain M, Moss B (2001) Vaccinia virus F13L protein with a conserved phospholipase catalytic motif induces colocalization of the B5R envelope glycoprotein in post-Golgi vesicles. J Virol 75:7528–7542

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Joklik WK (1962) The purification of four strains of poxvirus. Virology 18:9–18

    Article  CAS  PubMed  Google Scholar 

  44. Wolffe EJ, Vijaya S, Moss B (1995) A myristylated membrane protein encoded by the vaccinia virus L1R open reading frame is the target of potent neutralizing monoclonal antibodies. Virology 211:53–63

    Article  CAS  PubMed  Google Scholar 

  45. Smith GL, Law M (2004) The exit of vaccinia virus from infected cells. Virus Res 106:189–197

    Article  CAS  PubMed  Google Scholar 

  46. Deacon SW, Beeser A, Fukui JA, Rennefahrt UE, Myers C, Chernoff J et al (2008) An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase. Chem Biol 15:322–331

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Soares JA, Leite FG, Andrade LG, Torres AA, de Sousa LP, Barcelos LS et al (2009) Activation of the PI3K/Akt pathway early during vaccinia and cowpox virus infection is required for both host survival and viral replication. J Virol 83:6883–6899

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Amstutz B, Gastaldelli M, Kalin S, Imelli N, Boucke K, Wandeler E et al (2008) Subversion of CtBP1-controlled macropinocytosis by human adenovirus serotype 3. Embo J 27:956–969

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Karjalainen M, Kakkonen E, Upla P, Paloranta H, Kankaanpaa P, Liberali P et al (2008) A raft-derived, Pak1-regulated entry participates in alpha2beta1 integrin-dependent sorting to caveosomes. Mol Biol Cell 19:2857–2869

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Sanchez EG, Quintas A, Perez-Nunez D, Nogal M, Barroso S, Carrascosa AL et al (2012) African swine fever virus uses macropinocytosis to enter host cells. PLoS Pathog 8:e1002754

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Schelhaas M, Shah B, Holzer M, Blattmann P, Kuhling L, Day PM et al (2012) Entry of human papillomavirus type 16 by actin-dependent, clathrin- and lipid raft-independent endocytosis. PLoS Pathog 8:e1002657

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Van den Broeke C, Deruelle M, Nauwynck HJ, Coller KE, Smith GA, Van Doorsselaere J et al (2009) The kinase activity of pseudorabies virus US3 is required for modulation of the actin cytoskeleton. Virology 385:155–160

    Article  PubMed  Google Scholar 

  53. Villa NY, Bartee E, Mohamed MR, Rahman MM, Barrett JW, McFadden G (2010) Myxoma and vaccinia viruses exploit different mechanisms to enter and infect human cancer cells. Virology 401:266–279

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Nikolic DS, Lehmann M, Felts R, Garcia E, Blanchet FP, Subramaniam S et al (2011) HIV-1 activates Cdc42 and induces membrane extensions in immature dendritic cells to facilitate cell-to-cell virus propagation. Blood 118:4841–4852

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Abella JV, Galloni C, Pernier J, Barry DJ, Kjær S, Carlier MF, Way M (2016) Isoform diversity in the Arp2/3 complex determines actin filament dynamics. Nat Cell Biol 18:76–86

    Article  CAS  PubMed  Google Scholar 

  56. Bokoch GM (2003) Biology of the p21-activated kinases. Annu Rev Biochem 72:743–781

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors are grateful to Dr. Jonathan Chernoff (Tumor Cell Biology Program, Fox Chase Cancer Center, Philadelphia, USA), who kindly provided us with wild-type (WT) and pak1 knockout (PAK1-/-) mouse embryonic fibroblasts (MEFs). We also thank Dr. B. Moss (NIAID, Bethesda, MD, USA) for providing us with VACV F13L-GFP and antibody against viral protein L1R, and Dr. C. Jungwirth (Universität Würzburg, Germany), who provided us with VACV WR. We are indebted to Gisele C. dos Santos, Paula Marinho, João R. Santos and Alice A. Torres (from Viruses Laboratory) for excellent technical support. We are also grateful to Stephanie Lamb, Jia Liu, Masmudur Rahman, Dorothy Smith and Sherin Smallwood (from G.M.’s laboratory) for helpful discussions and assistance. This work was supported by grants from The Minas Gerais State’s Foundation for Research Support (FAPEMIG), Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), and the National Council for Scientific and Technological Development (CNPq). L.G.A and J.D.A were recipients of pre-doctoral fellowships from CNPq. J.D.A. was also a recipient of a fellowship from the International Program for doctoral training (PDSE/CAPES). F.L.B.M was the recipient of a Scientific Initiation Fellowship from CNPq. C.A.B, E.G.K, F.G.F. and G.B.M. are recipients of research fellowships from CNPq. G.M.’s laboratory is supported by NIH grant R01 AI080607. Confocal microscopy and scanning electron microscopy were performed at the Institute of Biological Sciences’ Center for Electron Microscopy (CEMEL) and at the UFMG’s Microscopy Center (CM-UFMG), respectively.

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Correspondence to Cláudio A. Bonjardim.

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This study was funded by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and National Institutes of Health (NIH) (grant number R01 AI080607).

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The authors declare that they have no conflict of interest.

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Luciana G. Andrade and Jonas D. Albarnaz contributed equally to this study.

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Andrade, L.G., Albarnaz, J.D., Mügge, F.L.B. et al. Vaccinia virus dissemination requires p21-activated kinase 1. Arch Virol 161, 2991–3002 (2016). https://doi.org/10.1007/s00705-016-2996-3

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