[en] Marek’s Disease Virus (MDV) is an avian alpha-herpesvirus that only spreads from cell-to-cell in cell culture. While its cell-tocell spread has been shown to be dependent on actin filament dynamics, the mechanisms regulating this spread remain largely unknown. Using a recombinant BAC20 virus expressing an EGFPVP22 tegument protein, we found that the actin cytoskeleton arrangements and cell-cell contacts differ in the center and periphery of MDV infection plaques, with cells in the latter areas showing stress fibers and rare cellular projections. Using specific inhibitors and activators, we determined that Rho-ROCK pathway, known to regulate stress fiber formation, and Rac-PAK, known to promote lamellipodia formation and destabilize stress fibers, had strong contrasting effects on MDV cell-to-cell spread in primary chicken embryo skin cells (CESCs). Inhibition of Rho and its ROCKs effectors led to reduced plaque sizes whereas inhibition of Rac or its group I-PAKs effectors had the adverse effect. Importantly, we observed that the shape of MDV plaques is related to the semi-ordered arrangement of the elongated cells, at the monolayer level in the vicinity of the plaques. Inhibition of Rho-ROCK signaling also resulted in a perturbation of the cell arrangement and a rounding of plaques. These opposing effects of Rho and Rac pathways in MDV cell-to-cell spread were validated for two parental MDV recombinant viruses with different ex vivo spread efficiencies. Finally, we demonstrated that Rho/Rac pathways have opposing effects on the accumulation of N-cadherin at cell-cell contact regions between CESCs, and defined these contacts as adherens junctions. Considering the importance of adherens junctions in HSV-1 cell-to-cell spread in some cell types, this result makes of adherens junctions maintenance one potential and attractive hypothesis to explain the Rho/Rac effects on MDV cell-to-cell spread. Our study provides the first evidence that MDV cell-to-cell spread is regulated by Rho/Rac signaling.
Disciplines :
Life sciences: Multidisciplinary, general & others
Author, co-author :
Richerioux, Nicolas ✱; Institut Scientifique de Recherche Agronomique - INRA > Sante Animale > UMR1282, Infectious Diseases and Public Health, ISP, BIOVA team, Tours-Nouzilly
Blondeau, Caroline ✱; University College London - UCL > Infection and Immunity > Medical Research Council Centre for Medical Molecular Virology
Wiedemann, Agnes ✱; Institut Scientifique de Recherche Agronomique - INRA > Sante Animale > UMR1282, Infectious Diseases and Public Health, ISP, SPVB team, Tours-Nouzilly
Remy, Sylvie; Institut Scientifique de Recherche Agronomique - INRA > Sante Animale > UMR1282, Infectious Diseases and Public Health, ISP, BIOVA team, Tours-Nouzilly
Vautherot, Jean-Francois; Institut Scientifique de Recherche Agronomique - INRA > Sante Animale > UMR1282, Infectious Diseases and Public Health, ISP, BIOVA team, Tours-Nouzilly
Denesvre, Caroline; Institut Scientifique de Recherche Agronomique - INRA > Sante Animale > UMR1282, Infectious Diseases and Public Health, ISP, BIOVA team, Tours-Nouzilly
✱ These authors have contributed equally to this work.
Language :
English
Title :
Rho-ROCK and Rac-PAK Signaling Pathways Have Opposing Effects on the Cell-to-Cell Spread of Marek’s Disease Virus
Publication date :
2012
Journal title :
PLoS ONE
eISSN :
1932-6203
Publisher :
Public Library of Science, San Franscisco, United States - California
Zelnik V (2004) Diagnosis of Marek's disease. In: Davison F, Nair VK, editors. Marek's disease An evolving problem. Compton, UK: Elsevier Academic Press.
Biggs PM, Payne LN, (1967) Studies on Marek's disease. I. Experimental transmission. J Natl Cancer Inst 39: 267-280.
Denesvre C, Blondeau C, Lemesle M, Le Vern Y, Vautherot D, et al. (2007) Morphogenesis of a highly replicative EGFPVP22 recombinant Marek's disease virus in cell culture. J Virol 81: 12348-12359.
Nazerian K, Burmester BR, (1968) Electron microscopy of a herpes virus associated with the agent of Marek's disease in cell culture. Cancer Res 28: 2454-2462.
Sattentau Q, (2008) Avoiding the void: cell-to-cell spread of human viruses. Nat Rev Microbiol 6: 815-826.
Aubert M, Yoon M, Sloan DD, Spear PG, Jerome KR, (2009) The virological synapse facilitates herpes simplex virus entry into T cells. J Virol 83: 6171-6183.
Favoreel HW, van Minnebruggen G, Adriaensen D, Nauwynck HJ, (2005) Cytoskeletal rearrangements and cell extensions induced by the US3 kinase of an alphaherpesvirus are associated with enhanced spread. Proc Natl Acad Sci U S A 102: 8990-8995.
Gill MB, Edgar R, May JS, Stevenson PG, (2008) A gamma-herpesvirus glycoprotein complex manipulates actin to promote viral spread. PLoS One 3: e1808.
Johnson DC, Webb M, Wisner TW, Brunetti C, (2001) Herpes simplex virus gE/gI sorts nascent virions to epithelial cell junctions, promoting virus spread. J Virol 75: 821-833.
Jolly C, Kashefi K, Hollinshead M, Sattentau QJ, (2004) HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse. J Exp Med 199: 283-293.
Sherer NM, Lehmann MJ, Jimenez-Soto LF, Horensavitz C, Pypaert M, et al. (2007) Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission. Nat Cell Biol 9: 310-315.
Sowinski S, Jolly C, Berninghausen O, Purbhoo MA, Chauveau A, et al. (2008) Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission. Nat Cell Biol 10: 211-219.
Dixit R, Tiwari V, Shukla D, (2008) Herpes simplex virus type 1 induces filopodia in differentiated P19 neural cells to facilitate viral spread. Neurosci Lett 440: 113-118.
Oh MJ, Akhtar J, Desai P, Shukla D, (2010) A role for heparan sulfate in viral surfing. Biochem Biophys Res Commun 391: 176-181.
May JS, Walker J, Colaco S, Stevenson PG, (2005) The murine gammaherpesvirus 68 ORF27 gene product contributes to intercellular viral spread. J Virol 79: 5059-5068.
Spear PG, Eisenberg RJ, Cohen GH, (2000) Three classes of cell surface receptors for alphaherpesvirus entry. Virology 275: 1-8.
Yoon M, Spear PG, (2002) Disruption of adherens junctions liberates nectin-1 to serve as receptor for herpes simplex virus and pseudorabies virus entry. J Virol 76: 7203-7208.
Hall A, (1998) Rho GTPases and the actin cytoskeleton. Science 279: 509-514.
Etienne-Manneville S, Hall A, (2002) Rho GTPases in cell biology. Nature 420: 629-635.
Takai Y, Sasaki T, Matozaki T, (2001) Small GTP-binding proteins. Physiol Rev 81: 153-208.
Kaverina I, Krylyshkina O, Small JV, (2002) Regulation of substrate adhesion dynamics during cell motility. Int J Biochem Cell Biol 34: 746-761.
Miyake Y, Inoue N, Nishimura K, Kinoshita N, Hosoya H, et al. (2006) Actomyosin tension is required for correct recruitment of adherens junction components and zonula occludens formation. Exp Cell Res 312: 1637-1650.
Burridge K, Wennerberg K, (2004) Rho and Rac take center stage. Cell 116: 167-179.
Kozma R, Ahmed S, Best A, Lim L, (1995) The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol Cell Biol 15: 1942-1952.
Bishop AL, Hall A, (2000) Rho GTPases and their effector proteins. Biochem J 348: 241-255.
Lammers M, Meyer S, Kuhlmann D, Wittinghofer A, (2008) Specificity of interactions between mDia isoforms and Rho proteins. J Biol Chem 283: 35236-35246.
Wheeler AP, Ridley AJ, (2004) Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility. Exp Cell Res 301: 43-49.
Schonichen A, Geyer M, (2010) Fifteen formins for an actin filament: a molecular view on the regulation of human formins. Biochim Biophys Acta 1803: 152-163.
Sanders LC, Matsumura F, Bokoch GM, de Lanerolle P, (1999) Inhibition of myosin light chain kinase by p21-activated kinase. Science 283: 2083-2085.
Schumacher D, Tischer BK, Trapp S, Osterrieder N, (2005) The Protein Encoded by the US3 Orthologue of Marek's Disease Virus Is Required for Efficient De-Envelopment of Perinuclear Virions and Involved in Actin Stress Fiber Breakdown. J Virol 79: 3987-3997.
Blondeau C, Marc D, Courvoisier K, Vautherot JF, Denesvre C, (2008) Functional homologies between avian and human alphaherpesvirus VP22 proteins in cell-to-cell spreading as revealed by a new cis-complementation assay. J Virol 82: 9278-9282.
Cooper JA, (1987) Effects of cytochalasin and phalloidin on actin. J Cell Biol 105: 1473-1478.
Holzinger A, (2009) Jasplakinolide: an actin-specific reagent that promotes actin polymerization. Methods Mol Biol 586: 71-87.
Bubb MR, Spector I, Beyer BB, Fosen KM, (2000) Effects of jasplakinolide on the kinetics of actin polymerization. An explanation for certain in vivo observations. J Biol Chem 275: 5163-5170.
Lazaro-Dieguez F, Aguado C, Mato E, Sanchez-Ruiz Y, Esteban I, et al. (2008) Dynamics of an F-actin aggresome generated by the actin-stabilizing toxin jasplakinolide. J Cell Sci 121: 1415-1425.
Mege RM, Gavard J, Lambert M, (2006) Regulation of cell-cell junctions by the cytoskeleton. Curr Opin Cell Biol 18: 541-548.
Yonemura S, Itoh M, Nagafuchi A, Tsukita S, (1995) Cell-to-cell adherens junction formation and actin filament organization: similarities and differences between non-polarized fibroblasts and polarized epithelial cells. J Cell Sci 108 (Pt 1): 127-142.
Aktories K, Braun U, Rosener S, Just I, Hall A, (1989) The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3. Biochem Biophys Res Commun 158: 209-213.
Sekine A, Fujiwara M, Narumiya S, (1989) Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase. J Biol Chem 264: 8602-8605.
Sahai E, Olson MF, (2006) Purification of TAT-C3 exoenzyme. Methods Enzymol 406: 128-140.
Paterson HF, Self AJ, Garrett MD, Just I, Aktories K, et al. (1990) Microinjection of recombinant p21rho induces rapid changes in cell morphology. J Cell Biol 111: 1001-1007.
Wiegers W, Just I, Muller H, Hellwig A, Traub P, et al. (1991) Alteration of the cytoskeleton of mammalian cells cultured in vitro by Clostridium botulinum C2 toxin and C3 ADP-ribosyltransferase. Eur J Cell Biol 54: 237-245.
Narumiya S, Ishizaki T, Uehata M, (2000) Use and properties of ROCK-specific inhibitor Y-27632. Methods Enzymol 325: 273-284.
Davies SP, Reddy H, Caivano M, Cohen P, (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95-105.
Sward K, Dreja K, Susnjar M, Hellstrand P, Hartshorne DJ, et al. (2000) Inhibition of Rho-associated kinase blocks agonist-induced Ca2+ sensitization of myosin phosphorylation and force in guinea-pig ileum. J Physiol 522 Pt 1: 33-49.
Kadir S, Astin JW, Tahtamouni L, Martin P, Nobes CD, (2011) Microtubule remodelling is required for the front-rear polarity switch during contact inhibition of locomotion. J Cell Sci 124: 2642-2653.
Straight AF, Cheung A, Limouze J, Chen I, Westwood NJ, et al. (2003) Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science 299: 1743-1747.
Albertinazzi C, Cattelino A, de Curtis I, (1999) Rac GTPases localize at sites of actin reorganization during dynamic remodeling of the cytoskeleton of normal embryonic fibroblasts. J Cell Sci 112 (Pt 21): 3821-3831.
Manser E, Huang HY, Loo TH, Chen XQ, Dong JM, et al. (1997) Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes. Mol Cell Biol 17: 1129-1143.
Gao Y, Dickerson JB, Guo F, Zheng J, Zheng Y, (2004) Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci U S A 101: 7618-7623.
Andor A, Trulzsch K, Essler M, Roggenkamp A, Wiedemann A, et al. (2001) YopE of Yersinia, a GAP for Rho GTPases, selectively modulates Rac-dependent actin structures in endothelial cells. Cell Microbiol 3: 301-310.
Deacon SW, Beeser A, Fukui JA, Rennefahrt UE, Myers C, et al. (2008) An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase. Chem Biol 15: 322-331.
Radeff-Huang J, Seasholtz TM, Matteo RG, Brown JH, (2004) G protein mediated signaling pathways in lysophospholipid induced cell proliferation and survival. J Cell Biochem 92: 949-966.
Jarosinski KW, Margulis NG, Kamil JP, Spatz SJ, Nair VK, et al. (2007) Horizontal transmission of Marek's disease virus requires US2, the UL13 protein kinase, and gC. J Virol 81: 10575-10587.
Comunale F, Causeret M, Favard C, Cau J, Taulet N, et al. (2007) Rac1 and RhoA GTPases have antagonistic functions during N-cadherin-dependent cell-cell contact formation in C2C12 myoblasts. Biol Cell 99: 503-517.
Vaezi A, Bauer C, Vasioukhin V, Fuchs E, (2002) Actin cable dynamics and Rho/Rock orchestrate a polarized cytoskeletal architecture in the early steps of assembling a stratified epithelium. Dev Cell 3: 367-381.
Chan MW, El Sayegh TY, Arora PD, Laschinger CA, Overall CM, et al. (2004) Regulation of intercellular adhesion strength in fibroblasts. J Biol Chem 279: 41047-41057.
Mary S, Charrasse S, Meriane M, Comunale F, Travo P, et al. (2002) Biogenesis of N-cadherin-dependent cell-cell contacts in living fibroblasts is a microtubule-dependent kinesin-driven mechanism. Mol Biol Cell 13: 285-301.
Schumacher D, McKinney C, Kaufer BB, Osterrieder N, (2008) Enzymatically inactive U(S)3 protein kinase of Marek's disease virus (MDV) is capable of depolymerizing F-actin but results in accumulation of virions in perinuclear invaginations and reduced virus growth. Virology 375: 37-47.
van den Broeke C, Radu M, Deruelle M, Nauwynck H, Hofmann C, et al. (2009) Alphaherpesvirus US3-mediated reorganization of the actin cytoskeleton is mediated by group A p21-activated kinases. Proc Natl Acad Sci U S A 106: 8707-8712.
Favoreel HW, Enquist LW, Feierbach B, (2007) Actin and Rho GTPases in Herpesvirus biology. Trends Microbiol 15: 426-433.
Favoreel HW, van den Broeke C, Desplanques A, Deruelle M, van Minnebruggen G, et al. (2010) Alphaherpesvirus use and misuse of cellular actin and cholesterol. Vet Microbiol 143: 2-7.
Lyman MG, Enquist LW, (2009) Herpesvirus interactions with the host cytoskeleton. J Virol 83: 2058-2066.
Taylor MP, Koyuncu OO, Enquist LW, (2011) Subversion of the actin cytoskeleton during viral infection. Nat Rev Microbiol 9: 427-439.
van den Broeke C, Favoreel HW, (2011) Actin' up: herpesvirus interactions with Rho GTPase signaling. Viruses 3: 278-292.
Clement C, Tiwari V, Scanlan PM, Valyi-Nagy T, Yue BY, et al. (2006) A novel role for phagocytosis-like uptake in herpes simplex virus entry. J Cell Biol 174: 1009-1021.
Frampton AR Jr, Stolz DB, Uchida H, Goins WF, Cohen JB, et al. (2007) Equine herpesvirus 1 enters cells by two different pathways, and infection requires the activation of the cellular kinase ROCK1. J Virol 81: 10879-10889.
Naranatt PP, Krishnan HH, Smith MS, Chandran B, (2005) Kaposi's sarcoma-associated herpesvirus modulates microtubule dynamics via RhoA-GTP-diaphanous 2 signaling and utilizes the dynein motors to deliver its DNA to the nucleus. J Virol 79: 1191-1206.
Veettil MV, Sharma-Walia N, Sadagopan S, Raghu H, Sivakumar R, et al. (2006) RhoA-GTPase facilitates entry of Kaposi's sarcoma-associated herpesvirus into adherent target cells in a Src-dependent manner. J Virol 80: 11432-11446.
Mercer J, Helenius A, (2009) Virus entry by macropinocytosis. Nat Cell Biol 11: 510-520.
van Leeuwen H, Elliott G, O'Hare P, (2002) Evidence of a role for nonmuscle myosin II in herpes simplex virus type 1 egress. J Virol 76: 3471-3481.
Cheung A, Dantzig JA, Hollingworth S, Baylor SM, Goldman YE, et al. (2002) A small-molecule inhibitor of skeletal muscle myosin II. Nat Cell Biol 4: 83-88.
Ostap EM, (2002) 2,3-Butanedione monoxime (BDM) as a myosin inhibitor. J Mus Res Cell Motil 23: 305-308.
Loubery S, Coudrier E, (2008) Myosins in the secretory pathway: tethers or transporters? Cell Mol Life Sci 65: 2790-2800.
Ren XD, Kiosses WB, Schwartz MA, (1999) Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 18: 578-585.
Schauer K, Duong T, Bleakley K, Bardin S, Bornens M, et al. (2010) Probabilistic density maps to study global endomembrane organization. Nat Methods 7: 560-566.
Keyser J, Lorger M, Pavlovic J, Radziwill G, Moelling K, (2007) Role of AF6 protein in cell-to-cell spread of Herpes simplex virus 1. FEBS Lett 581: 5349-5354.
Dorange F, El Mehdaoui S, Pichon C, Coursaget P, Vautherot JF, (2000) Marek's disease virus (MDV) homologues of herpes simplex virus type 1 UL49 (VP22) and UL48 (VP16) genes: high-level expression and characterization of MDV-1 VP22 and VP16. J Gen Virol 81: 2219-2230.
Hase H, Hirayama E, Kim J, (1996) Further investigation of some inhibitors on myogenic differentiation: mechanism of inhibition with HMBA on quail myoblasts transformed with Rous sarcoma virus. Cell Struct Funct 21: 17-25.
Petherbridge L, Brown AC, Baigent SJ, Howes K, Sacco MA, et al. (2004) Oncogenicity of virulent Marek's disease virus cloned as bacterial artificial chromosomes. J Virol 78: 13376-13380.
Schumacher D, Tischer BK, Fuchs W, Osterrieder N, (2000) Reconstitution of Marek's disease virus serotype 1 (MDV-1) from DNA cloned as a bacterial artificial chromosome and characterization of a glycoprotein B-negative MDV-1 mutant. J Virol 74: 11088-11098.
Sebbagh M, Renvoize C, Hamelin J, Riche N, Bertoglio J, et al. (2001) Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat Cell Biol 3: 346-352.
Rosselin M, Virlogeux-Payant I, Roy C, Bottreau E, Sizaret PY, et al. (2010) Rck of Salmonella enterica, subspecies enterica serovar Enteritidis, mediates Zipper-like internalization. Cell Res 20: 647-664.
Blondeau C, Chbab N, Beaumont C, Courvoisier K, Osterrieder N, et al. (2007) A full UL13 open reading frame in Marek's disease virus (MDV) is dispensable for tumor formation and feather follicle tropism and cannot restore horizontal virus transmission of rRB-1B in vivo. Vet Res 38: 419-433.
