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Journal of Virology, August 2002, p. 8335-8346, Vol. 76, No. 16
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.16.8335-8346.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Critical Role for Protein Tyrosine Phosphatase SHP-1 in Controlling Infection of Central Nervous System Glia and Demyelination by Theiler's Murine Encephalomyelitis Virus

Paul T. Massa,* Stacie L. Ropka, Sucharita Saha, Karen L. Fecenko, and Kathryn L. Beuler

Department of Neurology and Department of Microbiology and Immunology, Upstate Medical University, State University of New York, Syracuse, New York 13210

Received 25 February 2002/ Accepted 17 May 2002


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ABSTRACT
 
We previously characterized the expression and function of the protein tyrosine phosphatase SHP-1 in the glia of the central nervous system (CNS). In the present study, we describe the role of SHP-1 in virus infection of glia and virus-induced demyelination in the CNS. For in vivo studies, SHP-1-deficient mice and their normal littermates received an intracerebral inoculation of an attenuated strain of Theiler's murine encephalomyelitis virus (TMEV). At various times after infection, virus replication, TMEV antigen expression, and demyelination were monitored. It was found that the CNS of SHP-1-deficient mice uniquely displayed demyelination and contained substantially higher levels of virus than did that of normal littermate mice. Many infected astrocytes and oligodendrocytes were detected in both brains and spinal cords of SHP-1-deficient but not normal littermate mice, showing that the virus replicated and spread at a much higher rate in the glia of SHP-1-deficient animals. To ascertain whether the lack of SHP-1 in the glia was primarily responsible for these differences, glial samples from these mice were cultured in vitro and infected with TMEV. As in vivo, infected astrocytes and oligodendrocytes of SHP-1-deficient mice were much more numerous and produced more virus than did those of normal littermate mice. These findings indicate that SHP-1 is a critical factor in controlling virus replication in the CNS glia and virus-induced demyelination.


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INTRODUCTION
 
Neurotropic viruses that infect astrocytes and myelin-forming oligodendrocytes often lead to demyelinating disease similar to that seen in multiple sclerosis (11, 43, 56). Demyelination in rodent models for multiple sclerosis results in inefficient saltatory conduction of nerve fibers with accompanying motor deficits and limb paralysis (61, 62). Recent research has centered on understanding the mechanisms responsible for virus-induced demyelination in these animals and the genetic susceptibility to disease (3, 7, 8, 12, 20, 29, 44, 48). These studies have indicated that damage to oligodendrocytes and myelin may occur by multiple distinct pathways. Depending on the particular virus, these pathways include direct cytopathic effects of the virus in oligodendrocytes, virus-induced inflammatory immune responses promoted by infected glia in the white matter, or molecular mimicry between virus and myelin antigens (43, 56, 57). In each of these responses, the activities of proinflammatory cytokines, interferons, and virus-induced genes play an important role in promoting or protecting against oligodendrocyte pathology (6, 40, 44, 45, 51, 65). Therefore, the regulation of these activities in central nervous system (CNS) cells may be particularly important in controlling virus replication and virus-induced demyelinating processes. However, many of the host genes that control virus infection and demyelination in the CNS through multiple intracellular signaling pathways have not been identified.

Virus-induced genes provide for a rapid innate response to control virus replication at the earliest stages of infection. The activities of virus-induced cellular proteins, including interferons, cytokines, and intracellular signaling molecules, are controlled at multiple levels to provide for modulation of the antiviral state and inflammation (9, 19, 30, 38, 42, 55). Although these regulatory pathways have been extensively studied, such mechanisms in neural cells have been less well studied and may be unique. For instance, it was recently reported that interferons protected CNS neurons from virus infection but were unable to stimulate the expression of major histocompatibility complex class I genes in these cells (36). Multiple mechanisms likely are responsible for mediating tissue-specific antiviral responses in the CNS, but one such regulatory mechanism appears to involve SHP-1, a cytosolic protein tyrosine phosphatase that controls interferon and virus-induced signaling in the glia (16, 34, 37, 38, 66).

SHP-1 has been characterized as a key functional modulator of cytokine responses in hematopoietic and neural cells (9, 17, 19, 34, 42). The physiological ramifications of SHP-1 loss in animals have been extensively studied by using two independent strains of mice with natural mutations in the SHP-1 gene (53). Moth-eaten (me/me) mice have a single nucleotide deletion mutation which generates a cryptic mRNA splice donor site, a resulting frameshift, and a complete loss of SHP-1 protein expression (53). Viable moth-eaten (me[supi]v/mev) mice have a T-to-A transversion mutation in a splice donor that leads to the usage of cryptic donors on either side of the mutation. This situation results in an in-frame deletion and an insertion in the mRNAs, which encode a slightly smaller or larger SHP-1 protein with activity reduced to approximately 10% that in normal mice. Moth-eaten animals display a number of well-characterized hematopoietic abnormalities (32, 52); however, the regulatory role of SHP-1 in cells of epithelial origin, including glia, and the pathological consequences of SHP-1 loss in these cells have only recently been investigated (5, 16, 31, 42, 66, 67).

Massa and colleagues previously described the expression and functions of SHP-1 in astrocytes and oligodendrocytes, which represent the major macroglial populations of the CNS (34, 37, 38). By examination of glia from SHP-1-deficient mice (either moth-eaten or viable moth-eaten mice), they showed that SHP-1 controls gene expression induced by the proinflammatory cytokines gamma interferon (38) and interleukin-6 (34), both of which have been implicated in the pathogenesis of virus-induced demyelinating disease. Others have shown that SHP-1 also controls alpha/beta interferon signaling through alpha/beta interferon receptors in hematopoietic cells (9). Similar findings for oligodendrocytes have been reported elsewhere (P. T. Massa, S. L. Ropka, and S. Saha, abstract Immunology 12-16 May 2000, FASEB J. 14:A1084, 2000). Furthermore, it has been shown that SHP-1 controls the direct activation of NF-{kappa}B in astrocytes by viral mimetic double-stranded RNA (dsRNA) (37), which occurs as a consequence of the activation of the virus-inducible antiviral gene product double-stranded RNA-activated protein kinase (68). Taken together, these observations indicate that SHP-1 may control antiviral signaling pathways in the CNS glia. However, the biological significance of the regulation of virus-induced responses by SHP-1 has not been demonstrated. It was therefore of interest to determine whether SHP-1 controls the susceptibility of the CNS glia to infection by neurotropic viruses. To do this, we analyzed the susceptibility of SHP-1-deficient mice to a paralytic virus-induced demyelinating disease following infection with Theiler's murine encephalomyelitis virus (TMEV) (2, 47, 49, 59, 69).

We found that astrocytes and oligodendrocytes of mice lacking SHP-1 are extremely susceptible to TMEV infection both in vivo and in vitro and that these mice are highly susceptible to TMEV-induced demyelinating disease. In vitro, astrocytes and oligodendrocytes of SHP-1-deficient mice had a higher rate of infection and produced larger amounts of virus. We therefore propose that the susceptibility of astrocytes and myelin-forming oligodendrocytes to TMEV infection is controlled by innate antiviral responses mediated by SHP-1 within the CNS glia.


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MATERIALS AND METHODS
 
Mice. SHP-1-deficient moth-eaten (me/me) mice (C3HeB/FeJLe-a/a background) and viable moth-eaten (mev/mev) mice (C57BL/6 background) (53) and their phenotypically normal littermates (designated +/- for either me/+ or mev/+ and +/+) were produced from heterozygous breeding pairs obtained from Jackson Laboratories (Bar Harbor, Maine). Strain designations for heterozygous breeders are C3FeLe.B6-a/a Hcphme/+ (stock no. 000225) for moth-eaten mice and C57BL/6J Hcphmev/+ stock no. 000811) for viable moth-eaten mice.

Glial cultures. Glial cultures containing astrocytes and oligodendrocytes were produced from newborn mice as previously described (38). Cerebral hemispheres were used for cultures, and cerebella were used to probe SHP-1 in Western immunoblots to identify either moth-eaten or normal littermate mice. Genomic DNA was isolated from cerebellar tissue for verification of normal and mutant SHP-1 gene structures of moth-eaten mice as previously described (53). Brains from littermates of heterozygous breeding pairs having the moth-eaten mutation of the SHP-1 gene (either moth eaten or viable moth eaten) were dissected and mechanically dissociated for separate cultures. Cells in approximately 10 ml of Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum per brain were plated on 60-mm culture dishes and then fed with fresh medium consisting of DMEM containing 10% heat-inactivated horse serum at 5 days after plating. Glial cultures were used at 14 days postplating to analyze TMEV replication.

Virus and cell lines. The attenuated strain of TMEV, BeAn 8386, was obtained from the American Type Culture Collection (ATCC), Manassas, Va. (ATCC VR-995, originally contributed by H. L. Lipton) (27, 28). BeAn 8386 was prepared by propagating in BHK-21 cells (ATCC CCL-10) and harvesting in tissue culture supernatant at 2 x 105 PFU/ml. Purified virus stock was prepared from the tissue culture supernatant by polyethylene glycol precipitation and sucrose density gradient centrifugation. Briefly, virus-containing culture supernatant was clarified by centrifugation at 2,500 x g for 20 min. Virus was precipitated with 8% polyethylene glycol in 1.6 M NaCl (50). The concentrated viral lysate was then treated with 1% sodium dodecyl sulfate for 10 min and centrifuged over a 20 to 70% continuous sucrose gradient at 160,000 x g in a Beckman SW41 rotor. This purified stock contained 7.4 x 106 PFU/ml in BHK-21 cells.

Virus inoculation. Weanling mice were anesthetized with methoxyflurane and inoculated intracerebrally (i.c.) in the left hemisphere with 1.48 x 103 PFU of BeAn 8386 in a volume of 0.02 ml. Mice were observed on a daily basis for signs of paralysis. Paralyzed moth-eaten mice were euthanatized with age-matched normal littermates, and the brains and spinal cords were removed and stored at -80°C until assayed for viral infectious units. Additionally, normal littermate and diseased moth-eaten mice were anesthetized and perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS), and the brains and spinal cords were embedded in paraffin for immunohistochemical analysis. For in vitro studies, glial cell cultures grown on 60-mm plates or plated on glass chamber slides were inoculated with purified virus stock (3.7 x 106 PFU [total]/dish) at a multiplicity of infection of 1.0 and incubated for 1 h at 37°C. Afterward, the inoculum was removed, and the cultures were rinsed with PBS and refed with DMEM containing 10% normal horse serum. The cultures were then incubated for 3 and 6 days, triplicate cell supernatants and cell lysates were harvested, and virus titers were determined by plaque assays on BHK-21 cells.

Virus plaque assays. To quantify the production of TMEV, brains (brain plus brain stem) and spinal cords were prepared separately as 10% homogenates in DMEM and clarified by centrifugation as described above. The resulting supernatants were assayed for virus titers. Both in vivo and in vitro samples were assayed for virus titers by standard plaque assays (14). Briefly, BHK-21 cell monolayers were grown on 60-mm dishes. Cell monolayers were inoculated with virus-containing samples (tissue homogenates or cell supernatants) and incubated for 1 h at 37°C. Inoculated cell monolayers were overlaid with 1% agar in DMEM containing 2% fetal bovine serum. Four days after infection, the agar layer was removed, and the cell monolayers were fixed with methanol and then stained with 1% crystal violet in 20% ethanol. Plaques per dish were counted, and PFU per milliliter or per gram were determined.

Immunohistochemical analysis. Double immunohistochemical analysis of myelin basic protein (MBP), myelin proteolipid protein (PLP), or glial fibrillary acidic protein (GFAP) and TMEV antigens was performed by deparaffinizing microtome sections of brains and spinal cords, rehydrating the samples in graded ethanols, and blocking the samples in 10% normal horse serum. The spinal cord sections were incubated with monoclonal antibodies to either MBP (rat monoclonal immunoglobulin G [IgG] against MBP; MCA 409; Accurate Chemical and Scientific Corp., Westbury, N.Y.), PLP (mouse monoclonal IgG against myelin PLP; clone plpc1; Oncogene Research Products, Cambridge, Mass.), or GFAP (rat monoclonal IgG; Zymed Laboratories, South San Francisco, Calif.) overnight; this step was followed by rinsing and incubating the sections in goat anti-rat or anti-mouse IgG conjugated to tetramethyl rhodamine isothiocyanate (TRITC; Zymed). The sections were further incubated overnight with rabbit antiserum against TMEV strain BeAn 8386 (provided by H. L. Lipton, Northwestern University) at a 1:2,000 dilution in 10% normal horse serum-PBS. After being rinsed, the cultures were incubated in goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC; Zymed). Coverslips were mounted with fluorescence mounting medium (Dako Corp., Carpinteria, Calif.) and viewed by epifluorescence microscopy with a Zeiss Axioskop microscope.

To visualize demyelination and infection in individual adjacent sections, spinal cord sections were stained for MBP or TMEV as described above, but the biotin-avidin-alkaline phosphatase technique with the blue 5-bromo-4-chloro-3-indolylphosphate (BCIP)-nitroblue tetrazolium product was used for detection. Alternatively, sections were incubated with goat anti-rat IgG conjugated to FITC as a secondary detection reagent. Inflammatory infiltrates were detected by staining adjacent sections of spinal cords and brain with hematoxylin and eosin (H+E). To visualize infection of oligodendrocytes in vitro by double immunofluorescence, live infected and noninfected cells were incubated at 4°C with mouse monoclonal antibody to oligodendrocyte-specific O1 antigens (4, 54); this step was followed by fixation and incubation with goat anti-mouse IgG conjugated to FITC. The cells were then permeabilized with 0.25% Triton X-100, incubated with anti-TMEV antibodies, and finally incubated with goat anti-rabbit antibodies conjugated to TRITC.


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RESULTS
 
Increased paralysis in TMEV-infected SHP-1-deficient mice. Moth-eaten (me/me) mice and normal littermate mice (C3FeLe.B6 background) infected with attenuated TMEV strain BeAn 8386 by i.c. injection were monitored daily for clinical signs, including limb paralysis. Moth-eaten (me/me) mice first appeared lethargic at approximately 3 to 4 days after infection, while normal littermates remained healthy (Table 1). By day 10, a majority of infected moth-eaten mice displayed clinical signs of spastic limb paralysis that rapidly progressed to quadriplegia and a moribund state in all infected animals. In distinction, normal littermates were only partially susceptible, with a third of the animals developing paralytic disease but at much later times after infection (4 to 5 weeks) (Table 1), compared to their moth-eaten littermates. Like moth-eaten mice, viable moth-eaten mice (mev/mev) also showed complete susceptibility to TMEV. However, their normal littermates (C57BL/6 background) were entirely resistant for up to 6 months after infection (Table 1). Infected mev/mev mice generally developed disease a few days later than me/me mice (10 to 15 days after infection).


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  		TABLE 1. Incidence of spastic paralysis in normal, moth-eaten, and viable moth-eaten mice infected with TMEV and concurrent oligodendrocyte infection and demyelination

Immunohistochemical examination of demyelination in moth-eaten mice. The ability of TMEV to produce spastic limb paralysis at early times after infection in moth-eaten mice was surprising, because this type of paralysis is a relatively late event in wild-type susceptible mice (27, 28, 60, 63). Because limb spasticity suggested possible demyelination in the spinal cords of moth-eaten mice (39), the ability of TMEV infection to cause spinal cord demyelination was assessed with immunohistological sections stained for MBP (Fig. 1 and 2). Uninfected moth-eaten mice showed the expected distribution of MBP staining in white matter tracts of the spinal cord (Fig. 1C). In TMEV-infected moth-eaten mice, MBP staining was sharply reduced in both white matter and gray matter, with some white matter regions of both dorsal (Fig. 1D) and ventral (Fig. 2A) tracts displaying extensive areas of focal demyelination. Some lesions were obviously hemorrhagic, with conspicuous red blood cells at the center of the lesions (Fig. 1D), features not seen in normal littermate or uninfected moth-eaten mice (Fig. 1A to C). In H+E-stained adjacent sections, areas of demyelination showed considerable white matter cellular infiltrates, especially in the vicinity of blood vessels (Fig. 2C). In contrast to what was seen in the spinal cord, no such large areas of focal demyelination or inflammation were detected in sagittal midline sections of the brain (data not shown), indicating that the spinal cord was particularly sensitive to virus-induced demyelination.



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  		FIG. 1. Focal demyelinating lesions in dorsal cervical spinal cords of moth-eaten (me/me) mice at 5 days after i.c. inoculation with the attenuated BeAn 8386 strain of TMEV. Five-micrometer paraffin sections were stained with rat monoclonal antibody to MBP and labeled with FITC-conjugated secondary antibody. Sections were photographed with color film by double exposure under both FITC (green) and red fluorescence filter sets. Green fluorescence labels MBP. Red profiles above the background in panel D indicate autofluorescent red blood cells in focal hemorrhagic lesions in regions of demyelination in the parenchyma of the spinal cord. The dorsal funiculus of the cervical spinal cord is shown in either TMEV-infected (B and D) or sham-infected (A and C) normal littermate mice (A and B) and me/me mice (C and D).



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  		FIG. 2. Demyelination, virus infection, and inflammation in the ventral cervical spinal cords of me/me mice 5 days after intracerebral inoculation with TMEV. Immunohistochemical staining of MBP (A) and TMEV (B) and H+E staining (C) in adjacent 5-µm paraffin sections are shown.

Immunofluorescence identification of infected cells in spinal cords and brains of TMEV-infected moth-eaten mice. Inspection of the most severely demyelinated areas in adjacent sections of the spinal cord by using an antiserum to TMEV showed numerous virus antigen-containing cells in both gray matter and white matter regions (Fig. 2B). Some cells in the gray matter could be morphologically identified as large dorsal horn motoneurons (Fig. 3 and 4). However, many other smaller cells containing TMEV antigens were scattered throughout the spinal cord and could not be morphologically identified. In the brain, many infected cells were detected in the white matter (Fig. 5), but none were detected in distinct neuronal layers in the cerebral cortex, hippocampus, or brain stem nuclei (data not shown). Of particular note, almost all of the TMEV antigen was localized to small cells in white matter tracts, especially in the corpus callosum. Taken together, these observations indicated that TMEV-infected cells in white matter regions occurred in both brains and spinal cords of SHP-1-deficient animals and that demyelination was extensive in the spinal cord in areas of TMEV infection.



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  		FIG. 3. Double immunofluorescence of TMEV (A, C, and E; FITC) and PLP (B, D, and F; TRITC) in the ventral cervical spinal cords of me/me (A to D) mice 5 days after inoculation with TMEV. (A and B) Doubly labeled cells (arrows) in the ventral funiculus at the interface between the white matter and the gray matter of the medial nuclei. A large motoneuron in the gray matter also contains TMEV antigen (arrowhead). (C and D) Doubly labeled cells (arrows) in a demyelinated region of the ventral funiculus adjacent to the ventral median fissure (*). (E and F) No TMEV antigens are seen in cervical spinal cords of +/- animals infected with TMEV.



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  		FIG. 4. TMEV infection of GFAP-positive astrocytes in moth-eaten mouse cervical spinal cords (A and B) and corpus callosa (C and D) 5 days after inoculation with TMEV. Arrows indicate astrocytes doubly labeled for TMEV (FITC) and GFAP (TRITC). The arrowheads in panels A and B indicate TMEV-infected neurons in the spinal cord gray matter that are not stained for GFAP. (E and F) Normal littermate mouse cervical spinal cords show no TMEV-infected cells.



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  		FIG. 5. Double immunofluorescence of MBP (TRITC; red) and TMEV (FITC; green) in the brain 5 days after inoculation with TMEV. Five-micrometer paraffin sections were stained with rat monoclonal antibody to MBP and labeled with TRITC-conjugated secondary antibody. TMEV antibodies were detected with FITC-conjugated antibodies. Sections were photographed with color film by double exposure of the same frame under both FITC and TRITC filter sets. (A) x400 magnification of the corpus callosum (lower) at the interface with the cerebral cortex (upper). (B) Higher magnification (x630) of an area similar to that in panel A but centered at the interface, where myelination is sparse, to allow resolution of doubly labeled cells (brownish yellow cell bodies indicated by arrows).

To determine whether some of the small infected cells in the spinal cord and brain white matter regions were glial cells, sections were doubly labeled for TMEV and oligodendrocyte-specific antigens. While cell bodies in the spinal cord were not discernibly stained with MBP antibodies, many cells in the spinal cord were doubly labeled for both TMEV and oligodendrocyte-specific PLP, especially in areas of diffuse myelin in demyelinating lesions (Fig. 3). In the brain, infected oligodendrocytes were doubly labeled for MBP and TMEV at the interface of the corpus callosum and cerebral cortex, where myelination was sparse enough to allow resolution of the cell bodies in magnified micrographs (Fig. 5B). Nonetheless, not all infected cells in the white matter were labeled with PLP or MBP (Fig. 5). Many of these infected cells were doubly labeled for GFAP in both the spinal cord (Fig. 4A and B) and the brain (Fig. 4C and D), indicating that astrocytes were also productively infected in moth-eaten mice. In contrast, no infected cells were detected in the spinal cords or brains of age-matched normal littermate mice that had received TMEV inoculation (Fig. 3 and 4). Taken together, these data indicated that astrocytes and oligodendrocytes of SHP-1-deficient animals were particularly susceptible to TMEV infection and that TMEV rapidly spread from the brain to the glia in the spinal cord.

Virus spread and replication in moth-eaten mice. As noted above, TMEV-infected astrocytes and oligodendrocytes were detected in both brains and spinal cords of moth-eaten mice but not in infected normal littermate mice. We reasoned that the replication and spread of TMEV may be much greater in moth-eaten mice. Therefore, we assayed infectious virus in brains and spinal cords of moth-eaten and normal littermate mice after infection. For these studies, moth-eaten (me/me), viable moth-eaten (mev/mev), and normal littermate mice were infected i.c. with 1.5 x 103 PFU/brain. Normal littermates of viable moth-eaten mice had essentially no detectable infectious virus in the brain, on average (0.6 PFU/g of brain); however, diseased viable moth-eaten mice contained an average of 1.4 x 105 PFU/g of brain, constituting approximately a million more virus particles per gram of tissue than the levels found in normal mouse brain (Fig. 6A). Consistent with the latter results, viable moth-eaten mice had nearly 1,000-fold more virus particles per gram of tissue in the spinal cord than did normal littermates (Fig. 6B). Moth-eaten (me/me) mice also contained higher virus titers in brains and spinal cords than did their normal littermates (Fig. 6C and D). However, unlike normal littermates of viable moth-eaten mice (C57BL/6 background), normal littermates of moth-eaten mice (C3FeLe.B6 background) had substantial virus titers in both brains and spinal cords, indicating differences in background susceptibility, in agreement with the data in Table 1. Despite this level of virus replication, repeated immunohistochemical analysis was not able to detect virus antigen-containing cells in C3FeLe.B6 normal littermates, perhaps due to the lower sensitivity of this assay. Nonetheless, plaque assays indicated that both replication and spread of TMEV were clearly increased in the two strains of SHP-1-deficient mice, in accord with their increased susceptibility to clinical disease compared to the status of their normal littermates.



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  		FIG. 6. TMEV titers in brains and spinal cords of infected mev/mev and me/me mice and normal littermate (+/- Control) mice. Brains (A and C) and spinal cords (B and D) were harvested from paralyzed viable moth-eaten (mev/mev) (A and B) or moth-eaten (me/me) (C and D) animals along with age-matched sham-infected normal littermate animals (+/- Control). Error bars indicate standard errors of the means. Numbers in the histograms are for control PFU per gram of brain or cord where these cannot be read from the ordinate. Differences in the means between normal littermate mice and either moth-eaten or viable moth-eaten mice were significant (P < 0004) for both brains and spinal cords.

Analysis of TMEV infection of moth-eaten mouse glia in vitro. The increased infection of astrocytes and oligodendrocytes and the concomitant demyelination in SHP-1-deficient mice suggested a possible alteration in direct virus-oligodendrocyte interactions dependent on SHP-1 in these cells. To test this possibility, we analyzed TMEV replication in glial cell cultures containing astrocytes and oligodendrocytes produced from moth-eaten and normal littermate mice. Glial cell cultures were inoculated with 7.4 x 106 PFU of TMEV/ml (multiplicity of infection, 1.0) and then incubated for 3 days after inoculation. We first analyzed the numbers and types of glial cells infected by using double immunohistochemical analysis. To analyze oligodendrocyte infection, cultures were stained with oligodendrocyte-specific antibody to O1 antigens and subsequently for intracellular TMEV antigens. O1 antigen-positive oligodendrocytes expressing TMEV antigens were readily identified in both moth-eaten and normal littermate glial cell cultures (Fig. 7A); however, the number of oligodendrocytes infected was much higher (approximately 10-fold) in moth-eaten mouse cultures (Fig. 7B). Additionally, many O1 antigen-negative cells in moth-eaten mouse cultures contained TMEV antigens and had an astrocytic morphology. To ascertain whether these cells were astrocytes, cultures were doubly labeled for GFAP and TMEV antigens. Many GFAP-positive astrocytes were found to contain TMEV antigens in moth-eaten mouse cultures, but none were seen in normal littermate cultures (Fig. 8). Taken together, the immunofluorescence data showed that TMEV produced much more infection of O1 antigen-positive oligodendrocytes and GFAP-positive astrocytes in moth-eaten than in wild-type mouse glial cell cultures.



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  		FIG. 7. Frequency of oligodendrocyte infection in vitro. (A) Double-immunofluorescence analysis of TMEV infection of O1 antigen-positive oligodendrocytes in moth-eaten and normal littermate mice 2 days after inoculation. Individual cells plated on glass chamber slides were labeled for TMEV (TRITC), and O1 antigens (FITC) were photographed at a x630 magnification. (B) Histogram of doubly labeled TMEV-positive, O1 antigen-positive oligodendrocytes counted in random fields at a x120 magnification in +/-and me/me cultures infected for 2 days with TMEV. O1 antigen-positive oligodendrocytes (TMEV positive plus TMEV negative) were present at the same densities in the two samples. Error bars indicate standard errors of the means.



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  		FIG. 8. Double-immunofluorescence analysis of TMEV infection of GFAP-positive astrocytes in moth-eaten and normal littermate mice 2 days after inoculation. Glial cultures of +/- mice (A to D) or me/me mice (E to H) were either inoculated with TMEV (C, D, G, and H) or sham inoculated (A, B, E, and F). The left panels were stained for TMEV antigens, and the corresponding fields represented in the right panels were stained for GFAP.

Virus production in the glia of normal and moth-eaten mice. To ascertain whether moth-eaten mouse glial cells produced higher virus titers than normal littermate glial cells, infectious virus in supernatants and cell lysates from the above-described infected glial cell cultures was assessed at 3 and 6 days after infection. Mean virus titers (PFU per milliliter of supernatant) were significantly higher (approximately fivefold) in me/me mouse cultures than in normal littermate mouse cultures (Fig. 9). Analysis of lysates of infected cells at 3 days after infection indicated that the majority of the virus was cell associated in both me/me glia and normal littermate glia and that me/me glia contained significantly more virus particles than normal littermate glia. At 6 days after infection, the ratio between released virus and cell-associated virus was increased in both me/me and normal littermate glial cell cultures, indicating that a higher proportion of virus was being released from the cells as the infection progressed. Nonetheless, the difference in the amounts of released virus and cell-associated virus between me/me glia and normal littermate glia increased over time (Fig. 9). Taken together with the immunohistochemical data, these data indicated that moth-eaten mouse astrocytes and oligodendrocytes sustained a higher level of virus production than did the glia of normal littermates.



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  		FIG. 9. TMEV titers in vitro. Glial cultures of either me/me mice or normal littermate mice (+/- Control) were inoculated with TMEV. Virus was harvested in both supernatants (released) and cell lysates (cell associated) at 3 and 6 days after infection (P.I.) and quantified by plaque assays. Histograms indicate the mean PFU per milliliter in supernatants and PFU per cell based on protein content in cell lysates. Statistical differences between specimens were measured by Student's t test (one tailed). Each experiment was performed in triplicate. Error bars indicate standard errors of the means. Differences in the means between normal littermate (+/- Control) and moth-eaten (me/me) glial cultures were significant at each time after infection for both released and cell-associated viruses (P < 0.001).


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DISCUSSION
 
In the present report, we have shown that SHP-1 is a critical determinant in controlling virus replication in the glia of the CNS. Further, in vitro studies suggested that SHP-1 may control virus replication at least in part at the level of direct virus-cell interactions. However, the way in which SHP-1 may function to control virus replication in these cells and whether direct virus-cell interactions controlled by SHP-1 fully account for increased virus growth in SHP-1-deficient mice in vivo are not known. To address possible alterations in virus-cell interactions, we are currently investigating multiple antiviral pathways that are likely to be affected by SHP-1 in the CNS glia in vitro. For instance, the role of an innate antiviral state including the interferon system has been shown to be critically important for controlling infection by TMEV in the CNS (12). Such studies may be relevant to the possible role of SHP-1 in controlling TMEV infection, because previous studies showed that SHP-1 altered STAT1 activation in response to interferons (9, 38, 42). One possibility is that increased induction of STAT1 enhances the expression of proapoptotic genes (22), which may increase virus replication in the CNS, as recently described for Sindbis virus infections of neurons (25). However, other distinct antiviral pathways may be directly affected by the loss of SHP-1 in the CNS glia. The role of SHP-1 in controlling NF-{kappa}B activation by dsRNA in astrocytes was recently described. It is known that NF-{kappa}B activation by dsRNA is mediated by the antiviral gene product double-stranded RNA-activated protein kinase (68). Additionally, dsRNA is known to affect the expression of other virus-induced transcription factors and genes (13, 23) that may also be modulated by SHP-1 activity in virus-infected cells. Finally, it was recently shown that SHP-1 is required for the induction of neuronal nitric oxide synthetase (NOS1) activity in nonhematopoietic cells (31), and NOS1 activity has been shown to be a critical antiviral activity for controlling CNS virus infections (21, 46). Future studies will be aimed at determining the role of SHP-1 in regulating these multiple antiviral pathways in the CNS.

In a number of models of virus-induced demyelinating disease, infection often involves astrocytes and oligodendrocytes in the white matter in the vicinity of demyelinating lesions (1, 2, 18, 24, 44, 47, 49, 69). Demyelination caused by virus infection in CNS white matter can result from at least two mechanisms that are relevant to the present study. One is direct cytopathic effects of the virus on oligodendrocytes, and the second is an indirect immunopathologic response to the virus or autoantigens involving inflammation and oligodendrocyte pathology. Of particular note, the latter is often promoted by proinflammatory cytokine secretion and major histocompatibility complex expression induced by viruses in astrocytes (10, 26, 33, 35, 57, 58). The relative contributions of these mechanisms to the demyelinating process in TMEV-infected moth-eaten mice are presently unknown. Nonetheless, increased virus replication in oligodendrocytes and astrocytes is likely to promote both pathways to myelin degeneration. With respect to possible immunopathology in the demyelinating process, extensive demyelinating lesions in spinal cords showed increased levels of cellular infiltrates indicative of an inflammatory component. However, inflammation may occur as a secondary event in the removal of myelin debris by phagocytic cells following demyelination, such as is seen in toxin-induced models (15). Therefore, the mechanism of virus-induced demyelination in mice lacking SHP-1 remains to be determined but is most likely controlled by both direct and indirect consequences of increased virus replication in astrocytes and oligodendrocytes in the white matter of these mice.

The present and previous studies on virus-induced demyelination indicate that mechanisms of demyelination are complex and are controlled by multiple genes that regulate innate, adaptive, and autoimmune responses (11, 41, 56, 64). In the present report, we have focused on a genetic alteration that may act at the level of the CNS glia for controlling TMEV replication. We have found that, compared to normal littermates, mice lacking SHP-1 produce more virus in brains and spinal cords after TMEV infection, succumb to a rapid-onset demyelinating disease, and display early spastic limb paralysis. We believe that disease in mice with a genetic deficiency in SHP-1 activity is caused by a specific defect in innate antiviral responses against TMEV in the CNS glia. Our current studies are directed at discovering virus-glia interactions that are altered in the absence of SHP-1 and that lead to increased virus replication, oligodendrocyte pathology, and demyelination.


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ACKNOWLEDGMENTS
 
This work was supported by grants from the National Institutes of Health (NS41593-01) and from the National Multiple Sclerosis Society (RG 2569B4/5).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Neurology and Department of Microbiology and Immunology, Upstate Medical University, State University of New York, 750 E. Adams St., Syracuse, NY 13210. Phone: (315) 464-7606. Fax: (315) 464-6402. E-mail: massap{at}upstate.edu. Back


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Journal of Virology, August 2002, p. 8335-8346, Vol. 76, No. 16
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.16.8335-8346.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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