Nonstructural Protein {sigma}1s Is a Determinant of Reovirus Virulence and Influences the Kinetics and Severity of Apoptosis Induction in the Heart and Central Nervous System

The mechanisms by which viruses kill susceptible cells in targetorgans and ultimately produce disease in the infected host remainpoorly understood. Dependent upon the site of inoculation andstrain of virus, experimental infection of neonatal mice withreoviruses can induce fatal encephalitis or myocarditis. Reovirus-inducedapoptosis is a major mechanism of tissue injury, leading todisease development in both the brain and heart. In culturedcells, differences in the capacity of reovirus strains to induceapoptosis are determined by the S1 gene segment, which alsoplays a major role as a determinant of viral pathogenesis inboth the heart and the central nervous system (CNS) in vivo.The S1 gene is bicistronic, encoding both the viral attachmentprotein sigma-1 and the nonstructural protein sigma-1-small( ${sigma}$ 1s). Although ${sigma}$ 1s is dispensable for viral replication in vitro,we wished to investigate the expression of ${sigma}$ 1s in the infectedheart and brain and its potential role in reovirus pathogenesisin vivo. Two-day-old mice were inoculated intramuscularly orintracerebrally with either ${sigma}$ 1s^– or ${sigma}$ 1s⁺ reovirus strains.While viral replication in target organs did not differ between ${sigma}$ 1s^– and ${sigma}$ 1s⁺ viral strains, virus-induced caspase-3 activationand resultant histological tissue injury in both the heart andbrain were significantly reduced in ${sigma}$ 1s^– reovirus-infectedanimals. These results demonstrate that ${sigma}$ 1s is a determinantof the magnitude and extent of reovirus-induced apoptosis inboth the heart and CNS and thereby contributes to reovirus pathogenesisand virulence.

INTRODUCTION

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Introduction

Experimental infection of neonatal mice with reovirus has providedimportant new insights into the mechanisms of viral entry, spread,tissue tropism, and disease pathogenesis in the infected host.Reoviruses infect and produce tissue injury in multiple organsystems, including the heart and central nervous system (CNS)(28, 31). Reovirus-induced apoptosis is a major mechanism ofcell death, tissue injury, and ensuing disease in infected neonatalmice (8, 9, 19, 24, 25). In both the infected heart and brain,reovirus antigen, areas of histological injury, and apoptosisall colocalize (8, 19, 25). In the brain, type 3 (T3) reovirusescause fatal encephalitis associated with neuronal injury andvirus-induced apoptosis in neurons of the cortex, thalamus,and hippocampus (19, 24, 25). The viral S1 gene is a key determinantof the pattern of viral injury in the CNS (37, 38), althoughother viral genes play contributory roles as well (14). In theheart, reovirus-induced apoptosis appears to be the major mechanismfor virus-induced myocardial injury and consequential death(8, 9). Reovirus genetics has identified multiple viral genes,including M1, L1, L2, and S1, as determinants of reovirus-inducedacute myocarditis (28). S1 has been implicated as a major determinantof cytopathic effects in cardiomyocytes (3). The S1 gene isalso a major determinant of differences in the capacity of reovirusstrains to induce apoptosis in a variety of cultured cells (33,34).

The S1 double-stranded RNA gene segment is bicistronic, encodingboth the viral attachment protein sigma-1 ( ${sigma}$ 1) and the nonstructuralprotein sigma-1-small ( ${sigma}$ 1s) from overlapping but out-of-sequenceopen reading frames (11, 15, 27). ${sigma}$ 1 is a structural proteinlocated at the icosahedral vertices of the viral outer capsidand functions as the viral cell attachment protein (17). ${sigma}$ 1 mayinfluence virulence through its role in host cell receptor bindingto mediate viral entry or through receptor-triggered intracellularsignaling pathways (6, 20, 32). The second S1-encoded protein, ${sigma}$ 1s, is a nonstructural protein and is a key determinant of thecapacity of reoviruses to induce a G₂/M cell cycle arrest ininfected cells (22, 23). We have recently shown that ${sigma}$ 1s containsa functional nuclear localization signal and undergoes activesignal-mediated nuclear import in both transfected and infectedcells, resulting in profound disruption of nuclear architecture(13). Although a ${sigma}$ 1s-null mutant replicates as well as wild-typeT3 reovirus in cultured L929 and MDCK cells (26), the fact that ${sigma}$ 1s is conserved in all known reovirus field isolates (5, 10)suggests that it plays an important role during viral pathogenesisin vivo. In order to evaluate the potential role of ${sigma}$ 1s duringpathogenesis, we compared heart and CNS disease following infectionof neonatal mice with a ${sigma}$ 1s-null reovirus mutant (C84-MA) tothat following infection with ${sigma}$ 1s-positive control strains.

We show that a ${sigma}$ 1s deficiency does not affect viral growth intarget tissues but profoundly reduces the capacity of reovirusto induce apoptosis and tissue injury in both the heart andthe CNS. These studies indicate that although ${sigma}$ 1s may not berequired for viral replication in cultured cells, it plays amajor role in viral virulence and disease outcome in vivo.

MATERIALS AND METHODS

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Materials and Methods

Mice. Swiss-Webster mouse litters were housed in individual filter-toppedcages in an American Association for Laboratory Animal Care-accreditedanimal facility. All animal procedures were performed underprotocols approved by the appropriate institutional animal careand use committees.

Mouse inoculations. Two-day-old Swiss-Webster mice (Harlan Sprague-Dawley, Indianapolis,Ind.) were inoculated either intramuscularly (hind limb) orintracranially (right cerebral hemisphere) with 10⁵ or 10³ PFU(as specified) of the indicated reovirus strain in a 20-µl(intramuscular inoculation) or 10-µl (intracranial inoculation)volume of gel saline (137 mM NaCl, 0.2 mM CaCl₂, 0.8 mM MgCl₂,19 mM H₃BO₃, 0.1 mM Na₂B₄O₇, 0.3% gelatin).

Viruses. The ${sigma}$ 1s and ${sigma}$ 1 protein sequences of the viruses used are diagrammedin Fig. 1. All viruses utilized were from laboratory stocks.T3C84 and T3C84-MA were originally provided by T. Dermody (VanderbiltUniversity, Nashville, Tenn.). T3 C84-MA is a ${sigma}$ 1s-null mutantthat was originally isolated following serial passage of theT3C84 (C84) strain through murine erythroleukemia cells (26).C84-MA contains a tryptophan (W)-to-arginine (R) mutation atposition 202 which enables it to bind terminal sialic acid (SA)on glycosylated cellular proteins (SA⁺), whereas C84 cannot(SA^–) (Fig. 1). C84-MA also contains an S1 gene mutationwhich results in a lysine (K)-to-isoleucine (I) substitutionat amino acid 26 in ${sigma}$ 1 (Fig. 1). This mutation also introducesa premature stop codon in ${sigma}$ 1s. Since SA binding plays a significantrole in reovirus pathogenesis (1, 2, 7), we used reovirus T3clone 93 (C93) and the prototype T3 reovirus strain Abney (T3A),both of which bind SA, as additional controls for C84-MA. C93expresses ${sigma}$ 1s, binds SA coreceptors, and is the most closelyrelated SA⁺ field isolate to C84-MA in terms of its ${sigma}$ 1 aminoacid sequence. T3A expresses ${sigma}$ 1s and binds to SA residues ontarget cells (Fig. 1).

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FIG. 1. ${sigma}$ 1s^– and ${sigma}$ 1s⁺ T3 reoviruses used in this study. Shown are representations of the 120-amino-acid ${sigma}$ 1s protein sequence (stippled bars) and the 455-amino-acid ${sigma}$ 1 protein sequence (white bars) for each viral strain used. C84-MA does not express ${sigma}$ 1s due to an S1 gene mutation (black square). C84-MA, C93, and T3A bind SA (black triangles). C84 does not bind SA due to a mutation in ${sigma}$ 1 (open triangle). C93 and T3A contain differences in the ${sigma}$ 1 protein sequence compared to C84-MA (dotted boxes). Noted to the right is the ${sigma}$ 1s expression status and SA binding ability of each strain.

Viral titer. Mice were sacrificed at the indicated times postinfection, andwhole hearts or brains were placed in gel saline and immediatelyfrozen at –70°C. After three freeze (–70°C)-thaw(37°C) cycles, tissues were sonicated for 30 s with a microtipprobe (Heat Systems model XL2020) until a homogenate solutionwas formed. Viral suspensions were serially diluted in gel salinein 10-fold steps. Monolayers of L929 cells were infected withdiluted virus in duplicate for plaque assay as previously described(36).

Histological analysis. Mice were sacrificed at the indicated times postinfection, andindividual organs were immediately immersed in 10% bufferedformalin solution for 24 h and then transferred to 70% ethanolfor histological preparation. For the brain, paraffin-embeddedtissues were cut to 4-µm coronal sections, showing cingulatecortex, frontoparietal cortex, hippocampus, and thalamus. Sectionswere stained with hematoxylin and eosin (H&E) for analysisof tissue pathology and quantitative scoring. Scoring of theextent of neuropathologic damage was based on a previously validatedblinded grading system in which the extent of morphologicalchanges and the area affected within specific brain regionswere graded on a scale of 0 to 4 (30) (Table 1). For the heart,paraffin-embedded tissues were transversely cut to 4-µmsections. Quantitative scoring of histological injury in theheart was based on a previously validated blinded grading systemin which the number and size of specific heart lesions wereevaluated on a scale of 0 to 4 (8, 29) (Table 1). Histologicalinjury in both the heart and brain was evaluated and scoredfrom either a single coronal H&E-stained section per mouseor a single H&E-stained transverse heart section per mousefrom multiple animals. For caspase-3 immunohistochemistry, 4-µmparaffin-embedded tissue sections were retrieved in a decloaker(BioCare, Vista, Calif.) in citrate buffer, followed by stainingwith rabbit anti-mouse polyclonal antibody directed againstactivated caspase-3 (Cell Signaling Technologies, Beverly, Mass.)at a 1:25 dilution, followed by detection by using an automatedstaining system with the Ventana (Tucson Ariz.) detection system.Quantitative scoring of caspase-3-stained tissue sections wasbased on a blinded grading system in which the number of caspase-3-positivecells and the size of caspase-3-positive areas were graded ona scale of 0 to 3 (Table 1). For ${sigma}$ 1s immunohistochemistry inthe brain, 4-µm paraffin-embedded coronal tissue sectionswere deparaffinized and retrieved in antigen-unmasking solution(Vector Laboratories, Burlingame, Calif.) according to the high-temperatureunmasking technique provided by the manufacturer. After an overnightpermeabilization with Neuropore at 4°C (Trevigen, Gaithersburg,Md.), goat anti-mouse monoclonal antibody directed against ${sigma}$ 1s(3E2) was used at 200 µg/ml diluted into 3% bovine serumalbumin (BSA)-Tris-buffered saline (TBS) plus 0.1% Tween (TBST)overnight at 4°C. As a secondary antibody, biotinylatedgoat anti-mouse antibody (Vector Laboratories) was used at 1:100in 3% BSA-TBST for 2 h at 25°C. Detection was monitoredby a diaminobenzidine tetrahydrochloride-based immunohistochemistryprotocol according to the suggestions of the manufacturer (VectorLaboratories). Dehydration was carried out in a series of gradedethanol solutions, followed by clarification in xylene. Slideswere mounted with Vectamount (Vector Laboratories) and storedat 25°C. A similar procedure was used to evaluate ${sigma}$ 1s expressionin cardiac tissue sections, with the following exceptions: monoclonalantibody 2F4 was used as the primary antibody at a concentrationof 100 µg/ml, diluted into 3% BSA-TBST, overnight at 4°C.The hybridoma cell lines synthesizing the anti- ${sigma}$ 1s antibodies,2F4 and 3E2, were generous gifts (26). Anti- ${sigma}$ 1s antibodies werepurified on protein A columns based on the recommended protocoldescribed by the manufacturer (Pierce, Rockford, Ill.). Forreovirus antigen detection (reovirus structural proteins), deparaffinizedtissue sections were retrieved in antigen-unmasking solution(Vector Laboratories) according to the high-temperature unmaskingtechnique provided by the manufacturer and permeabilized inNeuropore (Trevigen) at 4°C overnight. Rabbit polyclonalreovirus antiserum was used at 1:200 in 5% normal goat serum-TBSTat 25°C for 1 h. Extensive washing in TBST was followedby incubation in fluorescein isothiocyanate-conjugated goatanti-rabbit immunoglobulin G (Vector Laboratories) diluted at1:100 in 3% BSA-TBST. After extensive washing in TBST and TBS,nuclei were visualized with Hoechst 33342 (Molecular Probes,Eugene, Oreg.). Stained sections were mounted with Vectashield(Vector Laboratories) and stored at 4°C.

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TABLE 1. Tissue section grading systems for apoptosis and histopathological damage in ${sigma}$ 1s^– and ${sigma}$ 1s⁺ reovirus-infected animals

Statistics. All statistical analyses were performed with In Stat version3.0 (Graph Pad, San Diego, Calif.).

RESULTS

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Results

${sigma}$ 1s enhances reovirus virulence following intramuscular inoculation. In order to evaluate the role of ${sigma}$ 1s in the induction of reovirus-inducedmyocarditis, 2-day-old neonatal mice were inoculated intramuscularlywith 10⁵ PFU of reovirus strain C84-MA ( ${sigma}$ 1s^– SA⁺), C93( ${sigma}$ 1s⁺ SA⁺), T3A ( ${sigma}$ 1s⁺ SA⁺), or C84 ( ${sigma}$ 1s⁺ SA^–). Infected animalswere examined daily to assess health and progression of disease(Fig. 2). All C84-MA ( ${sigma}$ 1s^– SA⁺)-infected mice survivedvirus challenge, whereas all mice infected with the controlT3A ( ${sigma}$ 1s⁺ SA⁺) or C93 ( ${sigma}$ 1s⁺ SA⁺) strain appeared to be sick atapproximately 6 days postinfection and died within 14 days ofinfection (mean day of death ± standard error of themean, 9.9 ± 1.2 days or 10.4 ± 0.5, respectively)(Fig. 2). The ${sigma}$ 1s⁺ SA^– strain C84 showed an intermediatephenotype, with 30% mortality (Fig. 2). The observed differencesin patterns of lethality between ${sigma}$ 1s^– and ${sigma}$ 1s⁺ strains indicatedthat ${sigma}$ 1s was an important determinant of virulence followingperipheral intramuscular inoculation. The intermediate phenotypeof the SA^– strain C84 indicated that in addition to thepresence or absence of ${sigma}$ 1s, SA binding is likely to be an importantdeterminant of reovirus virulence following intramuscular inoculation.

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FIG. 2. ${sigma}$ 1s confers enhanced virulence following intramuscular reovirus inoculation. Neonatal mice (n = 7 to 16) were inoculated intramuscularly with C84-MA ( ${sigma}$ 1s^– SA⁺), C93 ( ${sigma}$ 1s⁺ SA⁺), T3A ( ${sigma}$ 1s⁺ SA⁺), or C84 ( ${sigma}$ 1s⁺ SA^–).

${sigma}$ 1s is not required for viral replication in the heart. Following intramuscular inoculation of either ${sigma}$ 1s^– or ${sigma}$ 1s⁺reovirus strains, the viral titer in the hearts of infectedanimals was determined at 8 days postinfection. There were nosignificant differences in titer observed between virus strainsin the heart (P > 0.05 for all interstrain comparisons) (Fig.3). ${sigma}$ 1s was therefore not required for either efficient spreadto or growth within the heart. Similarly, no significant differencesin titer were observed between SA⁺ and SA^– viruses (Fig.3), suggesting that SA binding was also not a significant determinantof spread to or growth within the heart in infected animalsfollowing intramuscular peripheral inoculation.

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FIG. 3. ${sigma}$ 1s does not influence viral growth in the heart. Viral titers in infected hearts were not significantly different (P > 0.05) between ${sigma}$ 1s^– and all ${sigma}$ 1s⁺ viral strains. Each bar represents the average viral titer from 5 to 10 mice at 8 days post-intramuscular inoculation. Error bars indicate standard errors of the means.

${sigma}$ 1s is a determinant of apoptosis and pathogenesis in the heart. Having shown that ${sigma}$ 1s is a determinant of lethality followingintramuscular reovirus inoculation, we next determined the effectof ${sigma}$ 1s on viral pathogenesis in hearts of reovirus-infected animals.To evaluate overall myocardial tissue injury, H&E-stainedtransverse cardiac sections were evaluated via light microscopy,and the degree of injury was quantified by using a previouslyvalidated scoring system (8, 29) (Table 1 and Fig. 4). At 8days postinfection, prominent myocardial disruption, numerousapoptotic bodies, and evidence of pyknotic nuclei were observedin hearts of ${sigma}$ 1s⁺ virus-infected mice regardless of SA bindingability, while very minor myocardial injury was noted in ${sigma}$ 1s^–reovirus-infected animals (Fig. 4).

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FIG. 4. ${sigma}$ 1s is required for maximal myocardial tissue injury. The degree of injury in the myocardium, as measured by quantifying H&E-stained heart sections, was significantly reduced in ${sigma}$ 1s^– (C84-MA) virus-infected mice. Each bar represents the average score from 10 to 13 mice. Error bars indicate standard errors of the means. *, P < 0.05.

Immunohistochemical detection of the activated form of caspase-3was used as a marker of apoptosis in reovirus-infected hearts.Dark brown staining indicates active caspase-3 apoptotic lesionswithin the myocardium (Fig. 5B and C). Mice infected with the ${sigma}$ 1s^– strain C84-MA showed a significant decrease in boththe number and size of apoptotic lesions in the heart comparedto each of the ${sigma}$ 1s⁺ control viruses at 8 days postinfection (Fig.5A). In contrast, all ${sigma}$ 1s⁺ reoviruses produced severe myocardialinjury with extensive areas of apoptosis analogous to thosewe have previously described for the prototypic myocarditis-inducingstrain, 8B (8). The rare apoptotic lesions that were observedin ${sigma}$ 1s^– virus-infected hearts appeared similar in cellularcomposition and caspase-3 staining to heart lesions seen in ${sigma}$ 1s⁺ virus-infected hearts (data not shown).

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FIG. 5. ${sigma}$ 1s is required for maximal apoptosis induction in the heart. Immunohistochemical staining of activated caspase-3 in whole hearts (B and C) (original magnification, x25) demonstrates that loss of ${sigma}$ 1s expression significantly reduces reovirus-induced apoptosis (A). In panel A, each bar represents the average score from four to eight mice. Error bars indicate standard errors of the means. *, P < 0.05.

Animals which survived C84-MA ( ${sigma}$ 1s^– SA⁺) virus challengewere sacrificed at 50 days postinfection and evaluated for myocardialinjury. H&E-stained heart sections did not show any myocarditicdamage, and no infectious virus was detectable by plaque assayin the heart or other organs (data not shown).

Taken together, these studies indicate that ${sigma}$ 1s was a determinantof the extent and severity of myocardial injury and apoptosisbut not of viral growth in the hearts of infected mice. We nextwished to determine whether these same features occurred inCNS infection.

${sigma}$ 1s is a determinant of T3 reovirus virulence following intracranial inoculation. In order to study the role of ${sigma}$ 1s in pathogenesis of reovirus-inducedCNS disease, 2-day-old mice were intracerebrally inoculatedwith 10³ PFU of reovirus strain C84-MA ( ${sigma}$ 1s^– SA⁺), C93( ${sigma}$ 1s⁺ SA⁺), T3A ( ${sigma}$ 1s⁺ SA⁺), or C84 ( ${sigma}$ 1s⁺ SA^–). Mortalitywas monitored for 30 days following inoculation (Fig. 6). Onehundred percent of mice infected with either C93 or T3A died(mean day of death ± standard error of the mean, 9.1± 0.8 and 8.9 ± 0.3, respectively), compared toonly 67% of C84-MA-infected mice (mean day of death ±standard error of the mean, 14.3 ± 1.2) (P < 0.001).The SA^– strain C84 also showed enhanced survival comparedto T3A and C93 (Fig. 6).

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FIG. 6. ${sigma}$ 1s confers enhanced virulence following intracranial inoculation. Neonatal mice (n = 7 to 11) were inoculated intracerebrally with 10³ PFU of C84-MA ( ${sigma}$ 1s^– SA⁺), C93 ( ${sigma}$ 1s⁺ SA⁺), T3A ( ${sigma}$ 1s⁺ SA⁺), or C84 ( ${sigma}$ 1s⁺ SA^–) per mouse.

In an effort to see whether the attenuated phenotype of C84-MA( ${sigma}$ 1s^– SA⁺) would persist after massive viral challenge,mice were injected intracranially with 10⁵ PFU (approximately10,000 times the intracranial 50% lethal dose) of the test virusesper mouse. All mice died at this high challenge dose, althoughthe mean day of death for ${sigma}$ 1s^– virus (C84-MA)-infectedmice was significantly (P < 0.001) prolonged (11.2 ±0.3 days) compared to that for either ${sigma}$ 1s⁺ T3A (8.3 ±0.3 days)- or ${sigma}$ 1s⁺ C93 (9 ± 0 days)-infected animals.

These results indicate that ${sigma}$ 1s is a determinant of neurovirulencefollowing intracerebral inoculation of reovirus. The enhancedsurvival in mice infected with the SA^– strain C84 comparedto the SA⁺ strains T3A and C93 also indicate that SA bindingplays a role in neurovirulence.

${sigma}$ 1s is not required for viral replication in the brain. Having shown that ${sigma}$ 1s influenced neurovirulence after intracranialinoculation, we next wished to determine whether this was dueto an effect on viral growth in the CNS. Neonatal mice wereintracerebrally infected with 10⁵ PFU of the various reovirusstrains. At 8 days postinfection brain tissue was removed fromeuthanatized animals, and the viral titer was determined byplaque assay of brain homogenates (Fig. 7). As seen in the heartmodel, the presence or absence of ${sigma}$ 1s did not significantly affectviral growth in the brain (P > 0.05 for all interstrain comparisonsexcept that between the SA^– virus C84 and the SA⁺ virusT3A, for which P < 0.01). These results indicate that ${sigma}$ 1sdoes not influence viral growth in the brain.

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FIG. 7. ${sigma}$ 1s does not influence viral growth in the brain. Viral titers were not significantly different between ${sigma}$ 1s^– and all ${sigma}$ 1s⁺ viral strains (P > 0.05). T3A and C84 viral titers were significantly different (**, P < 0.01). Each bar represents the average viral titer from three to six mice 8 days post-intracranial inoculation. Error bars indicate standard errors of the means.

${sigma}$ 1s is a determinant of apoptosis and pathogenesis in the brain. We next wished to determine whether ${sigma}$ 1s influenced the extentof viral injury in the CNS. At 8 days following infection, micewere sacrificed and neuropathologic injury was evaluated. The ${sigma}$ 1s^– strain C84-MA (Fig. 8A to C and J) induced significantlyless tissue injury in the cortex, thalamus, and hippocampusthan its ${sigma}$ 1s⁺ SA⁺ counterparts (Fig. 8D to I and J). By contrast,large areas in the thalamus and cortex of ${sigma}$ 1s⁺ SA⁺ (C93 and T3A)virus-infected animals were destroyed and contained numerousapoptotic bodies (Fig. 8D and E). Extensive neuronal death wasalso evident in the hippocampi of these animals (Fig. 8F). ${sigma}$ 1s^–(C84-MA) virus-infected mice, examined when moribund (day 11),did show substantial CNS injury (data not shown), indicatingthat loss of ${sigma}$ 1s delayed but did not prevent the appearance ofCNS injury following high-dose virus challenge. The SA^–virus (C84) also showed markedly reduced tissue injury (Fig.8G to J) compared to SA⁺ ${sigma}$ 1s⁺ virus C93 (Fig. 8D to F) or T3A(data not shown). When moribund, C84-infected mice displayeddelayed CNS injury (data not shown).

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FIG. 8. ${sigma}$ 1s determines the extent and severity of reovirus-induced CNS injury. Representative H&E-stained coronal sections of the cortex, thalamus, and hippocampus are shown for C84-MA (A to C), C93 (D to F), and C84 (G to I) (original magnification, x400). Tissue injury was quantified by using a previously validated neuropathology grading system (J). Each bar represents the average score from four to nine mice. Error bars indicate standard errors of the means. Statistical comparisons are shown in below the corresponding areas of the graph. NS, not significant.

Immunohistochemical detection of the activated form of caspase-3was used as a marker for reovirus-induced apoptosis in the brain.Mice infected with ${sigma}$ 1s^– virus (C84-MA) had no detectableapoptosis in the cortex, thalamus, or hippocampus at 8 dayspostinfection (Fig. 9B). Conversely, ${sigma}$ 1s⁺ control strains C93(Fig. 9D) and T3A (data not shown) displayed high levels ofcaspase-3 activation in all three areas. The SA^– virusC84 also displayed substantially reduced apoptosis in the cortex,thalamus, and hippocampus (Fig. 9C). As was the case for histologicalinjury, C84-MA-infected mice examined when moribund (day 11)showed substantial caspase-3 activation (data not shown), indicatingthat loss of ${sigma}$ 1s served to delay but not prevent apoptosis followinghigh-dose viral challenge.

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FIG. 9. ${sigma}$ 1s determines the extent and severity of reovirus-induced apoptosis in the brain. The cortex, hippocampus, and thalamus were examined at 8 days post-intracranial inoculation (A). Active, cleaved caspase-3 was quantified by the number and size of caspase-3-positive apoptotic areas (E). C84-MA ( ${sigma}$ 1s^– SA⁺)-infected mice displayed reduced caspase-3 activation in the brain compared to ${sigma}$ 1s⁺ control viruses (B to E) (original magnification, x25). In panel E, each bar represents the average score from four to seven mice. Error bars indicate standard errors of the means. Statistical comparisons are shown in below the corresponding areas of the graph. NS, not significant.

By contrast to the results seen following high-dose viral challenge,long-term survivors of low-dose intracerebral challenge with ${sigma}$ 1s^– reovirus (Fig. 6) sacrificed at 30 days postinfectiondid not show any histopathological indication of viral damage,and no infectious virus was detectable by plaque assay in thebrain or other organs (data not shown). Similar results wereobtained with survivors of SA^– (C84) infection as well(data not shown).

Expression of ${sigma}$ 1s in vivo by reovirus strains. We next wished to evaluate ${sigma}$ 1s expression by C84-MA ( ${sigma}$ 1s^–SA⁺) and the ${sigma}$ 1s⁺ strains T3A, C93, and C84 following both intramuscular(Fig. 10A to F) and intracranial (Fig. 10G to L) inoculationof neonatal mice. In vivo ${sigma}$ 1s expression was evaluated by stainingcardiac and coronal sections from ${sigma}$ 1s^– and ${sigma}$ 1s⁺ virus-infectedanimals with monoclonal antibodies directed against T3 ${sigma}$ 1s. Expressionof reovirus structural proteins was evaluated by using polyclonalantisera in adjacent sections.

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FIG. 10. ${sigma}$ 1s is expressed in vivo following both intramuscular and intracranial inoculation with 10⁵ PFU of T3A ( ${sigma}$ 1s⁺ SA⁺) (A and G) and C84 ( ${sigma}$ 1s⁺ SA^–) (B and H), but not C84-MA ( ${sigma}$ 1s^– SA⁺) (C and I), per mouse. In vivo ${sigma}$ 1s expression in both the hearts and brains of infected animals was evaluated by diaminobenzidine tetrahydrochloride immunohistochemistry with monoclonal antibodies specific for T3 ${sigma}$ 1s. Cardiac sections from intramuscularly inoculated mice (T3A, C84, and C84-MA) were stained with monoclonal antibody 2F4 directed against ${sigma}$ 1s (A to C) (original magnification, x200) and polyclonal antisera directed against reovirus structural proteins (D to F) (original magnification, x100). Coronal sections from intracranially inoculated mice (T3A, C84, and C84-MA) were stained with monoclonal antibody 3E2 directed against ${sigma}$ 1s (G to I) (original magnification, x200) and polyclonal antisera directed against reovirus structural proteins (J to L) (original magnification, x400). Polyclonal antiserum directed against T3 reovirus structural proteins was used as a control for the presence of reovirus in both cardiac (D to F) and brain (J to L) tissues.

As expected, both heart and brain tissues from C84-MA ( ${sigma}$ 1s^–SA⁺)-infected mice did not show positive ${sigma}$ 1s staining (Fig. 10C and I)despite detectable expression of reovirus structuralproteins (Fig. 10F and L). These data indicate that reoviruswas present in both the infected hearts and brains of C84-MAmice but was not expressing ${sigma}$ 1s.

In contrast, coronal sections from ${sigma}$ 1s⁺ SA⁺ (T3A) virus-infectedmice displayed prominent expression of ${sigma}$ 1s (Fig. 10G) and structuralproteins (Fig. 10J) within the cortex, as well as the thalamusand hippocampus (data not shown). In the heart, the ${sigma}$ 1s⁺ strainT3A displayed high levels of ${sigma}$ 1s staining (Fig. 10A) and structuralproteins (Fig. 10D) within areas of cardiac lesions. The ${sigma}$ 1s⁺SA⁺ strain C93 displayed staining patterns similar to thoseof T3A in both the heart and brain (data not shown). In contrastto the exclusively cytoplasmic localization of reovirus structuralproteins in infected cardiomyocyte neurons, ${sigma}$ 1s was observedin both the cytoplasm and nuclei of infected cells, thus corroboratingin vivo our previous in vitro data indicating that ${sigma}$ 1s localizesto both the cytoplasm and nuclei of infected cells (13).

The ${sigma}$ 1s⁺ SA^– strain C84 expressed ${sigma}$ 1s in both the heartand CNS (Fig. 10B and H). In the brain, reovirus structuralproteins were observed throughout the cortex (Fig. 10K), thalamus(data not shown), and hippocampus (data not shown). The relativeamount of ${sigma}$ 1s expression compared to structural protein expressionappeared to be lower in C84-infected mice than in T3A- or C93-infectedmice, particularly in the CNS (Fig. 10G, J, H, and K). In theheart, the ratio of ${sigma}$ 1s expression to expression of reovirusstructural proteins seemed to approximate that for T3A (Fig.10 A, D, B, and E). Although these data are not quantitative,the reduced level of ${sigma}$ 1s expression compared to that of reovirusstructural protein expression observed in C84 infected tissuessuggests that C84 may express ${sigma}$ 1s at reduced levels comparedto ${sigma}$ 1s⁺ SA⁺ strains, a result also noted in vitro (26).

DISCUSSION

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Discussion

We now show that the reovirus nonstructural ${sigma}$ 1s protein is adeterminant of viral injury in the hearts and brains of infectedneonatal mice. Although viral growth in the brain and heartwas not affected by the presence or absence of ${sigma}$ 1s, the extentof reovirus-induced apoptosis induction and severity of ensuingtissue damage in both organs was significantly influenced by ${sigma}$ 1s expression. In addition, ${sigma}$ 1s^– SA⁺ (C84-MA) virus-infectedanimals displayed enhanced survival following either intramuscularor intracranial challenge compared to mice injected with ${sigma}$ 1s⁺SA⁺ viral strains. These studies provide the first demonstrationthat ${sigma}$ 1s is expressed following intramuscular or intracranialinoculation and plays a significant role in reovirus pathogenesisin vivo.

Our results demonstrate that ${sigma}$ 1s expression does not influenceviral growth in the heart or brain and supports previous invitro data indicating that ${sigma}$ 1s is not required for reovirus replicationin either L929 or MDCK cells (26). We further demonstrate that ${sigma}$ 1s influences the rate and extent of apoptosis induction invivo, a phenomenon not obvious in vitro (26). This is not surprising,as there are many host factors that may operate in concert with ${sigma}$ 1s or other reovirus proteins to affect reovirus-induced diseasein vivo (35). For example, ${sigma}$ 1s elicits a strong and specificcytotoxic-T-lymphocyte response (12) which may contribute tothe ability of ${sigma}$ 1s to modulate the extent and severity of apoptosisinduction that we observed in vivo. The conservation of ${sigma}$ 1s openreading frames in all reovirus field isolates provides furthersupport for the conclusion that ${sigma}$ 1s plays an important role duringviral pathogenesis in vivo.

We have recently shown that ${sigma}$ 1s actively localizes to the nucleusin infected cells and induces severe disruptions in nucleararchitecture, including perturbation of the A-type nuclear laminanetwork (LaA/C) and induction of nuclear herniations (13). Therelationship, if any, between ${sigma}$ 1s-induced nuclear architecturechanges observed in vitro and the described effects on pathogenesisin vivo are unknown. However, recent studies report that disruptionof LaA/C in myocardiocytes weakens nuclear structural mechanicsand renders cells more sensitive to apoptosis following mechanicalstress, such as that generated by the beating myocardium (16).This suggests the possibility that ${sigma}$ 1s-induced LaA/C defectsmay also function in vivo to render infected myocardiocytesmore susceptible to apoptosis induction by reovirus.

The apoptosis-enhancing effects of LaA/C disruption are lesslikely the potential mechanism of action for ${sigma}$ 1s in the CNS dueto the lack of mechanical strain in brain tissue. Other cytopathiceffects of ${sigma}$ 1s may possibly influence the extent and severityof apoptosis induction in the brain. A growing body of evidencesuggests that aberrant reentry into the cell cycle or abnormalexpression of key cell cycle regulatory proteins representsa common mechanism of neuronal apoptosis (4). ${sigma}$ 1s modulates inductionof reovirus-induced G₂/M cell cycle arrest in infected cellsand inhibits p34 (cdc2) kinase activity (22). In addition, reovirusinfection alters the expression profiles of key cell cycle proteins(21). Given these data, it is plausible that in reovirus-infectedneurons, ${sigma}$ 1s perturbation of cell cycle regulatory proteins mayexacerbate reovirus-induced neuronal apoptosis.

SA binding optimizes induction of apoptosis in a variety ofcultured cells (7, 20) and is required for full expression ofcertain disease phenotypes, including reovirus-induced biliaryatresia (2). Our studies suggest that SA binding is not essentialfor reovirus spread to and growth within the heart followingperipheral inoculation. SA binding is not required for apoptosisinduction in the heart or development of myocarditis, as theSA^– strain (C84) was efficiently myocarditic and inducedapoptosis. SA binding may be more important in CNS pathogenesisthan in the heart, as the SA^– strain (C84) showed reducedCNS tissue injury and apoptosis compared to the control ${sigma}$ 1s⁺SA⁺ strains (T3A and C93).

An additional factor that may influence pathogenesis is differencesin the amount of ${sigma}$ 1s expression in vivo. C84-MA ( ${sigma}$ 1s^– SA⁺)does not express ${sigma}$ 1s in vivo and is significantly less pathogenicin both the heart and the CNS. The SA^– strain C84 alsoappeared to have lower levels of ${sigma}$ 1s expression in vivo thanother ${sigma}$ 1s⁺ strains. Although not quantitative, our data supportprevious in vitro data (26) and suggest that the altered phenotypeof C84 could also reflect reduced ${sigma}$ 1s expression.

Our findings represent the first evidence of a role for thenonstructural reovirus protein ${sigma}$ 1s in viral pathogenesis in vivo.The onset of apoptosis induction and severity of virus-inducedtissue injury are both dependent on the expression of ${sigma}$ 1s. Theevidence presented here is all the more provocative consideringthe implication of a role for ${sigma}$ 1s in virus-induced pathogenesisindependent of any effects on viral growth.

ACKNOWLEDGMENTS

This work was supported by Merit and REAP grants from the Departmentof Veterans Affairs NIH NINDS 1ROINS50138 (to K.L.T.), U.S.Army Medical Research and Material Command grant DAMD17-98-1-8614(to K.L.T.), and the Reuler-Lewin Family Professorship of Neurology(to K.L.T.).

We acknowledge the University of Colorado Health Sciences CenterHistology Services for assisting with histological preparation.

FOOTNOTES

* Corresponding author. Mailing address: Department of Neurology (B-182), University of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262. Phone: (303) 393-2874. Fax: (303) 393-4686. E-mail: Ken.Tyler{at}uchsc.edu.

REFERENCES

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