Background Echovirus 30 (Echo30) is one of the most frequently identified human enteroviruses (EVs) causing aseptic meningitis and encephalitis. However the mechanism underlying the pathogenesis of Echo30 infection with significant clinical outcomes is not completely understood. The aim of this investigation is to illustrate molecular pathologic alteration in neuronal cells induced by Echo30 infection using clinical isolate from young patient with neurologic involvement. Methodology/Principal Findings To characterize the neuronal cellular response to Echo30 infection, we performed a proteomic analysis based on two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF Mass Spectrophotometric (MS) analysis. We identified significant alteration of several protein expression levels in Echo30-infected SK-N-SH cells. Among these proteins, we focused on an outstanding up-regulation of Triple functional domain (TRIO) in Echo30-infected SK-N-SH cells. Generally, TRIO acts as a key component in the regulation of axon guidance and cell migration. In this study, we determined that TRIO plays a role in the novel pathways in Echo30 induced neuronal cell death. Conclusions/Significance Our finding shows that TRIO plays a critical role in neuronal cell death by Echo30 infection. Echo30 infection activates TRIO-guanine nucleotide exchange factor (GEF) domains (GEFD2) and RhoA signaling in turn. These results suggest that Echo30 infection induced neuronal cell death by activation of the TRIO-RhoA signaling. We expect the regulation of TRIO-RhoA signaling may represent a new therapeutic approach in treating aseptic meningitis and encephalitis induced by Echo30.
References
[1]
Choi YJ, Park KS, Baek KA, Jung EH, Nam HS, et al. (2010) Molecular characterization of Echovirus 30-associated outbreak of aseptic meningitis in Korea in 2008. J Microbiol Biotechnol 20: 643–649.
[2]
Dalwai A, Ahmad S, Al-Nakib W (2010) Echoviruses are a major cause of aseptic meningitis in infants and young children in Kuwait. Virol J 16: 236–241.
[3]
Baek KA, Yeo SG, Lee BH, Park KS, Song JH, et al. (2011) Epidemics of enterovirus infection in Chungnam Korea, 2008 and 2009. Virol J 8: 297–305.
[4]
Palacios G, Casas I, Cisterna D, Trallero G, Tenorio A, et al. (2002) Molecular epidemiology of Echovirus 30: Temporal circulation and prevalence of single lineages. J Virol 76: 4940–4949.
[5]
McWilliam EC, Bendig J, Cabrerizo M, Cardosa J, Hyypi？ T, et al. (2009) Transmission networks and population turnover of Echovirus 30. J Virol 83: 2109–2118.
[6]
Weng KF, Chen LL, Huang PN, Shih SR (2010) Neural pathogenesis of enterovirus 71 infection. Microbes Infect 12: 505–510.
[7]
Chen CS, Yao YC, Lin SC, Lee YP, Wang YF, et al. (2007) Retrograde axonal transport: a major transmission route of Enterovirus 71 in mice. J Virol 81: 8996–9003.
[8]
Brunel D, Lévêque N, Jacques J, Renois F, Motte J, et al. (2008) Clinical and virological features of an aseptic meningitis outbreak in North-Eastern France, 2005. J Clin Virol 42: 225–228.
[9]
Frantzidou F, Kamaria F, Dumaidi K, Skoura L, Antoniadis A, et al. (2008) Aseptic meningitis and encephalitis because of herpesviruses and enteroviruses in an immunocompetent adult population. Eur J Neurol 15: 995–997.
[10]
Leong WF, Chow VT (2006) Transcriptomic and proteomic analyses of rhadomyosarcoma cells reveal differential cellular gene expression in response to enterovirus 71 infection. Cell Microbiol 8: 565–580.
[11]
Peng YJ, He WQ, Tang J, Tao T, Chen C, et al. (2010) TRIO is a key quanine nucleotide exchange factor coordinating regulation of the migration and morphogenesis of granule cells in the developing cerebellum. J Biol Chem 28: 24834–24844.
[12]
Ferraro F, Ma XM, Sobota JA, Eipper BA, Mains RE (2007) Kalirin/TRIO Rho guanine nucleotide exchange factors regulate a novel step in secretory granule maturation. Mol Biol Cell 18: 4813–4825.
[13]
Portales-Casamar E, Brian？on-Marjollet A, Fromont S, Triboulet R, Debant A (2006) Identification of novel neuronal isoforms of the Rho-GEF TRIO. Biol Cell 98: 183–193.
[14]
Bellanger JM, Lazaro JB, Diriong S, Fernandez A, Lamb N (1998) The two guanine nucleotide exchange factor domains of TRIO link the Rac1 and the RhoA pathways in vivo. Oncogene 16: 147–152.
[15]
Chhatriwala MK, Betts L, Worthylake DK, Sondek J (2007) The DH and PH domains of TRIO coordinately engage Rho GTPases for their efficient activattion. J Mol Biol 368: 1307–1320.
[16]
Dyer JO, Demarco RS, Lundquist EA (2010) Distinct roles of Rac GTPases and the UNC-73/TRIO and PIX-1 Rac GTP exchange factors in neuroblast protrusion and migration in C.elegans. Small GTPases 1: 44–61.
[17]
Bateman J, Van Vactor D (2001) The TRIO family of guanine-nucleotide-exchange factors: regulators of axon guidance. J Cell Sci 114: 1973–1980.
Neubrand VE, Thomas C, Schmidt S, Debant A, Schiavo G (2010) Kidins220/ARMS regulates Rac1-dependent neurite outgrowth by direct interaction with the RhoGEF TRIO. J Cell Sci 123: 2111–2123.
[20]
Henson PM (2005) Engulfment: Ingestion and Migration with Rac, Rho and TRIO. Curr Biol 15: 29–30.
[21]
Brian？on-Marjollet A, Ghogha A, Nawabi H, Triki I, Auziol C, et al. (2008) TRIO mediates Netrin-1-induced Rac1 activation in axon outgrowth and guidance. Mol Cell Biol 28: 2314–2323.
[22]
Bouquier N, Vignal E, Charrasse S, Weill M, Schimidt S, et al. (2009) A cell active chemical GEF inhibitor selectively targets the TRIO/RhoG/Rac1 signaling pathway. Chem Biol 16: 657–666.
[23]
Ma XM, Huang JP, Eipper BA, Mains RE (2005) Expression of TRIO, a member of the Dbl family of Rho GEFs in the developing rat brain. J Comp Neurol 482: 333–348.
[24]
Schmidt S, Diriong S, Méry J, Fabbrizio E, Debant A (2002) Identification of the first Rho-GEF inhibitor, TRIPα, which targets the RhoA-specific GEF domain of TRIO. FEBS Lett 523: 35–42.
[25]
Medley QG, Serra-Pagès C, Iannotti E, Seipel K, Tang M, et al. (2000) The trio guanine nucleotide exchange factor is a RhoA target. J Biol Chem 275: 36116–36123.
[26]
Ruiz-Loredo AY, López E, López-colome AM (2011) Thrombin promotes actin stress fiber formation in RPE through Rho/ROCK-mediated MLC phosphorylation. J Cell Physiol 226: 414–423.
[27]
Xu D, Kishi H, Kawamichi H, Kajiya K, Takada Y, et al. (2012) Sphingosylphosphoryl-choline induces stress fiber formation via activation of Fyn-RhoA-ROCK signaling pathway in fibroblasts. Cell Signal 24: 282–289.
[28]
Kadi A, de Isla N, Lacolley P, Stolz JF, Menu P (2007) Potential relation between cytoskeleton reorganization and e-NOS activity in sheared endothelial cells (Effect of rate and time exposure). Clin Hemortheol Microcirc 37: 131–140.
[29]
Allen C, Srivastava K, Bayraktutan U (2010) Small GTPase RhoA and its effector rho kinase mediate oxygen glucose deprivation-evoked in vitro cerebral barrier dysfunction. Stroke 41: 2056–2063.
[30]
Laufs U, Endres M, Stagliano N, Amin-Hanjani S, Chui DS, et al. (2000) Neuroprotection mediated by changes in the endothelial actin cytoskeleton. J Clin Invest 106: 15–24.
[31]
O'Brien SP, Seipel K, Medley QG, Bronson R, Segal R, et al. (2000) Skeletal muscle deformity and neuronal disorder in TRIO exchange factor-deficient mouse embryos. Proc Natl Acad Sci USA 97: 12074–12078.
[32]
Estrach S, Schmidt S, Diriong S, Penna A, Blangy A, et al. (2002) The human Rho-GEF trio and its target GTPase RhoG are involved in the NGF pathway, leading to neurite outgrowth. Curr Biol 12: 307–312.
[33]
Amano M, Chihara K, Kimura K, Fukata Y, Nakamura N, et al. (1997) Formation of actin stress fibers and forcal adhesions enhanced by Rho-kinase. Science 28: 1308–1311.
[34]
Amano M, Ito M, Kimura K, Fukata Y, Chihara K, et al. (1996) Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem 271: 20246–20249.
[35]
Chrzanowska-Wodnicka M, Burridge K (1996) Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J Cell Biol 133: 1403–1415.
[36]
Lee JS, Kang Decker N, Chatterjee S, Yao J, Friedman S, et al. (2005) Mechanisms of nitric oxide interplay with Rho GTPase family members in modulation of actin membrane dynamics in pericytes and fibroblasts. Am J Pathol 166: 1861–1870.
[37]
Leong SK, Ruan RS, Zhang Z (2002) A critical assessment of the neurodestructive and neuroprotective effects of nitric oxide. Ann N Y Acard Sci 292: 161–181.
[38]
Aghajanian A, Wittchen ES, Campbell SL, Burridge K (2009) Direct activation of RhoA by reactive oxygen species requires a redox-sensitive motif. PLoS One 4: e8045.
[39]
Kim HJ, Kang BH, Hwang SY, Hong JY, Kim KS, Cheon DS (2012) Epidemics of viral meningitis caused by Echovirus 6 and Echovirus 30 in Korea in 2008. Virol J 9: 38.
[40]
Nix WA, Oberste MS, Pallansch MA (2006) Sensitive, seminested PCR amplification of VP1 sequences for direct identification of all enterovirus serotypes from original clinical specimens. J Clin Microbiol 44: 2698–2704.
