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Journal of Virology, May 2009, p. 5035-5045, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02433-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Reovirus Activates Transforming Growth Factor β and Bone Morphogenetic Protein Signaling Pathways in the Central Nervous System That Contribute to Neuronal Survival following Infection

J. David Beckham,^1,4 Kathryn Tuttle,² and Kenneth L. Tyler^1,2,3,4*

Departments of Medicine,¹ Neurology,² Microbiology, University of Colorado Denver, Denver, Colorado 80045,³ Denver Veteran Affairs Medical Center, Denver, Colorado 80220⁴

Received 25 November 2008/ Accepted 26 February 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Viral infections of the central nervous system (CNS) are important causes of worldwide morbidity and mortality, and understanding how viruses perturb host cell signaling pathways will facilitate identification of novel antiviral therapies. We now show that reovirus infection activates transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) signaling in a murine model of encephalitis in vivo. TGF-β receptor I (TGF-βRI) expression is increased and its downstream signaling factor, SMAD3, is activated in the brains of reovirus-infected mice. TGF-β signaling is neuroprotective, as inhibition with a TGF-βRI inhibitor increases death of infected neurons. Similarly, BMP receptor I expression is increased and its downstream signaling factor, SMAD1, is activated in reovirus-infected neurons in the brains of infected mice in vivo. Activated SMAD1 and SMAD3 were both detected in regions of brain infected by reovirus, but activated SMAD1 was found predominantly in uninfected neurons in close proximity to infected neurons. Treatment of reovirus-infected primary mouse cortical neurons with a BMP agonist reduced apoptosis. These data provide the first evidence for the activation of TGF-β and BMP signaling pathways following neurotropic viral infection and suggest that these signaling pathways normally function as part of the host's protective innate immune response against CNS viral infection.

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The transforming growth factor β (TGF-β) superfamily of growth factors regulates multiple cellular functions including inflammation, cell growth, differentiation, migration, and apoptosis (33). In excess of 30 genes represent the TGF-β superfamily in mammals including three TGF-β genes, four activin β-chains (nodal), 10 bone morphogenetic proteins (BMPs), and 11 growth and differentiation factors. The receptors for the TGF-β superfamily of ligands form the only known transmembrane Ser-Thr kinases (33). The signaling pathways are similar for all ligands. Briefly, a TGF-β ligand binds to and brings into proximity a TGF-β receptor type I (TGF-βRI) and a TGF-β receptor type II (TGF-βRII), assembling a heterotetrameric complex (45). The constitutively active type II receptor kinase phosphorylates the type I receptor at several serine and threonine residues in a glycine- and serine-rich juxtamembrane domain, resulting in the recruitment and phosphorylation at two C-terminal serine residues in the MH2 domain of the receptor-regulated SMADs (R-SMAD): SMAD1, SMAD2, SMAD3, SMAD5, and SMAD8 (33). Phosphorylated R-SMAD proteins form complexes with the common mediator SMAD4, translocate to the nucleus, and alter gene expression. Each type I receptor typically binds a specific TGF-β superfamily ligand and activates a subset of R-SMADs. The TGF-β-activin-nodal ligands signal through specific type I receptors to activate SMAD2 or SMAD3, and the BMP-growth and differentiation factor ligands signal through specific type I receptors and activate SMAD1, SMAD5, or SMAD8 (33).

Members of the TGF-β superfamily modulate innate immune responses to multiple infections by controlling inflammation and repair after injury (25). In addition, TGF-β signaling controls apoptosis and viral replication in several viral systems including polyomaviruses such as BK virus (1) and JC virus (16, 30), human immunodeficiency virus (16), Epstein-Barr virus reactivation (17), and hepatitis C virus (26). In the case of hepatitis C virus, the synergistic activation of BMP signaling and alpha interferon suppresses viral replication (35). In noninfectious models of disease, previous studies have shown that modulating TGF-β signaling is protective in a murine model of Alzheimer's disease (36), and augmenting BMP signal activation can protect cells and neurons following oxidative stress (15), stroke (40), or other cellular injuries (3, 44). However, to our knowledge, the roles of TGF-β and BMP signaling have not been studied following acute viral infection in the central nervous system (CNS).

Reovirus infection is a well-characterized experimental system utilized to study viral pathogenesis. Serotype 3 strains of reovirus (Abney [T3A] and Dearing [T3D]) induce apoptosis in vitro and in vivo by activating caspase-3-dependent cell death (4, 28). Reovirus-induced encephalitis in vivo is largely a result of virus-induced apoptosis with little associated infiltrate of inflammatory cells. Caspase 3 activation is initiated by reovirus-induced activation of death receptors and is augmented by mitochondrial apoptotic signaling (6, 24, 31). Previous studies have also demonstrated that virus-induced signaling events affect cell survival and cell death. Reovirus-induced selective activation of mitogen-activated protein kinases such as c-Jun N-terminal kinase (JNK) are vital to apoptosis in vitro and in a murine model of reovirus-induced encephalitis (2, 9). Similarly, the activation and subsequent inhibition of NF-B signaling are important determinants of apoptosis (5, 7, 10). These pathways are likely to act in part by regulating critical components of either death receptor or mitochondrial apoptotic signaling. For example, reovirus-induced inhibition of NF-B activation decreases cellular levels of c-FLIP, a caspase 8 inhibitor, and inhibition of JNK signaling decreases mitochondrial release of proapoptotic proteins cytochrome c and SMAC (5, 8). While many of these signaling pathways modulate apoptosis, the reovirus model of pathogenesis has been utilized to understand the interferon response to viral infection in cell culture, in myocardial cells, and in the CNS as well (18, 22, 34). Understanding the cellular response to viral infection will lead to the identification of new targets for antiviral therapy.

Studies of neuroinvasive viral infections including those with Sindbis virus, West Nile virus, herpes simplex virus, and cytomegalovirus have shown that apoptosis is an important mechanism of neuronal cell death (11, 20, 27, 32). In many cases of neuroinvasive viral infection, exemplified by West Nile virus, viremia has ended by the time that the patient presents with acute symptoms; yet, ongoing virus-induced injury in the CNS results in significant morbidity and mortality (13, 21). There are currently no proven effective therapies for acute CNS viral infections other than acyclovir therapy for herpes simplex virus encephalitis, and even with optimal treatment of herpes simplex virus encephalitis, morbidity and mortality remain significant. The goal of our studies is to utilize the reovirus system to identify potential novel therapeutic targets that will enhance neuroprotection following CNS viral infection.

We show here for the first time that TGF-β and BMP are activated in response to viral infection in a model of murine viral encephalitis in vivo. We extend these findings by showing that virus-activated BMP signaling protects mouse cortical neurons from cell death.

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Virus stocks. Reovirus serotype 3 strains Abney (T3A) and Dearing (T3D) are laboratory stocks (37) purified from the supernatant in a sucrose gradient. Briefly, laboratory stocks of P2 reovirus (T3A or T3D) were layered over a 30% sucrose gradient followed by ultracentrifugation at 4°C at 24,000 rpm overnight. Virus was collected and suspended in phosphate-buffered saline (PBS), and titers were determined using standard plaque assay techniques (see below). All in vitro studies were completed with a multiplicity of infection (MOI) of 100 to maximize the infection and facilitate synchronous viral replication. Reovirus infection efficiency in primary mouse cortical neuronal cultures ranges from 30 to 40% at day 3 postinfection and increases thereafter.

Primary cell culture. Cultures of primary neurons (mouse cortical cultures [MCCs]) were prepared from the cortices of embryonic day 16 Swiss Webster Hsd:nd4 mice (Harlan-Sprague Dawley, Indianapolis, IN) as described previously (19). Briefly, cortices were dissected in cation-free Hanks balanced salt solution containing glucose (10 g/liter) and then digested in 0.125% Trypsin at 37°C followed by three washes in neurobasal medium supplemented with 10% fetal bovine serum (FBS). MCCs were mechanically dissociated by trituration with a Pasteur pipette. Primary neurons were plated on cell culture dishes coated with poly-D-lysine/laminin (Biocoat; Becton Dickinson, Franklin Lakes, NJ) in neurobasal medium containing 10% FBS, B-27 (2%, vol/vol), antibiotic (penicillin-streptomycin; 1,000 units/ml), and L-glutamine (0.6 mM). MCCs were infected for 1 hour followed by replacement of medium with neurobasal medium (B-27 [2%, vol/vol], penicillin-streptomycin [1,000 units/ml], uridine [10 µM], and L-glutamine [0.6 mM]). All studies were performed at day 1 in vitro. All culture media and supplements were purchased from Invitrogen unless stated otherwise.

Immunocytochemistry analysis of MCCs characterized each culture with a monoclonal antibody specific for microtubule-associated protein 2 (1:100; Abcam). Primary cortical cultures were comprised of 90 to 95% neurons, harvested at days 4 and 5 postinfection, and fixed in 4% paraformaldehyde, as previously described (19, 31). Coverslips were blocked and permeabilized and then incubated with primary antibodies (anti-cleaved caspase 3 [Cell Signaling; 1:400], microtubule-associated protein 2, and anti-3 reovirus [4F-2; 1:100]) per the manufacturer's instructions.

In vitro experiments. HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% FBS, 4 mM L-glutamine, sodium pyruvate (132 mg/liter), and penicillin-streptomycin (1,000 units/ml). Cells were treated with vehicle controls, chemical inhibitors, or recombinant proteins and infected 30 min later with T3A unless otherwise specified. Chemical inhibitors and recombinant proteins included SB432542 (Tocris; 10 µM), TGF-βRI inhibitor III (Calbiochem; 10 µM), SMAD3 inhibitor (Calbiochem; 10 µM), BMP inhibitor (43) (Dorsomorphin; Calbiochem; 10 µM), and recombinant human BMP-6 (R&D Systems; 50 ng/ml).

In vivo studies. Two-day-old Swiss Webster pups were inoculated (intracerebrally [i.c.]) with 1,000 PFU of reovirus (T3A or T3D) in a 10-µl volume as described previously (31). For in vivo pharmacologic treatments, mice were intraperitoneally injected at the time of infection with TGF-βRI inhibitor III (Calbiochem; 20 mg/kg of body weight) or vehicle control (PBS) in a volume of 10 µl followed by repeated daily treatment. All experiments were approved by the IACUC and performed in accordance with national PHS guidelines on the ethical use of animals, and all studies were completed with multiple replicate animals from at least three separate litters.

Western blotting. Whole-brain lysates were prepared as previously described (19). Lysates were loaded onto a 10% polyacrylamide-Tricine gel (Hoefer Pharmacia Biotech, San Francisco, CA), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a Hybond C nitrocellulose membrane (Amersham Biosciences). Immunoblotting was performed as described previously (29). Membranes were probed with antibodies to phosphorylated (Ser463/465) SMAD3/SMAD1 (Cell Signaling; 1:1,000), total SMAD3 (Cell Signaling; 1:1,000), cleaved poly(ADP-ribose) polymerase (PARP) (Cell Signaling; 1:1,000), cleaved caspase 3 (Cell Signaling; 1:1,000), phosphorylated (Ser463/465) SMAD1/SMAD5/SMAD8 (Cell Signaling; 1:1,000), BMP receptor IB (BMPRIB)/ALK6 (R&D Systems; 1:1,000), and β-actin (Calbiochem; 1:15,000). Secondary antibodies used for Western blot assays included mouse- and rabbit-specific horseradish peroxidase-conjugated anti-immunoglobulin G (anti-IgG; Amersham Biosciences; 1:2,000). Image J software available through the NIH was used for densitometry measurements of Western blots. All densitometry values were divided by corresponding β-actin band density in order to normalize values to protein loading.

Histological studies. Brain tissue was formalin fixed, paraffin embedded, and cut into 4-µm-thick coronal brain sections. Sections were deparaffinized in xylene and rehydrated in sequential ethanol washes. Tissue was subjected to antigen retrieval (10 mM citrate buffer), permeabilization, and blocking (10% normal goat serum in PBS plus 0.3% Triton) followed by incubation with primary antibodies: monoclonal reovirus 3-reovirus antibody (4F2; 1:100), serine 423/425 phosphorylation-specific SMAD3 (Rockland, Gilbertsville, PA; 1:100), TGF-βRI (Abcam, Cambridge, MA; 1:100), phosphorylation-specific SMAD1 (Ser463/465)/SMAD5/SMAD8 (Cell Signaling Technology, Danvers, MA; 1:100), and mouse anti-neuronal nucleus antibody (NeuN; Chemicon; 1:100). Sections were incubated with secondary antibodies (Cy3-conjugated AffiniPure goat anti-rabbit IgG [Jackson ImmunoResearch; 1:300], Alexa Fluor 488-conjugated goat anti-mouse IgG [Invitrogen; 1:200], and biotinylated goat anti-rabbit antibody [Invitrogen; 1:100]), and nuclei were stained (1 µg/ml Hoechst 33342 [Invitrogen]) and were mounted (VectorShield [Vector Laboratories]) followed by visualization of immunofluorescence using a Zeiss Axioplan 2 digital deconvolution microscope with a Cooke Sensicam 12-bit camera.

Viral titer assays. L929 mouse fibroblasts were used for viral titer assays and were maintained in 2X199 medium (10% heat-inactivated FBS and 4 mM L-glutamine). Primary MCCs were infected with purified T3A (MOI of 100), and inoculum was removed and fed with serum-free medium plus vehicle control or subjected to BMP6 ligand treatment. MCCs were harvested as a cell pellet at day 4 postinfection, washed, and frozen followed by three freeze-thaw cycles and sonication. Serial dilutions of the homogenate were prepared in gel saline, and viral titers were determined by plaque assays as previously described (12).

Statistical analysis. All statistical analyses were performed using Instat and Prism (GraphPad Software Inc., San Diego, CA). Statistical comparisons were made using a two-tailed, unpaired t test with Welch correction.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In order to facilitate the discovery of novel signaling pathways associated with reovirus infection, we utilized Panomics protein/DNA array I to examine the pattern of transcription factor activation in primary neuronal cultures. Primary MCCs were infected with reovirus serotype 3 strain Abney (T3A) (MOI, 100) or were mock infected. Neurons were harvested at specific time points postinfection followed by extraction of nuclear subcellular fractions. Nuclear proteins were then analyzed for altered expression in transcription factors using the Panomics protein/DNA array I. These screening studies suggested that SMAD3 was upregulated in reovirus-infected MCCs compared to mock-infected controls (data not shown) and promoted a more-detailed investigation of signaling pathways involving SMAD proteins.

Reovirus activates TGF-β signaling in vivo. First, we characterized the TGF-β signaling pathway by examining the regulation of the TGF-βRI in vivo. We inoculated Swiss Webster pups at day 2 of life by i.c. injection with 1,000 PFU of reovirus (T3A or T3D) or PBS as a mock-infection control. At day 8 postinfection, mice were sacrificed and brains were analyzed for evidence of TGF-βRI upregulation using immunohistochemistry staining with an antibody specific for TGF-βRI. Analysis of three separate brains from each treatment group revealed upregulation of TGF-βRI in reovirus-infected mice compared to mock-infected controls in regions of the brain infected by reovirus: the cingulate cortex, hippocampus, and thalamus (Fig. 1A to F).

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FIG. 1. Reovirus infection upregulates TGF-βRI in vivo. Brains of mock-infected (A to C) and reovirus-infected (D to F) (T3D, 1,000 PFU, i.c.) Swiss Webster pups sacrificed at day 8 postinfection were paraffin embedded, permeabilized, and stained with an antibody specific for TGF-βRI (brown). Images shown are representative of three replicate individual brains per treatment group. Original magnification, x400.

Since a major receptor for TGF-β signaling was upregulated, we next wished to characterize the activation of the downstream SMAD proteins by evaluating the activation of SMAD3 in a model of viral encephalitis in vivo. We infected 2-day-old neonatal Swiss Webster pups with reovirus (T3A or T3D; 1,000 PFU i.c.) and sacrificed all mice at day 8 postinfection. Whole-brain lysates were prepared and analyzed with Western blotting using a phosphorylation-specific (Ser423/425) antibody to SMAD3 (pSMAD3). We found evidence of a significant (P < 0.05) fourfold upregulation of pSMAD3 at 8 days postinfection with reovirus compared to mock-infected brains (Fig. 2A and B). In general, the phosphorylated SMAD proteins often resolve as a doublet as shown elsewhere (41, 42). In order to identify the brain regions involved in pSMAD3 activation, we performed immunohistochemistry studies using an antibody specific to the phosphorylated form (Ser423/425) of SMAD3 (Rockland). We found that pSMAD3 was both upregulated and localized to the nucleus in neurons of reovirus-infected brains (Fig. 2F to H) compared to mock-infected brains (Fig. 2C to E). Additionally, upregulation of pSMAD3 was confined to the cingulate cortex, hippocampus, and thalamus. Activation of SMAD3 was not seen in brain regions not infected by reovirus. Despite changes in pSMAD3, Western blot analysis of whole-brain lysates from mice at day 8 post-infection with T3D or T3A (1,000 PFU, i.c.) revealed no significant changes in total SMAD3 protein expression in mice with viral encephalitis compared to mock-infected mice (Fig. 2I). Additionally, PCR studies for SMAD3 mRNA revealed no changes following reovirus infection (data not shown). Studies of SMAD3 activation were first demonstrated in vitro using reovirus-infected HEK293 cells. SMAD3 is activated at 24 and 48 h postinfection with reovirus (data not shown).

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FIG. 2. Reovirus activates TGF-β signaling in vivo. (A) Western blot analysis. Whole-brain lysates from mock-infected and reovirus-infected Swiss Webster pups (1,000 PFU, i.c., day 8 postinfection) were resolved on 10% polyacrylamide gels, transferred to a nitrocellulose membrane, and probed with an antibody specific for phosphorylated SMAD3 (pSMAD3) and with anti-β-actin. (B) Densitometry values (densitometry units/β-actin densitometry units) from six replicate individual brains per treatment group from separate litters indicate a fourfold increase in pSMAD3 expression (P < 0.05) with infection. (C to H) Immunohistochemistry studies. Brains from mock-infected (C to E) and reovirus-infected (F to H) (1,000 PFU, i.c.) mice (8 days postinfection) were stained with an antibody to pSMAD3 (brown). Images are representative of three replicates per treatment group. Original magnification, x400. (I) Whole-brain lysates from the same treatment groups were analyzed using Western blotting with antibodies to total SMAD3 (TSMAD3).

Inhibition of TGF-β signaling in virus-infected cells and neurons increases apoptosis in vivo. Having shown that TGF-β signaling is activated following reovirus infection in vivo, we next evaluated the role of TGF-β signaling in reovirus pathogenesis in vivo. Two-day-old Swiss Webster mice were infected with reovirus (T3D; 1,000 PFU, i.c.) and treated with TGF-βRI inhibitor (TGF-βRI inhibitor III; Calbiochem; 20 mg/kg) by intraperitoneal injection or vehicle control (equivalent volume, 10 µl) at the time of infection and then daily thereafter until all mice were sacrificed at day 8 postinfection. Whole-brain lysates were evaluated with Western blotting using antibodies specific for pSMAD3, cleaved (Asp214) PARP, and the large cleavage fragment (17/19 kDa) adjacent to Asp175 of caspase 3. Western blot analysis revealed increased apoptosis as indicated by increased caspase 3 cleavage and increased PARP cleavage in reovirus-infected mice treated with the TGF-βRI inhibitor compared to untreated, reovirus-infected mice (Fig. 3A). This experiment was replicated using six separate individuals per treatment group from three separate litters. Immunohistochemistry staining of brain sections from littermates of the same treatment groups shows that increases in apoptosis occur in the absence of significant effects on viral antigen (green) distribution (Fig. 3B to D).

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FIG. 3. Inhibition of reovirus-induced TGF-β signal activation increases apoptosis in vivo. (A) Whole-brain lysates from mock-infected and reovirus-infected (1,000 PFU, i.c. injection) Swiss Webster pups sacrificed at day 8 postinfection following daily intraperitoneal treatment with TGF-βRI inhibitor (TGF-βRI Inh III) or vehicle control were resolved on 10% polyacrylamide gels, transferred to nitrocellulose membranes, and probed with anti-pSMAD3, anti-cleaved caspase 3 (cl-Casp3), anti-cleaved PARP (cl-PARP), and anti-β-actin. Immunoblots are representative of three individual replicates per treatment group from multiple litters. (B to D) Immunohistochemistry staining of the same treatment groups revealed no differences in viral antigen (green) or in the distribution of cleaved caspase 3 (red) in animals treated with vehicle control or TGF-βRI inhibitor. Images shown are of the cingulate cortex. Original magnification, x400.

Prior to in vivo experiments, analysis was first completed in HEK293 cells (data not shown). Cells were treated with a TGF-βRI inhibitor (SB431542), TGF-βRI inhibitor III, SMAD3 inhibitor, or vehicle controls and infected 30 minutes later with reovirus (T3A; MOI, 100). Western blot analysis of whole-cell lysates demonstrated that inhibitors of TGF-β signaling (TGF-βRI inhibitor III, SB431542, and SMAD3 inhibitor) inhibited the activation of SMAD3 and resulted in a corresponding increase in apoptosis, as measured by an increase in the large cleavage fragment (89 kDa) adjacent to Asp214 of PARP, in virus-infected cells (data not shown).

Together, these data demonstrate that TGF-β signaling is activated following viral infection in an in vivo model of viral encephalitis. Additionally, inhibition of TGF-β signaling in vivo resulted in increased cell death, indicating that TGF-β signaling is involved in the protective innate immune response to viral CNS infection.

Reovirus infection activates BMP signaling in vivo. Next, we evaluated the other major group of R-SMAD proteins (SMAD1, SMAD5, and SMAD8) for evidence of activation following reovirus infection. As shown by Western blot analysis of whole-cell lysates, reovirus-infected HEK293 cells expressed increased phosphorylated (Ser463/465) SMAD1 (pSMAD1). We found that pSMAD1 is upregulated at 24 h postinfection following reovirus infection (data not shown). In order to further understand the role of activated SMAD1 in an in vivo model of viral encephalitis, we either mock infected 2-day-old Swiss Webster mice or infected them with reovirus (T3A or T3D; 1,000 PFU, i.c.) and sacrificed mice at days 4, 6, and 8 postinfection. Whole-brain lysates were analyzed by Western blotting using an antibody specific for the BMPRI. We found a significant (P = 0.016) sevenfold increase in BMPRI expression in reovirus-infected brains at 8 days postinfection (Fig. 4A and B). We then wanted to evaluate the activation of SMAD1 in the same mice. Whole-brain lysates from the same treatment groups were analyzed by Western blotting using an antibody specific to pSMAD1. We found a significant (P = 0.02) sixfold increase in pSMAD1 by day 6 postinfection (Fig. 4C and D). Results shown are representative of six replicates per time point. Changes in pSMAD1 occurred in the absence of alterations in total SMAD1 expression (Fig. 4E).

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FIG. 4. Reovirus upregulates BMP signaling in vivo. (A) Western blot analysis. Whole-brain lysates from mock-infected and reovirus-infected (T3D, 1,000 PFU, i.c.) Swiss Webster pups sacrificed at day 8 postinfection were resolved on an 8% polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with anti-BMPRI and anti-β-actin. (B) Densitometry results from five individual replicates indicate a sixfold increase (P = 0.016) in BMPRI expression in reovirus-infected mouse brains. (C) Additional whole-brain lysate samples from the same treatment groups were analyzed with Western blotting at 4 and 6 days postinfection and probed with anti-pSMAD1 and anti-β-actin. (D) Densitometry results from eight individual replicates indicate a sixfold increase in pSMAD1 activation (P = 0.02) at day 6 postinfection. (E) Whole-brain lysate samples from the same treatment groups at day 8 postinfection were probed with anti-total SMAD1 (TSMAD1).

Reovirus-induced SMAD1 activation occurs in uninfected neurons in close proximity to regions of virus infection. Having shown that pSMAD1 is activated in reovirus-infected mouse brains, we wished to understand the localization of pSMAD1 in the brain and the association between viral antigen and pSMAD1. Two-day-old Swiss Webster mice were infected with reovirus (T3D or T3A; 1,000 PFU, i.c.) or were mock infected and were sacrificed at days 6 and 8 postinfection. Brain tissue sections from mice sacrificed at day 6 postinfection were analyzed using immunohistochemistry and revealed upregulation of pSMAD1 (red) in the cingulate cortex, hippocampus, and thalamus (Fig. 5A to F). Brain regions not typically susceptible to reovirus infection, such as the parietal cortex, exhibited little evidence of pSMAD1 upregulation (Fig. 5G). Additionally, viral antigen (green) and pSMAD1 (red) did not typically colocalize in the same cell; instead, phosphorylated SMAD1 was commonly observed in close proximity to antigen-positive cells (Fig. 5E and F).

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FIG. 5. SMAD1 activation occurs in virus antigen-negative neurons in regions of reovirus infection. (A to G) Brains from mock-infected (A to C) and reovirus-infected (D to G) (T3A and T3D, 1,000 PFU, i.c. injection) Swiss Webster pups sacrificed at days 6 and 8 postinfection were paraffin embedded, sectioned, permeabilized, and stained for fluorescent immunohistochemical analysis with anti-pSMAD1 (red) and anti-reovirus 3 viral antigen (green). (G) Representative sample of a brain region not infected by reovirus (parietal cortex) from a reovirus-infected mouse brain. (H to M) Adjacent sections from the same treatment groups were stained for fluorescent immunohistochemical analysis with anti-pSMAD1 (red) and anti-neuronal nucleus (green) (neuronal nucleus is a neuronal marker). All images are representative of three individual replicate brains per treatment group. Original magnification, x400.

Reovirus infection in mouse brains is almost exclusively limited to neurons (31, 37). We therefore wanted to determine whether SMAD1 activation was occurring in neurons or in glial cells. Immunohistochemical analysis of reovirus (T3D or T3A; 1,000 PFU, i.c.)-infected mouse brains at day 6 postinfection showed that pSMAD1 (red) was expressed predominantly in neurons (green; Fig. 5H to M). These data indicate that SMAD1 activation occurred in neurons in close proximity to reovirus-infected neurons and raise the possibility that SMAD1 activation protects neurons from viral infection.

Inhibition of reovirus-induced BMP signaling increases apoptosis. Our studies suggested that SMAD1 activation may protect cells against virus-induced cell death. In order to test this possibility, we treated HEK293 cells with a BMP agonist (recombinant BMP6 ligand), a BMPRI inhibitor (43), both BMP6 ligand and BMPRI inhibitor, or vehicle control and then infected the cells 30 min later with reovirus (T3A; MOI, 100). Western blot analysis of whole-cell lysates demonstrated upregulation of pSMAD1 by 24 h postinfection following reovirus infection (Fig. 6). Inhibition of BMP signaling substantially decreased pSMAD1 upregulation following reovirus infection with or without addition of BMP6 ligand (Fig. 6). Inhibition of BMP signaling in reovirus-infected cells was associated with increased PARP cleavage at 8 and 24 h postinfection compared to mock-infected cells in the presence or absence of BMP6 ligand (Fig. 6). Addition of BMP6 ligand, BMP inhibitor, or both to mock-infected HEK293 cells did not increase PARP cleavage. The presence of PARP cleavage as early as 8 h postinfection in reovirus-infected cells treated with a BMP inhibitor is substantially earlier than when PARP cleavage occurs in the absence of a BMP inhibitor (23). Similar to the TGF-β signal transduction studies, these data demonstrate that BMP signaling is activated following reovirus infection and that inhibition of this activation enhances cell death, indicating that TGF-β and BMP signaling pathways are important host cell innate responses to viral infection.

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FIG. 6. Inhibition of BMP signaling increases reovirus-induced apoptosis in vitro. For Western blot analysis, whole-cell lysates from mock-infected and reovirus-infected (T3A, MOI of 100) HEK293 cells treated with vehicle control, BMP6 ligand, and/or BMPRI inhibitor (BMP Inh) were resolved on 10% polyacrylamide gels, transferred to a nitrocellulose membrane, and probed with anti-pSMAD1, anti-cleaved PARP (cl-PARP), and anti-β-actin. The Western blot shown is representative of three experimental replicates.

BMP6 treatment of reovirus-infected MCCs prevents apoptosis. Having shown that inhibition of BMP signaling increased reovirus-induced cell death in vitro, we evaluated the protective effects of a BMP agonist (BMP6 ligand) on reovirus infection in primary MCCs. We treated primary MCCs with recombinant BMP6 ligand (100 ng/ml) or vehicle control for 30 min followed by reovirus infection (MOI, 100) or mock infection. Then, primary MCCs received either BMP6 or vehicle control daily until cells were harvested at days 4 and 5 postinfection. Blinded cell counts of fluorescent immunocytochemistry revealed significantly decreased caspase 3 cleavage (green) in BMP6 ligand-treated, reovirus-infected MCCs compared to untreated, reovirus-infected MCCs (P < 0.01) (Fig. 7A to D). BMP6-treated, reovirus-infected MCCs maintained neuronal markers (microtubule-associated protein 2; red) and neuronal morphology compared to untreated, reovirus-infected MCCs (Fig. 7B and C).

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FIG. 7. BMP6 ligand treatment of reovirus-infected MCCs prevents apoptosis. (A to C) Mock-infected (A) or reovirus-infected (B and C) (MOI, 100) primary MCCs were treated daily with vehicle control (B) or BMP6 ligand (C) and analyzed at day 4 postinfection using immunocytochemistry with antibodies to cleaved caspase 3 (green) and microtubule-associated protein 2 (red). Nuclear staining (Hoechst) is shown in blue. (D) Blinded cell counts of anti-cleaved caspase 3 (anti-cl-caspase 3)-positive cells in three replicate experiments show a threefold (P < 0.01) decrease in cl-caspase 3 expression in BMP6 ligand-treated, reovirus-infected neurons compared to untreated, infected neurons. (E) Whole-cell lysates from mock-infected and reovirus-infected (T3A, MOI of 100) MCCs treated daily with vehicle control or BMP6 ligand were probed with antibodies to pSMAD1, cleaved caspase 3, and β-actin. (F) Densitometry analysis results for cleaved caspase 3 expression from four replicate experiments indicated a threefold (P < 0.001) decrease in cleaved caspase 3 expression in BMP6 ligand-treated, reovirus-infected neurons compared to untreated, infected neurons.

Using the same treatment groups as described above to evaluate the effect of a BMP agonist on reovirus-induced apoptosis, we examined whole-neuron cell lysates by Western blotting for evidence of caspase 3 cleavage. We found that BMP6 treatment of reovirus-infected neurons significantly (P < 0.001) decreased apoptosis to the levels found in mock-infected primary MCCs (Fig. 7E and F). Together, these data indicate that enhancing BMP activation can greatly reduce virus-induced cell death in vitro.

BMP6 treatment does not significantly alter viral titer in primary MCCs. We have shown that BMP signaling is an important cellular innate immune response and is protective in reovirus-infected HEK cells and primary MCCs. We have also shown that BMP6 treatment of reovirus-infected MCCs significantly reduced apoptosis in infected neurons (Fig. 7E and F). We therefore wanted to evaluate the effect of BMP treatment on viral replication in primary MCCs. Primary MCCs were mock infected or reovirus infected (T3A and T3D; MOI, 100) followed by treatment with vehicle control or BMP6 ligand daily until cells were harvested. Subsequent cell pellets were harvested and analyzed by standard plaque assay techniques for quantification of cell-associated virus. Viral titers did not significantly differ between untreated, reovirus-infected MCCs (log₁₀ 7.8 ± 7.2) and BMP6 ligand-treated, reovirus-infected MCCs (log₁₀ 7.8 ± 7; P = 0.8) (Fig. 8). Therefore, BMP6 treatment of primary MCCs decreases apoptosis in the absence of a direct effect on viral replication.

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FIG. 8. BMP6 ligand treatment of reovirus-infected MCCs does not alter viral titer. Mock-infected or reovirus-infected (MOI of 100) primary MCCs were treated daily with vehicle control or BMP6 ligand. MCC cell pellets were harvested and analyzed at day 4 postinfection for viral titer. Values are averaged from four replicate experiments.

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This is the first study to establish that both TGF-β and BMP signaling are activated in the brains of infected mice. We extended these studies by showing that addition of BMP6 ligand to virus-infected neurons induced activation of SMAD1, resulting in neuronal protection from apoptosis (Fig. 7E and F). Similar protective effects with BMP treatment have been noted in models of breast cancer (14) and noninfectious neuronal injury (3, 15, 39). However, this is the first study to show that BMP signaling acts to reduce apoptosis in neurons following viral infection. Interestingly, BMP6 ligand treatment of virus-infected neurons significantly reduced cell death while having no significant effect on cell-associated viral titer (Fig. 8). These results echo our prior studies in which we used an in vivo pharmacologic inhibitor of JNK activation that resulted in reduced cell death and tissue injury with no significant decrease in viral titer (2). These data suggest that viral replication and virus-induced cell death can be disassociated and that successful therapy of viral encephalitis will require inhibition of both viral replication and virus-induced cell death. Additionally, reovirus replicates in the cytoplasm but triggers many specific signaling events in the nucleus such as SMAD activation. The role of these nuclear signaling events is not well understood, but some of these nuclear proteins may function as an important cellular response to viral infection.

In reovirus-infected mice, the majority of SMAD1 activation did not colocalize with viral antigen (Fig. 5D to F). Since reovirus infects neurons almost exclusively in vivo (31, 38), we verified that SMAD1 activation was primarily occurring in neurons (Fig. 5C to F). The majority of neurons positive for reovirus antigen did not colocalize with activated SMAD1; yet, activated SMAD1 occurred in close proximity to viral antigen-positive neurons, implying that SMAD1 is activated via a paracrine response to infection. Together with our in vitro studies showing that an agonist of BMP signaling protects neurons from apoptosis, we conclude that the BMP signal transduction pathway plays an important role in protecting neurons from viral injury and may represent a novel therapeutic target for therapy of viral infections of the CNS.

When analyzing pSMAD1 distribution in reovirus-infected neurons, we noted that the distribution of activated SMAD1 was very similar to that of STAT1 activation in reovirus-infected brains (18). Like pSMAD1, the majority of activated STAT1 does not colocalize with reovirus antigen, suggesting that specific neuroprotective cell signaling events are suppressed by reovirus infection in order to successfully infect the cell and trigger apoptosis. Given that viral infection continues to progress despite the presence of host cell antiviral responses, virus must be able to downregulate or circumvent cellular protective responses, as the majority of virus-infected cells are negative for activated SMAD1. Further studies are required to more fully understand a potential antiviral mechanism associated with BMP signal transduction.

Previous studies have used BMP6 ligand treatment in rodents (40) but were completed following a single intracerebral injection of BMP agonist. In our prior in vivo models of therapy, treatment of viral infection in the CNS requires multiple injections via an intraperitoneal route. Further improvements in pharmacologic delivery of BMP6 are needed before the efficacy of this treatment can be evaluated in the CNS.

Like BMP signaling, we show that TGF-β signal transduction is an important cellular defense against cell death. By inhibiting the activation of TGF-βRI, we were able to decrease the activation of pSMAD3 in vitro and in vivo, resulting in a corresponding increase in apoptosis. Mice treated with TGF-βRI inhibitor did not display accelerated disease symptoms and displayed no evidence of earlier histologic injury (data not shown), implying that clinical onset of disease is restricted by the replication dynamics of the virus and not the time course of cell death. In order to extend these findings, we treated reovirus-infected primary MCCs with TGF-β1 ligand in order activate the TGF-β signaling pathway. However, addition of TGF-β1 ligand did not increase SMAD3 activation in primary mouse cortical neurons or in HEK293 cells. While TGF-β1 is a common agonist of TGF-βRI and SMAD3 activation, it is possible that another TGF-β ligand, nodal, or activin may be responsible for activating this pathway in vivo. Further studies are needed in order to identify the specific ligand responsible for TGF-β signal activation in vivo.

Our findings establish that TGF-β and BMP signaling are activated during viral encephalitis, and these signal transduction pathways act as important cellular responses to viral infection. Further understanding of how these signal transduction pathways interact with other important cell signaling pathways, such as interferon-induced signal transduction, will provide new potential targets for neuroprotective strategies and novel therapeutic strategies for a disease with few successful treatment options.

 
We express our thanks to Jennifer Smith Leser for technical support.

This work was supported in part by VA Career Development Award-2, VA Merit funding, NIH 5K08AI076518, NIH 5R01NS050138, NIH 1R01NS051403, and ASCI Young Investigator Award.

 
* Corresponding author. Mailing address: University of Colorado Denver, Dept. Neurology (B182), 12700 E. 19th Avenue, Research Complex 2-5th Floor, Aurora, CO 80045. Phone: (303) 393-2874. Fax: (303) 393-4686. E-mail: Ken.Tyler{at}uchsc.edu

Published ahead of print on 11 March 2009.

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Journal of Virology, May 2009, p. 5035-5045, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02433-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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