Alphaviruses, including Venezuelan Equine Encephalitis Virus (VEEV), cause disease in both equine and humans that exhibit overt encephalitis in a significant percentage of cases. Features of the host immune response and tissue-specific responses may contribute to fatal outcomes as well as the development of encephalitis. It has previously been shown that VEEV infection of mice induces transcription of pro-inflammatory cytokines genes (e.g., IFN-γ, IL-6, IL-12, iNOS and TNF-α) within 6 h. GSK-3β is a host protein that is known to modulate pro-inflammatory gene expression and has been a therapeutic target in neurodegenerative disorders such as Alzheimer's. Hence inhibition of GSK-3β in the context of encephalitic viral infections has been useful in a neuroprotective capacity. Small molecule GSK-3β inhibitors and GSK-3β siRNA experiments indicated that GSK-3β was important for VEEV replication. Thirty-eight second generation BIO derivatives were tested and BIOder was found to be the most potent inhibitor, with an IC50 of ~0.5 μM and a CC50 of >100 μM. BIOder was a more potent inhibitor of GSK-3β than BIO, as demonstrated through in vitro kinase assays from uninfected and infected cells. Size exclusion chromatography experiments demonstrated that GSK-3β is found in three distinct complexes in VEEV infected cells, whereas GSK-3β is only present in one complex in uninfected cells. Cells treated with BIOder demonstrated an increase in the anti-apoptotic gene, survivin, and a decrease in the pro-apoptotic gene, BID, suggesting that modulation of pro- and anti-apoptotic genes contributes to the protective effect of BIOder treatment. Finally, BIOder partially protected mice from VEEV induced mortality. Our studies demonstrate the utility of GSK-3β inhibitors for modulating VEEV infection.
References
[1]
Olival KJ, Daszak P (2005) The ecology of emerging neurotropic viruses. Journal of Neurovirology 11: 441–446.
[2]
Gubler DJ (2002) The global emergence/resurgence of arboviral diseases as public health problems. Archives of Medical Research 33: 330–342.
[3]
Zacks MA, Paessler S (2010) Encephalitic alphaviruses. Vet Microbiol 140: 281–286.
[4]
Weaver SC, Salas R, RicoHesse R, Ludwig GV, Oberste MS, et al. (1996) Re-emergence of epidemic Venezuelan equine encephalomyelitis in South America. Lancet 348: 436–440.
Leon CA (1975) Sequelae of Venezuelan equine encephalitis in humans: a four year follow-up. Int J Epidemiol 4: 131–140.
[8]
Steele KE, Twenhafel NA (2010) Pathology of Animal Models of Alphavirus Encephalitis. Veterinary Pathology 47: 790–805.
[9]
Reichert E, Clase A, Bacetty A, Larsen J (2009) Alphavirus antiviral drug development: scientific gap analysis and prospective research areas. Biosecur Bioterror 7: 413–427.
[10]
Strauss JH, Strauss EG (1994) The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 58: 491–562.
[11]
Shirako Y, Strauss JH (1994) Regulation of Sindbis Virus-Rna Replication - Uncleaved P123 and Nsp4 Function in Minus-Strand Rna-Synthesis, Whereas Cleaved Products from P123 Are Required for Efficient Plus-Strand Rna-Synthesis. Journal of Virology 68: 1874–1885.
[12]
Rikkonen M, Peranen J, Kaariainen L (1994) Atpase and Gtpase Activities Associated with Semliki Forest Virus Nonstructural Protein Nsp2. Journal of Virology 68: 5804–5810.
[13]
de Cedron MG, Ehsani N, Mikkola ML, Garcia JA, Kaariainen L (1999) RNA helicase activity of Semliki Forest virus replicase protein NSP2. FEBS Letters 448: 19–22.
[14]
Rubach JK, Wasik BR, Rupp JC, Kuhn RJ, Hardy RW, et al. (2009) Characterization of purified Sindbis virus nsP4 RNA-dependent RNA polymerase activity in vitro. Virology 384: 201–208.
[15]
Thal MA, Wasik BR, Posto J, Hardy RW (2007) Template requirements for recognition and copying by Sindbis virus RNA-dependent RNA polymerase. Virology 358: 221–232.
[16]
Garmashova N, Atasheva S, Kang W, Weaver SC, Frolova E, et al. (2007) Analysis of Venezuelan equine encephalitis virus capsid protein function in the inhibition of cellular transcription. J Virol 81: 13552–13565.
[17]
Atasheva S, Garmashova N, Frolov I, Frolova E (2008) Venezuelan equine encephalitis virus capsid protein inhibits nuclear import in mammalian but not in mosquito cells. Journal of Virology 82: 4028–4041.
[18]
Atasheva S, Fish A, Fornerod M, Frolova EI (2010) Venezuelan Equine Encephalitis Virus Capsid Protein Forms a Tetrameric Complex with CRM1 and Importin alpha/beta That Obstructs Nuclear Pore Complex Function. Journal of Virology 84: 4158–4171.
[19]
Griffin D, Levine B, Tyor W, Ubol S, Despres P (1997) The role of antibody in recovery from alphavirus encephalitis. Immunol Rev 159: 155–161.
[20]
Muehlenbein MP, Cogswell FB, James MA, Koterski J, Ludwig GV (2006) Testosterone correlates with Venezuelan equine encephalitis virus infection in macaques. Virol J 3: 19.
[21]
Schoneboom BA, Catlin KM, Marty AM, Grieder FB (2000) Inflammation is a component of neurodegeneration in response to Venezuelan equine encephalitis virus infection in mice. J Neuroimmunol 109: 132–146.
[22]
Schoneboom BA, Lee JS, Grieder FB (2000) Early expression of IFN-alpha/beta and iNOS in the brains of Venezuelan equine encephalitis virus-infected mice. J Interferon Cytokine Res 20: 205–215.
[23]
Martin M, Rehani K, Jope RS, Michalek SM (2005) Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nat Immunol 6: 777–784.
[24]
Frame S, Cohen P (2001) GSK3 takes centre stage more than 20 years after its discovery. Biochem J 359: 1–16.
[25]
Sutherland C (2011) What Are the bona fide GSK3 Substrates? Int J Alzheimers Dis 2011: 505607.
[26]
Dolan PJ, Johnson GV (2010) The role of tau kinases in Alzheimer's disease. Curr Opin Drug Discov Devel 13: 595–603.
[27]
Hanger DP, Anderton BH, Noble W (2009) Tau phosphorylation: the therapeutic challenge for neurodegenerative disease. Trends in Molecular Medicine 15: 112–119.
[28]
Wang H, Brown J, Gu Z, Garcia CA, Liang R, et al. (2011) Convergence of the mammalian target of rapamycin complex 1- and glycogen synthase kinase 3-beta-signaling pathways regulates the innate inflammatory response. J Immunol 186: 5217–5226.
[29]
Wang H, Brown J, Martin M (2011) Glycogen synthase kinase 3: a point of convergence for the host inflammatory response. Cytokine 53: 130–140.
[30]
Huang WC, Lin YS, Wang CY, Tsai CC, Tseng HC, et al. (2009) Glycogen synthase kinase-3 negatively regulates anti-inflammatory interleukin-10 for lipopolysaccharide-induced iNOS/NO biosynthesis and RANTES production in microglial cells. Immunology 128: e275–286.
[31]
Cross DA, Culbert AA, Chalmers KA, Facci L, Skaper SD, et al. (2001) Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J Neurochem 77: 94–102.
[32]
Kehn-Hall K, Guendel I, Carpio L, Skaltsounis L, Meijer L, et al. (2011) Inhibition of Tat-mediated HIV-1 replication and neurotoxicity by novel GSK3-beta inhibitors. Virology 415: 56–68.
[33]
Konig R, Stertz S, Zhou Y, Inoue A, Hoffmann HH, et al. (2010) Human host factors required for influenza virus replication. Nature 463: 813–817.
[34]
Berge TO, Banks IS, Tigertt WD (1961) Attenuation of Venezuelan equine encephalomyelitis virus by in vitro cultivation in guinea-pig heart cells. American Journal of Hygiene 73: 209–218.
[35]
Kinney RM, Chang GJ, Tsuchiya KR, Sneider JM, Roehrig JT, et al. (1993) Attenuation of Venezuelan equine encephalitis virus strain TC-83 is encoded by the 5′-noncoding region and the E2 envelope glycoprotein. J Virol 67: 1269–1277.
[36]
Julander JG, Skirpstunas R, Siddharthan V, Shafer K, Hoopes JD, et al. (2008) C3H/HeN mouse model for the evaluation of antiviral agents for the treatment of Venezuelan equine encephalitis virus infection. Antiviral Res 78: 230–241.
[37]
Meijer L, Skaltsounis AL, Magiatis P, Polychronopoulos P, Knockaert M, et al. (2003) GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol 10: 1255–1266.
[38]
MacAulay K, Hajduch E, Blair AS, Coghlan MP, Smith SA, et al. (2003) Use of lithium and SB-415286 to explore the role of glycogen synthase kinase-3 in the regulation of glucose transport and glycogen synthase. Eur J Biochem 270: 3829–3838.
[39]
Coghlan MP, Culbert AA, Cross DA, Corcoran SL, Yates JW, et al. (2000) Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol 7: 793–803.
[40]
Li M, Wang X, Meintzer MK, Laessig T, Birnbaum MJ, et al. (2000) Cyclic AMP promotes neuronal survival by phosphorylation of glycogen synthase kinase 3beta. Mol Cell Biol 20: 9356–9363.
[41]
Chin PC, Majdzadeh N, D'Mello SR (2005) Inhibition of GSK3beta is a common event in neuroprotection by different survival factors. Brain Res Mol Brain Res 137: 193–201.
[42]
Maurer MH, Bromme JO, Feldmann RE Jr, Jarve A, Sabouri F, et al. (2007) Glycogen synthase kinase 3beta (GSK3beta) regulates differentiation and proliferation in neural stem cells from the rat subventricular zone. J Proteome Res 6: 1198–1208.
[43]
Magiatis P, Polychronopoulos P, Skaltsounis AL, Lozach O, Meijer L, et al. (2010) Indirubins deplete striatal monoamines in the Intact and MPTP-treated mouse brain and block kainate-induced striatal astrogliosis. Neurotoxicol Teratol 32: 212–219.
[44]
Jackson AC, SenGupta SK, Smith JF (1991) Pathogenesis of Venezuelan equine encephalitis virus infection in mice and hamsters. Veterinary Pathology 28: 410–418.
[45]
Charles PC, Walters E, Margolis F, Johnston RE (1995) Mechanism of neuroinvasion of Venezuelan equine encephalitis virus in the mouse. Virology 208: 662–671.
[46]
Charles PC, Trgovcich J, Davis NL, Johnston RE (2001) Immunopathogenesis and immune modulation of Venezuelan equine encephalitis virus-induced disease in the mouse. Virology 284: 190–202.
[47]
Ryzhikov AB, Ryabchikova EI, Sergeev AN, Tkacheva NV (1995) Spread of Venezuelan equine encephalitis virus in mice olfactory tract. Arch Virol 140: 2243–2254.
[48]
Jackson AC, Rossiter JP (1997) Apoptotic cell death is an important cause of neuronal injury in experimental Venezuelan equine encephalitis virus infection of mice. Acta Neuropathologica 93: 349–353.
[49]
Steele KE, Davis KJ, Stephan K, Kell W, Vogel P, et al. (1998) Comparative neurovirulence and tissue tropism of wild-type and attenuated strains of Venezuelan equine encephalitis virus administered by aerosol in C3H/HeN and BALB/c mice. Veterinary Pathology 35: 386–397.
[50]
Schoneboom BA, Catlin KMK, Marty AM, Grieder FB (2000) Inflammation is a component of neurodegeneration in response to Venezuelan equine encephalitis virus infection in mice. Journal of Neuroimmunology 109: 132–146.
[51]
Gould TD, Picchini AM, Einat H, Manji HK (2006) Targeting glycogen synthase kinase-3 in the CNS: implications for the development of new treatments for mood disorders. Curr Drug Targets 7: 1399–1409.
[52]
Beurel E, Jope RS (2006) The paradoxical pro- and anti-apoptotic actions of GSK3 in the intrinsic and extrinsic apoptosis signaling pathways. Prog Neurobiol 79: 173–189.
[53]
Maurer U, Charvet C, Wagman AS, Dejardin E, Green DR (2006) Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1. Mol Cell 21: 749–760.
[54]
Ding Q, He X, Hsu JM, Xia W, Chen CT, et al. (2007) Degradation of Mcl-1 by beta-TrCP mediates glycogen synthase kinase 3-induced tumor suppression and chemosensitization. Mol Cell Biol 27: 4006–4017.
[55]
Morel C, Carlson SM, White FM, Davis RJ (2009) Mcl-1 integrates the opposing actions of signaling pathways that mediate survival and apoptosis. Mol Cell Biol 29: 3845–3852.
[56]
Charvet C, Wissler M, Brauns-Schubert P, Wang SJ, Tang Y, et al. (2011) Phosphorylation of Tip60 by GSK-3 determines the induction of PUMA and apoptosis by p53. Mol Cell 42: 584–596.
[57]
Beyaert R, Vanhaesebroeck B, Suffys P, Van Roy F, Fiers W (1989) Lithium chloride potentiates tumor necrosis factor-mediated cytotoxicity in vitro and in vivo. Proc Natl Acad Sci U S A 86: 9494–9498.
[58]
Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, et al. (2000) Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation. Nature 406: 86–90.
[59]
Schafer A, Whitmore AC, Konopka JL, Johnston RE (2009) Replicon particles of Venezuelan equine encephalitis virus as a reductionist murine model for encephalitis. J Virol 83: 4275–4286.
[60]
Schafer A, Brooke CB, Whitmore AC, Johnston RE (2011) The Role of the Blood-Brain Barrier during Venezuelan Equine Encephalitis Virus Infection. J Virol 85: 10682–10690.
[61]
Stamatovic SM, Shakui P, Keep RF, Moore BB, Kunkel SL, et al. (2005) Monocyte chemoattractant protein-1 regulation of blood-brain barrier permeability. J Cereb Blood Flow Metab 25: 593–606.
[62]
Eugenin EA, Berman JW (2003) Chemokine-dependent mechanisms of leukocyte trafficking across a model of the blood-brain barrier. Methods 29: 351–361.
[63]
Rosenberg GA (2002) Matrix metalloproteinases and neuroinflammation in multiple sclerosis. Neuroscientist 8: 586–595.
[64]
Agrawal S, Anderson P, Durbeej M, van Rooijen N, Ivars F, et al. (2006) Dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation in experimental autoimmune encephalomyelitis. J Exp Med 203: 1007–1019.
[65]
Ramirez SH, Fan S, Zhang M, Papugani A, Reichenbach N, et al. (2010) Inhibition of glycogen synthase kinase 3beta (GSK3beta) decreases inflammatory responses in brain endothelial cells. Am J Pathol 176: 881–892.
[66]
Beurel E, Jope R (2011) Linking Depression to Inflammation through Dysregulated Glycogen Synthase Kinase-3 (GSK3). Biological Psychiatry 69: 34s–34s.
[67]
Wang HZ, Brown J, Martin M (2011) Glycogen synthase kinase 3: A point of convergence for the host inflammatory response. Cytokine 53: 130–140.
[68]
Amanchy R, Periaswamy B, Mathivanan S, Reddy R, Tattikota SG, et al. (2007) A curated compendium of phosphorylation motifs. Nature Biotechnology 25: 285–286.
[69]
Kulasegaran-Shylini R, Thiviyanathan V, Gorenstein DG, Frolov I (2009) The 5′UTR-specific mutation in VEEV TC-83 genome has a strong effect on RNA replication and subgenomic RNA synthesis, but not on translation of the encoded proteins. Virology 387: 211–221.
[70]
Pardigon N, Lenches E, Strauss JH (1993) Multiple binding sites for cellular proteins in the 3′ end of Sindbis alphavirus minus-sense RNA. J Virol 67: 5003–5011.
[71]
Ludwig GV, Turell MJ, Vogel P, Kondig JP, Kell WK, et al. (2001) Comparative neurovirulence of attenuated and non-attenuated strains of Venezuelan equine encephalitis virus in mice. Am J Trop Med Hyg 64: 49–55.
