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JOURNAL OF VIROLOGY Dec 2011 p 12464 12473 Vol 85 No 23 0022 538X 11 12 00 doi 10 1128 JVI 05576 11 Copyright 2011 American Society for Microbiology All Rights Reserved Glutamate Excitotoxicity Is Involved in the Induction of Paralysis in Mice after Infection by a Human Coronavirus with a Single Point Mutation in Its Spike Protein H17188 Elodie Brison He le ne Jacomy Marc Desforges and Pierre J Talbot Laboratory of Neuroimmunovirology INRS Institut Armand Frappier 531 boulevard des Prairies Laval Que bec Canada H7V 1B7 Received 30 June 2011 Accepted 15 September 2011 Human coronaviruses HCoV are recognized respiratory pathogens and some strains including HCoV OC43 can infect human neuronal and glial cells of the central nervous system CNS and activate neuroin flammatory mechanisms Moreover HCoV OC43 is neuroinvasive neurotropic and neurovirulent in suscep tible mice where it induces chronic encephalitis Herein we show that a single point mutation in the viral spike S glycoprotein Y241H acquired during viral persistence in human neural cells led to a hind limb paralytic disease in infected mice Inhibition of glutamate excitotoxicity using a 2 amino 3 5 methyl 3 oxo 1 2 oxazol 4 yl propranoic acid AMPA receptor antagonist GYKI 52466 improved clinical scores related to the paralysis and motor disabilities in S mutant virus infected mice as well as protected the CNS from neuronal dysfunctions as illustrated by restoration of the phosphorylation state of neurofilaments Expression of the glial glutamate transporter GLT 1 responsible for glutamate homeostasis was downregulated following infection and GYKI 52466 also significantly restored its steady state expression level Finally GYKI 52466 treatment of S mutant virus infected mice led to reduced microglial activation which may lead to improvement in the regulation of CNS glutamate homeostasis Taken together our results strongly suggest an involvement of excitotoxicity in the paralysis associated neuropathology induced by an HCoV OC43 mutant which harbors a single point mutation in its spike protein that is acquired upon persistent virus infection Coronaviruses form a family of ubiquitous enveloped RNA viruses that induce respiratory enteric and neurological dis eases in several species 10 Human coronaviruses HCoV are respiratory pathogens responsible for upper and lower respiratory tract infections 49 and for severe acute respira tory syndrome SARS 41 Possible involvement of HCoV other than SARS CoV in more serious human pathologies was recently reviewed 49 Indeed HCoV have been associated over the years with the development of pneumonia myocardi tis and meningitis 16 39 and occasionally acute disseminated encephalitis 54 We have previously demonstrated that HCoV are neuroinvasive in humans can infect and persist in human neural cells and can activate glial cells to produce proinflammatory mediators 3 5 8 14 Moreover we have shown that wild type reference strain HCoV OC43 has neuro invasive properties in mice leading to chronic encephalitis 25 with accompanying disabilities 23 Given that murine coro navirus mouse hepatitis virus MHV a strain structurally related to HCoV OC43 can cause neurodegenerative and neuroinflammatory disease in mice and rats 10 we hypoth esized that HCoV OC43 might be associated with neuroin flammatory and or neurodegenerative human diseases We have recently reported that a viral variant with four point mutations in its surface spike S glycoprotein acquired during viral persistence in human neural cells 48 led to a drastically modified virus induced neuropathology in BALB c mice char acterized by a multiple sclerosis MS like flaccid paralysis and inflammatory demyelination 24 Glutamate is the major excitatory neurotransmitter of the central nervous system CNS that is involved in several neurophysiological functions A disruption of its homeostasis can damage neurons which may eventually lead to cell death 30 This pathological process designated excitotoxicity is able to induce degeneration of neural cells following an excessive stimulation of glutamate on its specific ionotropic receptors 2 amino 3 5 methyl 3 oxo 1 2 oxazol 4 yl propra noic acid receptor AMPAr and N methyl D aspartic acid re ceptor NMDAr 35 Activation of these receptors results in neural Ca 2H11001 influx which can mediate excitotoxicity by the means of a cascade of events involving free radical production mitochondrial dysfunction and the activation of several en zymes involved in normal cell development and function re sulting in damage to the cell membrane cytoskeleton and DNA 42 Interestingly excitotoxicity was reported to be in volved in several neurodegenerative diseases such as Alzhei mer s disease and MS in humans 13 Glutamate reuptake is necessary for the regulation of physiological extracellular glu tamate concentrations and is mediated mainly by high affinity sodium dependent transporters At least five different gluta mate transporters expressed on neuronal or glial cells GLT 1 GLAST EAAC1 EAAT4 and EAAT5 have been well char acterized 12 and up to 90 of the total glutamate reuptake in the adult CNS is achieved by the glutamate transporter 1 GLT 1 expressed mainly on astrocytes 50 In several neu rological diseases disruption of the GLT 1 expression level Corresponding author Mailing address Laboratory of Neuroim munovirology INRS Institut Armand Frappier 513 boulevard des Prairies Laval Que bec Canada H7V 1B7 Phone 450 687 5010 ext 4300 Fax 450 686 5501 E mail pierre talbot iaf inrs ca H17188 Published ahead of print on 28 September 2011 12464 on March 18 2015 by SUNY HSCB MEDICAL RESEARCH LIBRARY http jvi asm org Downloaded from was reported to be associated with alteration in glutamate uptake 52 Glutamate excitotoxicity can damage the cytoskeleton of axons in vivo which may result in the slowing of axonal trans port 1 31 Neurofilaments NF are intermediate filaments constituting the neuron cytoskeleton and are involved in the stability of mature axons and the regulation of axonal transport rate In physiological conditions the heavy neurofilament pro teins NF H are predominantly phosphorylated in axons and nonphosphorylated in neuronal soma and dendrites 19 A shift of this NF phosphorylation state which is represented by a loss of nonphosphorylated NF in soma and an increase of nonphosphorylated NF in axons is a sign of neuronal injury 44 Evaluation of the NF phosphorylation state is a useful tool to monitor progressive axonal disabilities considering that the NF H protein phosphorylation state has been shown to be a biomarker in neurodegeneration 37 We have already dem onstrated that the NF H phosphorylation state was altered following HCoV OC43 infection of mice 24 In the present study we demonstrate that only a single point mutation Y241H in the spike glycoprotein S of HCoV OC43 is sufficient for the induction of motor dysfunctions and a paralytic disease in infected mice Furthermore infection of mice with this HCoV OC43 S mutant induced a significantly strong neuronal dysfunction and a significant decrease in the expression of the glutamate transporter GLT 1 on astrocytes compared to those seen with sham and wild type virus infec tion Therefore the virus induced pathological process ap pears to be driven by a glutamate excitotoxic mechanism as blockade of the AMPA receptor attenuated clinical scores CS related to the virus induced paralysis and motor disabil ities partially restored the physiological NF phosphorylation state and GLT 1 expression and reduced microglial cell acti vation MATERIALS AND METHODS Viruses The wild type reference HCoV OC43 virus VR759 was obtained in the 1980s from the American Type Culture Collection ATCC The recombi nant virus of HCoV OC43 rOC ATCC was generated using the full length cDNA clone pBAC OC43 FL and displayed the same phenotypic properties as the wild type virus as previously described 47 This recombinant virus was used as the reference control virus for all experiments We introduced into the spike glycoprotein of HCoV OC43 one or two point mutations at a time the D24Y and S83T mutations corresponding recombinant virus designated rOC U S24 83 the H183R and Y241H mutations corresponding recombinant virus designated rOC U S183 241 the H183R mutation corresponding recombinant virus desig nated rOC U S183 or the Y241H mutation corresponding recombinant virus designated rOC U S241 mutations were introduced into the full length cDNA clone pBAC OC43 FL by site directed mutagenesis using a QuikChange Multi site directed mutagenesis kit Stratagene as recommended by the supplier Each cDNA clone was transfected into BHK 21 cells amplified by two passages in the HRT 18 cell line and sequenced to make sure that only the introduced H183R and or Y241H mutation was present and that no other mutations appeared Survival curves and clinical scores Female BALB c mice Jackson Labora tories aged 22 days postnatal dpn were inoculated by the intracerebral i c route with 10 2 5 50 tissue culture infective doses TCID 50 of recombinant virus as previously described 24 Groups of 10 mice infected by each recom binant virus were observed on a daily basis over a period of 21 days postinfection dpi and survival and clinical scores related to motor dysfunctions were eval uated Mice infected with rOC U S241 which presented motor dysfunctions were evaluated and scored according to a scale based on experimental allergic en cephalitis EAE clinical score CS evaluation 0 to 1 normal with no clinical signs 1 5 to 2 partial hind limb paralysis with a walk close to ground level 2 5 to 3 5 complete hind limb paralysis 4 to 5 moribund state and death Infectious virus assays For each experimental condition groups of three infected BALB c mice were selected randomly every 2 days and dissected to monitor infectious virus production in brains and spinal cords Tissues were processed for the presence and quantification of infectious virus by an indirect immunoperoxidase assay as previously described 27 AMPA receptor antagonist The specific noncompetitive AMPA receptor an tagonist GYKI 52466 1 4 aminophenyl 4 methyl 7 8 methylenedioxy 5H 2 3 benzodiazepine hydrochloride was obtained from Tocris Bioscience and dis solved in Hanks balanced salt solution HBSS Invitrogen at a final concentration of 300 H9262g ml of HBSS 46 To investigate the effect of treatment with GYKI 52466 two groups of 10 BALB c mice infected with rOC ATCC or rOC U S241 were treated intraperitoneally with 3 mg kg body weight of GYKI 52466 at 12 h postinfection and then twice daily for 3 weeks or with only 100 H9262l of HBSS vehicle to normalize experimental stress conditions twice daily over a period of 3 weeks To verify the noncytotoxic effect of the GYKI 52466 solution sham infected mice received the same dose of AMPA receptor antag onist As described in the literature this pharmacological dose has been reported to have no behavioral effects and to induce neuroprotective actions 20 40 Immunohistochemistry Groups of three BALB c mice either sham infected or infected with each virus and treated with GYKI 52466 antagonist or vehicle were selected randomly and perfused with a solution of 4 paraformaldehyde at 10 dpi which corresponds to the peak of viral replication in the spinal cord and the outcome of clinical scores related to paralytic disease Lumbar segments from spinal cords were cryoprotected in 30 wt vol sucrose frozen at H1100220 C and processed for sets of 8 H9262m section size with a cryostat HM 525 Microm Axonal damage was investigated by assessing the heavy neurofilament NF H phosphorylation state Tissue sections were incubated with a mouse anti non phosphorylated neurofilament monoclonal antibody MAb SMI 311 1 1 000 Covance a mouse anti phosphorylated neurofilament MAb SMI 312 1 1 000 Covance or Mac 2 rat MAb 1 200 ATCC Cedarlane for2hatroom tem perature Tissue sections were then washed and incubated with a secondary anti mouse or anti rat biotinylated antibody before being revealed with an ABC Vectastain kit Vector Laboratories as previously described 24 Double stain ing for astrocytes and glutamate transporter GLT 1 fluorescence was investi gated with the primary antibodies polyclonal rabbit anti glial fibrillary acidic protein GFAP 1 1 000 Dako and goat anti glutamate transporter GLT 1 K 16 sc 31582 1 500 Santa Cruz Biotechnology Spinal cord sections were blocked with horse serum in phosphate buffered saline PBS 1H11003 for1hatroom temperature Following incubation with both primary antibodies for2hatroom temperature sections were washed and then incubated in the dark for2hat room temperature with the secondary fluorescent antibodies Alexa Fluor 488 anti rabbit 1 1 000 Invitrogen and Alexa Fluor 568 anti goat 1 1 000 Invitro gen After final PBS 1H11003 washes tissue sections were incubated for 5 min at room temperature with 4H11032 6 diamidino 2 phenylindole DAPI 1 100 Poly sciences Inc and then mounted with Immuno mount and observed under a fluorescence microscope Protein extraction and Western blot analysis Spinal cords from groups of three mice selected randomly were homogenized in radioimmunoprecipitation assay RIPA buffer 150 mM NaCl 50 Mm Tris pH 7 4 1 vol vol NP 40 0 25 wt vol sodium deoxycholate 1 mM EDTA supplemented with the protease cocktail inhibitor catalog no P8340 Sigma Lysates were cleared by centrifugation for 5 min at 4 C at 17 000 H11003 g and supernatants were aliquoted and stored at H1100280 C A bicinchoninic acid BCA protein assay kit Novagen was used to determine protein concentration according to the manufacturer s protocol Proteins 10 H9262g per sample were separated on a Novex NuPage 4 to 12 gradient gel Invitrogen and transferred to a polyvinylidene difluoride PVDF membrane Immobilon P transfer membrane Millipore with a Bio Rad Trans Blot semidry transfer cell apparatus Membranes were blocked with Tris buffered saline containing 1 vol vol Tween TBS T and 5 wt vol nonfat milk at 4 C overnight and then membranes were incubated with poly clonal rabbit anti glial fibrillary acidic protein GFAP 1 2 000 Dako guinea pig anti glutamate transporter GLT 1 MAb AB1783 1 1 000 Millipore Mac 2 rat MAb 1 100 ATCC Cedarlane or rabbit anti GAPDH glyceraldehyde 3 phosphate dehydrogenase 1 1 000 Santa Cruz Biotechnology for1hatroom temperature Membranes were washed three times with TBS T and then incu bated for1hatroom temperature with secondary antibody anti rabbit 1 1 000 GE Healthcare UK anti guinea pig 1 1 000 Millipore or anti rat 1 1 000 Kirkegaard detection was made by chemiluminescence using a Bio Rad Immun Star HRP substrate kit Band detection and semiquantification were made using the GeneSnap software from a Chemi Genius Syngene apparatus Analysis of vari ance ANOVA tests followed by post hoc Tukey s analysis were performed to VOL 85 2011 HCoV OC43 INDUCED PARALYSIS MEDIATED BY EXCITOTOXICITY 12465 on March 18 2015 by SUNY HSCB MEDICAL RESEARCH LIBRARY http jvi asm org Downloaded from determine the statistical significances in the differences in protein expression between different groups of mice using SPSS software version 16 0 RESULTS BALB c mice infected with HCoV OC43 containing the Y241H mutation in the spike glycoprotein rOC U S241 de velop a hind limb paralytic disease We recently showed that a viral variant bearing four point mutations in the spike S glycoprotein D24Y S83T H183R and Y241H acquired during viral persistence in human neural cells 48 led to a modified virus induced neuropathology in BALB c mice com pared to that caused by the reference HCoV OC43 rOC ATCC virus This modified pathology was characterized by an MS like flaccid paralysis with areas of demyelination in the spinal cord whereas mice infected by HCoV OC43 rOC ATCC exhibited only encephalitis 24 In order to further pinpoint the viral molecular determinants responsible for this modulation of virus induced neuropathogenesis we generated recombinant viruses that contained two S mutations at a time D24Y and S83T designated rOC U S24 83 or H183R and Y241H designated rOC U S183 241 Fig 1A Like in infection with virus containing the four mutations described above in fection of BALB c mice by rOC U S183 241 led to a paralytic disease with small areas of demyelination in the spinal cord whereas rOC U S24 83 induced encephalitis like rOC ATCC In order to investigate whether only one of the remaining muta tions either H183R or Y241H was sufficient to induce the paralytic disease in mice we generated recombinant viruses that harbored only one mutation at a time either H183R designated rOC U S183 or Y241H designated rOC U S241 Fig 1A Whereas BALB c mice infected with rOC U S183 developed an encephalitis similar to what was observed after FIG 1 Schematic representation of the point mutations introduced within the viral spike glycoprotein and associated neurovirulence of the different recombinant viruses in mice A Schematic representation of the main structural domains of the S protein of HCoV OC43 and the approximate locations of the four point mutations D24Y S83T H183R and Y241H These mutations were introduced into the viral genome to generate rOC U S24 241 24 Alternatively only pairs of mutations were introduced to generate rOC U S24 83 and rOC U S183 241 Finally one single mutation was introduced at a time to generate rOC U S183 and rOC U S241 Asterisks indicate recombinant viruses which induced a paralytic disease following infection RBD putative cell receptor binding domain HVR hypervariable region HR heptad repeat region aa amino acids B Survival curves following intracerebral inoculation of the different HCoV OC43 recombinants harboring various mutations in the S protein compared to that following inoculation with reference rOC ATCC virus Whereas infection of mice with the reference virus rOC ATCC induced 15 mortality mice infected with rOC U S24 241 showed a mortality rate of 80 The relative survival rates of mice infected by different viruses containing one two or four mutations in the spike protein suggest a synergic effect of these mutations on mortality rate Results are representative of three independent experiments 12466 BRISON ET AL J VIROL on March 18 2015 by SUNY HSCB MEDICAL RESEARCH LIBRARY http jvi asm org Downloaded from rOC ATCC infection mice infected with rOC U S241 presented motor dysfunctions and paralytic disease Neurovirulence of all recombinant viruses was evaluated following intracerebral in oculation of BALB c mice Fig 1B The survival curves of mice infected by the recombinant viruses bearing one two or four point mutations within the viral spike glycoprotein sug gested a synergistic effect of these mutations on the mortality rate Histological examination of infected CNS cells revealed that the primary target cell of the infection by all of these recombinant viruses rOC U S183 241 rOC U S183 and rOC U S241 remained the neurons data not shown as previously described for rOC U S24 241 and rOC ATCC 24 The rOC U S241 recombinant virus was associated with motor dysfunc tions in infected mice Therefore we conclude that the Y241H single mutation was necessary and sufficient to generate the observed paralytic phenotype and focused the rest of the study on this recombinant virus to evaluate the involvement of this single Y241H mutation in paralytic disease in comparison to the reference virus rOC ATCC To determine whether the difference in pathology induced by rOC U S241 and the reference virus rOC ATCC could be related to differences in kinetics of replication in the CNS brains and spinal cords were harvested and infectious virus titers were assayed every 2 days for a period of 22 dpi Even though both viruses replicated to similar extents in the CNS and the highest level of infectious virions were found at 10 dpi in brains as well as in spinal cords rOC U S241 remained in the spinal cord for a longer period of time than rOC ATCC In deed infectious rOC U S241 virus was still detectable at 15