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Glycan Engagement Dictates Hydrocephalus Induction by Serotype 1 Reovirus

ABSTRACT Receptors expressed on the host cell surface adhere viruses to target cells and serve as determinants of viral tropism. Several viruses bind cell surface glycans to facilitate entry, but the contribution of specific glycan moieties to viral disease is incompletely understood. Reovirus provides a tractable experimental model for studies of viral neuropathogenesis. In newborn mice, serotype 1 (T1) reovirus causes hydrocephalus, whereas serotype 3 (T3) reovirus causes encephalitis. T1 and T3 reoviruses engage distinct glycans, suggesting that glycan-binding capacity contributes to these differences in pathogenesis. Using structure-guided mutagenesis, we engineered a mutant T1 reovirus incapable of binding the T1 reovirus-specific glycan receptor, GM2. The mutant virus induced substantially less hydrocephalus than wild-type virus, an effect phenocopied by wild-type virus infection of GM2-deficient mice. In comparison to wild-type virus, yields of mutant virus were diminished in cultured ependymal cells, the cell type that lines the brain ventricles. These findings suggest that GM2 engagement targets reovirus to ependymal cells in mice and illuminate the function of glycan engagement in reovirus serotype-dependent disease.

is found on glycoproteins and the GM2 ganglioside, and it is possible that T1 reovirus can bind GM2-conjugated glycoproteins, glycolipids, or both. T1 reovirus-GM2 binding is likely physiologic, as soluble GM2 glycan inhibits the capacity of T1 but not T3 reovirus to infect mouse embryonic fibroblasts (MEFs) (22). Conversely, T3 reovirus binds the GM3 glycan (23), which contains ␣2,3-linked sialic acid, as well as ␣2,6and ␣2,8-linked sialyloligosaccharides (23). Soluble GM3 glycan does not inhibit the capacity of T1 reovirus to infect MEFs (22), indicating that reovirusglycan interactions are serotype dependent. These observations suggest that differences in glycan utilization by T1 and T3 reoviruses contribute to serotype-specific disease. In support of this idea, sialic acid-binding T3 strains infect neurons more efficiently than do non-sialic acid-binding strains (24), and consequently, sialic acid engagement enhances the capacity of T3 reovirus to cause encephalitis (24). The contribution of T1 reovirus-glycan interactions to the pathogenesis of hydrocephalus is not known.
Reovirus attachment protein 1 is an elongated, trimeric fiber comprising tail, body, and head domains. Using X-ray crystallography and saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy, we found that the T1 1 head domain interacts with the Neu5Ac and GalNAc components of the GM2 glycan (22). Structure-guided mutagenesis revealed that residues Ser370, Gln371, and Val354 are required for functional GM2 engagement in cell culture. Viruses containing the S370P or Q371E mutation in 1 display an impaired capacity to agglutinate human erythrocytes and infect MEFs, which are most susceptible to GM2-binding T1 strains. Viruses containing these mutations replicate efficiently in L929 (L) cells, which support infection by strains incapable of binding sialic acid (22). Thus, the defect in infectivity in MEFs by strains altered in GM2 binding is linked to an altered capacity to engage glycans.
In this study, we engineered a virus containing both the S370P and Q371E mutations in the 1 attachment protein of prototype T1 strain type 1 Lang (T1L) for use in studies to determine the function of reovirus-GM2 interactions in disease. STD-NMR experiments demonstrate that in comparison to wild-type T1L 1, the S370P/Q371E mutant 1 does not bind GM2. We infected mice with T1L and the S370P/Q371E mutant virus and found that glycan-binding activity does not markedly influence replicative capacity in the brain. However, the degree of hydrocephalus induced by wild-type T1L is substantially greater than that induced by the S370P/Q371E mutant. Concordantly, T1L induces less severe hydrocephalus in mice that do not express GM2. These findings establish GM2 binding as an important determinant of serotype-specific reovirus disease and shed light on mechanisms of virus-induced hydrocephalus.

Molecular characterization of the GM2-binding mutant virus strain.
To engineer a T1 reovirus incapable of engaging GM2, we used site-directed mutagenesis to construct an S370P/Q371E double-residue mutant virus combining two single-residue T1 1 mutations that alter glycan-binding capacity (22). This doubleresidue mutant replicates comparably to wild-type T1L in L cells (see Fig. S1 in the supplemental material), which are equally susceptible to glycan-binding and non-glycan-binding reovirus strains (22,23). Particle-to-PFU ratios were comparable, averaging 162:1 and 148:1 for wild-type and mutant virus stocks, respectively. Additionally, both wild-type and S370P/Q371E virus strains are neutralized by monoclonal antibody (MAb) 5C6 (see Fig. S2 in the supplemental material), a conformation-specific antibody that binds to a trimeric form of the T1 1 head domain (2). Collectively, these data suggest that the engineered mutations do not alter the folding or encapsidation of the T1 1 molecule. T1 reovirus-mediated agglutination of human erythrocytes requires binding to carbohydrates on the erythrocyte surface (25). To test whether the S370P/Q371E mutation impairs glycanbinding capacity, we quantified the hemagglutination titers of wild-type and mutant virus using human erythrocytes. Although the mutant retained some hemagglutination capacity, hemagglutination titers of the S370P/Q371E mutant were 32-fold lower than those of wild-type T1L (Fig. 1A). To determine whether residual hemagglutination capacity reflects residual glycan binding, we treated erythrocytes with Arthrobacter ureafaciens neuraminidase, which removes cell surface sialic acid, or phosphate-buffered saline (PBS) as a control prior to incubation with strain T1L and the S370P/Q371E mutant. T1L-mediated hemagglutination was impaired following neuraminidase treatment, whereas S370P/ Q371E was not (Fig. 1B), indicating that the residual hemagglutination capacity of the S370P/Q371E mutant is not attributable to sialylated glycan engagement. As expected, hemagglutination activity of prototype T3 strain type 3 Dearing (T3D) was abolished by neuraminidase treatment of erythrocytes (26). Incubation of wild-type and mutant T1 reovirus strains with T1 1-specific MAb 5C6 prevented hemagglutination but had no effect on hemagglutination by strain T3D (Fig. 1B). These findings suggest that T1L, but not the S370P/Q371E mutant, binds sialic acid to agglutinate human erythrocytes.
To determine whether the S370P/Q371E 1 attachment protein retains any residual GM2-binding activity, we assessed the binding of wild-type T1L and mutant S370P/Q371E 1 proteins to GM2 by STD-NMR, a technique capable of assessing lowaffinity interactions between a large molecule and a small ligand (27), including virus-glycan interactions (28,29). STD-NMR is based on selective irradiation of protons in the protein and detection of the subsequent magnetization transfer from the protein to the ligand. Protons in the Neu5Ac and GalNAc moieties of GM2 were found to interact with wild-type T1L 1 (Fig. 1C), reflecting the binding contacts in the crystal structure of the glycan with the T1L 1 head (22). The S370P/Q371E double-residue mutant 1 protein did not interact with the GM2 glycan as assessed by STD-NMR (Fig. 1C), indicating that the S370P/Q371E virus is incapable of binding GM2.
To test whether the S370P/Q371E mutant virus displays an altered capacity to infect cells that are maximally susceptible to glycan-binding reovirus strains, we inoculated MEFs, which require binding to GM2 for optimal infection by T1 strains (22), with wild-type T1L and the S370P/Q371E mutant. The S370P/ Q371E mutant displayed a diminished capacity to infect these cells compared with T1L at all multiplicities of infection (MOIs) tested ( Fig. 2A). To establish whether the differences in infectivity displayed by wild-type and mutant viruses in MEFs result from compromised viral attachment, T1L and the S370P/Q371E mutant were adsorbed to MEFs and L cells at 4°C to prevent internalization, and the percentage of cells bound by virus and the mean fluorescence intensity (MFI) were quantified using flow cytometry. The S370P/Q371E mutant adhered much less efficiently to MEFs than did T1L ( Fig. 2B; also, see Fig. S3 in the supplemental material), whereas both strains bound comparably to L cells (see Fig. S4 in the supplemental material). Thus, the S370P/Q371E mutant has a defect in attachment to and infectivity of cells that require glycan engagement for optimal infection.
Contribution of glycan engagement to reovirus tropism. To determine whether T1 1 GM2-binding capacity influences T1 reovirus infection of the murine brain, wild-type mice were inoculated with either 10 2 or 10 8 PFU of T1L or the S370P/Q371E mutant intracranially, and viral titers in brain homogenates were quantified at 4, 8, and 12 days postinoculation by plaque assay. Following inoculation with 10 2 PFU of virus, T1L produced slightly higher titers in the brain than the S370P/Q371E mutant on days 4 and 8 postinoculation, and the difference reached statistical significance on day 8 (Fig. 3A). These findings suggest that T1 1-glycan interactions contribute to optimal viral replication in the murine brain. However, no significant differences in titer were observed following inoculation with 10 8 PFU of virus (Fig. 3B), suggesting that the replication capacity of T1L and S370P/Q371E in the brain is equalized following high-MOI inoculation.
To determine whether T1L and the S370P/Q371E mutant differ in cell tropism, we inoculated mice in the right cerebral hemisphere with 10 8 PFU of T1L or the S370P/Q371E mutant, which is a dose that produces comparable titers in the brain (Fig. 3B). At 4 and 12 days postinoculation, mice were euthanized, and brains were excised, sectioned, and stained for reovirus antigen. We did not detect major differences in cells infected by wild-type and mutant virus. However, ependymal denuding was observed in the brain tissues of mice inoculated with wild-type virus (Fig. 4). Less viral antigen was observed in the contralateral hemisphere of mice inoculated with the S370P/Q371E mutant virus compared with mice inoculated with T1L (Fig. 4). We did not detect hydrocephalus in mice inoculated with either virus strain at this time point.
At 12 days postinoculation, signs of pathological injury were clearly evident (Fig. 5). Histological evaluation of brain tissue from mice inoculated with either T1L or the S370P/Q371E mutant showed greater cell density within the ventricles than tissue from mice inoculated with PBS as a control. However, the cellular infiltrate in T1L-infected animals was greater than in those infected with the S370P/Q371E virus (Fig. 5). Staining with antibodies specific for CD3 and F4/80, which mark T cells and macrophages, respectively, demonstrated that the exudates in brain tissue of mice infected with either virus contained both of these cell types. These observations suggest that T1L induces more infiltrating inflammatory cells than S370P/Q371E in the cerebral ventricles of infected mice.

Reovirus-GM2 interactions influence hydrocephalus severity.
To test whether GM2-binding capacity contributes to hydrocephalus induction by T1 reovirus, mice were inoculated intracranially with either T1L or the S370P/Q371E mutant, and the magnitude of hydrocephalus was assessed using magnetic resonance imaging (MRI) scanning 21 days postinoculation (Fig. 6A). Mice infected with T1L developed much more substantial hydrocephalus than those infected with the S370P/Q371E mutant. The mean ventricular volume of T1L-infected animals was 69.2 mm 3 , whereas that in S370P/Q371E mutant-infected mice was 13.7 mm 3 (Fig. 6B). The ventricular volume in S370P/Q371Einfected mice did not differ appreciably from that in mock-infected animals, which was 9.4 mm 3 . These observations suggest that the capacity to bind GM2 enhances T1 reovirus-induced hydrocephalus.
To complement studies evaluating hydrocephalus induction by viruses that differ in the capacity to engage GM2, we assessed the magnitude of hydrocephalus using mice that differ in expression of the GM2 glycan. The galgt1 and galgt2 genes encode enzymes required to express the GM2 glycan on gangliosides and   T1L or the S370P/Q371E mutant. Newborn mice were inoculated intracranially with 10 8 PFU of T1L or the S370P/Q371E mutant. Four days postinoculation, mice were euthanized, and brains were excised, fixed, and paraffin embedded. Brain sections were stained with H&E to examine tissue pathology (top). In the section from the T1L-infected brain, arrowheads indicate areas of ependymal cell denuding. Arrows point to sloughed cells in the ventricles, which may contribute to hydrocephalus. In the section from the S370P/ Q371E-infected brain, arrows indicate regions of apoptotic neurons and glia with mild hemorrhage. Samples were stained with polyclonal reovirus antiserum to detect viral antigen (bottom). Viral antigen is dispersed in the T1Linfected brain but more localized to the inoculation site in the S370P/Q371Einfected brain.
glycoproteins, respectively (30). Thus, galgt1 ϫ galgt2 doubleknockout mice, herein referred to as GM2 Ϫ/Ϫ mice, lack GM2 glycan expression on both gangliosides and glycoproteins. We inoculated wild-type and GM2 Ϫ/Ϫ mice intracranially with T1L and assessed hydrocephalus using MRI scanning 21 days postinoculation. T1L-mediated hydrocephalus, evidenced by MRI results (Fig. 6A) and quantification of ventricular volume (Fig. 6B), was more severe in wild-type mice than in GM2 Ϫ/Ϫ mice. The mean ventricular volume in T1L-infected GM2 Ϫ/Ϫ mice was 11.14 mm 3 , approximating that of wild-type mice inoculated with the S370P/Q371E mutant. Taken together, these results demonstrate that disruption of the 1-GM2 interaction through manipulation of the viral GM2-binding site or host expression of GM2 diminishes the severity of hydrocephalus caused by T1 reovirus.
Glycan binding is required for maximal infection of ependymal cells in vitro. Damage to ependymal cells caused by infection with T1 reovirus plays a key role in the pathogenesis of hydrocephalus caused by this reovirus serotype (12). To evaluate whether glycan binding activity influences reovirus infection of ependymal cells, we tested T1L and the S370P/Q371E mutant for the capacity FIG 5 Brain histology 12 days following intracranial inoculation with either T1L or the S370P/Q371E mutant. Newborn mice were inoculated intracranially with PBS or 10 8 PFU of either T1L or the S370P/Q371E mutant. Twelve days postinoculation, mice were euthanized, and brains were excised, fixed, and paraffin embedded. Coronal brain sections were stained with H&E to examine tissue pathology and anti-CD3 and anti-F4/80 to mark T cells and macrophages, respectively. Boxes indicate portions of the lateral ventricles that are enlarged in the images on the right. to infect BXD-1425EPN ependymoma cells (31). T1L infected these cells much more efficiently than did the S370P/Q371E mutant at all MOIs tested (Fig. 7A). To determine whether the differences in infectivity displayed by wild-type and mutant virus in ependymal cells results from differences in viral attachment, T1L and the S370P/Q371E mutant were adsorbed to BXD-1425EPN cells at 4°C, and the percentage of cells bound by virus was quantified using flow cytometry. Concordant with the infectivity results, T1L adhered more efficiently to these cells than did the S370P/Q371E mutant (Fig. 7B). Incubation of virus with soluble GM2 glycan prior to adsorption diminished the capacity of wildtype but not mutant virus to infect these cells, as did pretreatment of cells with A. ureafaciens neuraminidase (Fig. 7C). Thus, the capacity of T1 reovirus to bind GM2 confers a replication advantage in ependymal cells that is associated with enhanced viral attachment.

DISCUSSION
An S370P/Q371E mutant virus was selected to establish the function of GM2 engagement in T1 reovirus disease. This doubleresidue mutant virus replicates with wild-type efficiency in L cells and is neutralized by a T1 1 conformation-specific antibody (see Fig. S1 and S2 in the supplemental material), suggesting that the mutations do not adversely affect the structural integrity of 1. However, the mutant virus displays impaired hemagglutination capacity (Fig. 1A) and infectivity in MEFs relative to wild-type T1L (Fig. 2). Moreover, the S370P/Q371E 1 protein does not interact with GM2 in solution as assessed by STD-NMR (Fig. 1C). Using this mutant virus, we found that T1 reovirus 1-GM2 interactions contribute to serotype-specific disease. Wild-type T1L induces more severe hydrocephalus in wild-type mice than does the S370P/Q371E virus. Concordantly, T1L induces less severe hydrocephalus in GM2 Ϫ/Ϫ mice than wild-type mice, mimicking the phenotype of limited hydrocephalus induced by the S370P/ Q371E mutant in wild-type mice (Fig. 6). These observations provide strong evidence that GM2-binding capacity is a T1 reovirus virulence determinant for hydrocephalus severity.
Infection of mice with viruses that differ in the capacity to engage GM2 resulted in marked differences in disease severity, yet titers of wild-type and mutant viruses in the brain were comparable. Interestingly, histological analysis suggests that T1L spreads more efficiently to the contralateral hemisphere than does the S370P/Q371E mutant following intracranial inoculation (Fig. 4), suggesting that glycan-binding capacity enhances dissemination within a tissue target.
Previous reports of reovirus-induced hydrocephalus noted cytoplasmic inclusion formation in ependymal cells accompanied by denuding of the ependymal layer (13,14,32). Inflammatory exudates appear in the ventricles, peaking between days 10 and 12 postinoculation. Reparative processes also are evident, including proliferation of astroglia and capillaries at sites of ependymal damage (13,14). Histological evaluation of brain tissue in our study is consistent with these previous reports. Reovirus titers 12 days postinoculation had subsided to levels undetectable by immunohistochemistry. However, we noted increased cell density in the ventricles of mice inoculated with wild-type T1L and to a lesser extent the S370P/Q371E mutant. Immunohistochemistry revealed that T cells and macrophages infiltrated the ventricles of animals infected with either virus (Fig. 5). Concordantly, T1L produced severe hydrocephalus in wild-type mice, but disease was minimal in mice inoculated with the mutant virus incapable of GM2 binding and in mice that do not express the GM2 glycan.
The brain is composed of several cell types, and one possibility is that some cell types, such as ependymal cells, display glycandependent susceptibility to T1 reovirus. We tested this hypothesis using an ependymal cell line and found that T1L binds and infects these cells more efficiently than does the S370P/Q371E virus ( Fig. 7A and B). Moreover, preincubation of virus with soluble GM2 or cells with neuraminidase decreased infectivity of T1L but Results are expressed as the percent infected cells from a representative experiment of two independent experiments, each performed using triplicate wells. Error bars represent standard deviations. *, P Ͻ 0.05, **, P Ͻ 0.01, and ***, P Ͻ 0.001, as determined by two-way ANOVA followed by Bonferroni's multiple comparison test. (B) BXD-1425EPN cells were adsorbed with the virus strains shown at an MOI of 5 ϫ 10 4 particles per cell at 4°C for 1 h. Cells were washed twice with PBS and stained with Alexa-488-labeled reovirus antiserum. The percentage of cells with bound virus was quantified using flow cytometry. Results are from a representative experiment of two independent experiments, each performed in triplicate. Error bars represent standard deviations. ***, P Ͻ 0.001, as determined by two-tailed Student's t test. (C) T1L and the S370P/Q371E mutant at an MOI of 1 FFU/cell were incubated with the GM2 glycan at a final concentration of 2 mM for 1 h prior to adsorption to BXD-1425EPN cells at room temperature for 1 h. Cells were incubated with PBS or A. ureafaciens neuraminidase for 1 h prior to viral adsorption. Following adsorption, cells were washed twice with PBS, and fresh medium was added. After incubation at 37°C for 20 h, cells were fixed, and reovirus antigen was detected by indirect immunofluorescence. The percentage of infected cells in three fields of view per well was determined. Results are expressed as the percent infected cells from the combined means of three independent experiments, each performed using duplicate wells. Error bars represent standard errors of the means. *, P Ͻ 0.05, and **, P Ͻ 0.01, as determined by two-way ANOVA followed by Bonferroni's multiple comparison test. not the S370P/Q371E mutant (Fig. 7C). As ependymal cells constitute only a small fraction of total brain tissue, assessment of viral load in whole-brain homogenates by plaque assay may not be sufficiently sensitive to detect differences in replication at such discrete foci of infection, including the ependymal cell layer. Taken together, we think that T1 reovirus causes hydrocephalus by GM2-dependent infection of ependymal cells, which are damaged as a consequence of viral infection or virus-induced inflammation. Such damaged ependymal cells could be sloughed into the ventricles and obstruct cerebrospinal fluid flow or fail to efficiently reabsorb cerebrospinal fluid, resulting in ventricle enlargement.
An alternative possibility, which is not mutually exclusive with the one above, is that the T1L-GM2 interaction somehow interferes with normal brain activities. The neurobiological function of cell surface glycans is not completely understood. Gangliosides modulate several important properties, including leukocyte activation, migration, and differentiation (33)(34)(35). T1L cross-linking of GM2 could inhibit physiologic functions dependent on GM2, although we would then expect to see similar alterations in mice lacking GM2, and we did not. However, T1L-GM2 engagement may trigger unknown effects not observed following infection of either wild-type mice with the mutant incapable of GM2 binding or GM2 Ϫ/Ϫ mice with T1L. Many proteins, including sialic acidbinding immunoglobulin-like lectins (siglecs), interact with sialylated glycans (36) and trigger signaling cascades with immunological effects. Since glycans, including GM2, bind siglecs (34), it is possible that the T1L-GM2 interaction interferes with normal GM2-siglec interactions. Alternatively, gangliosides are required for tissue repair, including axon regeneration in the brain (37). Ligands that bind gangliosides, including GD1a-specific antibody, interfere with axon regeneration following injury (38). Thus, it is possible that T1L-GM2 cross-linking somehow impedes the damage repair response in the brain.
Differences in serotype-specific neurologic disease caused by reovirus segregate with the viral S1 gene and are thought to be attributable to differences in receptor engagement. Results from our study, coupled with previous reports (24,39), support this hypothesis. Our data demonstrate that T1L induces more severe hydrocephalus than does the S370P/Q371E mutant, which is deficient in glycan binding. Additionally, hydrocephalus is less severe following T1L infection of mice lacking the GM2 glycan than in wild-type mice. A function for glycan engagement also is apparent in T3 reovirus pathogenesis. T3 reoviruses differing only in the capacity to engage sialic acid differ in the capacity to infect neurons and cause lethal encephalitis (24). Thus, glycan binding contributes to reovirus serotype-specific tropism and virulence. T1 reovirus interactions with GM2 likely function in an adhesion-strengthening mechanism, similar to the process used by T3 reovirus to infect cells (1). In this mechanism, binding to an abundantly expressed glycan with low affinity precedes binding to a relatively low-abundance protein receptor with high affinity. The STD-NMR studies of 1-GM2 binding (Fig. 1C), together with the high doses of GM2 required to block T1 reovirus infection (22) (Fig. 7C), suggest that the virus binds GM2 with relatively low affinity compared with the high-affinity binding of reovirus to JAM-A (8). In further support of this idea, glycan binding is required for T1 reovirus infection of MEFs, which express modest levels of JAM-A, and dispensable for infection of L cells, which express significantly higher JAM-A levels (22). Ac-cordingly, the S370P/Q371E mutant virus displays an impaired capacity to infect MEFs yet is fully replication competent in L cells. The modest differences in viral titer in the ependymal cell line and striking differences in hydrocephalus induction by the wild-type and mutant viruses in our study provides further support for the hypothesis that GM2 functions in adhesion strengthening and is not the sole receptor responsible for disease. Since JAM-A is dispensable for T1L replication in the murine brain (15), we think that serotype-specific glycan-binding preferences dictate neurotropism by facilitating binding to yet-unknown cell-specific protein or nonsialylated glycan receptors.
We were unable to compare wild-type and GM2 Ϫ/Ϫ mice for the capacity to support infection by the reovirus strains used in our study. Unfortunately, GM2 Ϫ/Ϫ mice breed exceedingly poorly, yielding numbers of pups sufficient only for the hydrocephalus experiments. Nonetheless, the limited studies conducted using these mice complement experiments using the S370P/ Q371E virus, thereby strengthening the overall conclusions.
Reovirus is being developed as an oncolytic therapeutic due to its preferential replication in transformed cells (40)(41)(42)(43) and relative avirulence in adult humans. Reovirus strain T3D (Reolysin) is now undergoing evaluation in clinical trials for the treatment of several types of human cancer (44)(45)(46). The specific targeting of reovirus to transformed cells is not completely understood, but cancer cells display increased expression of sialylated glycans (47), suggesting that glycan engagement influences infectivity. GM2 antibodies show promise in killing lung cancer (48) and glioma (49) cells in culture and demonstrate therapeutic efficacy in a mouse model of lung cancer (50). Understanding reovirus-glycan interactions may help target reovirus to cancer cells to initiate preferential lysis. In support of this idea, a T3 reovirus that lacks the JAM-A-binding 1 head domain yet retains the glycanbinding region in the body domain of this serotype is less toxic than the native virus but retains oncolytic potential (51). Moreover, human glioblastoma cells grown under standard culture conditions require JAM-A binding for reovirus infection, but in spheroid cultures of these cells, which more closely resemble tumors in humans, reovirus infection is independent of JAM-A expression (52), suggesting that other factors, such as sialylated glycans, mediate reovirus attachment. T1 reovirus has not been tested for oncolytic capacity, but altered glycan expression by certain tumor cells (47,53) provides sufficient rationale to initiate such experiments. Another possible approach would be to engineer an oncolytic T3 reovirus that contains the T1 GM2-binding site. Such a virus might display increased infection of tumors that express GM2. Thus, understanding the molecular basis of reovirus-glycan interactions has enhanced knowledge of mechanisms used by reovirus to cause hydrocephalus and may improve the design of oncolytic vectors.
Infectivity studies. MEFs were adsorbed at room temperature for 1 h with virus at various MOIs determined using L cells. The inoculum was removed, and cells were washed twice with PBS and incubated at 37°C for 20 h. Cells were fixed in methanol and visualized by indirect immunofluorescence (22). T1L 1 protein expression and purification. Construct 1 long , comprising the three most C-terminal ␤-spirals of T1L 1 and the entire head domain (amino acids 261 to 470), was purified as described previously (22) and used in STD-NMR experiments. Mutations were generated using the GeneArt site-directed mutagenesis PLUS system (Invitrogen) along with AccuPrime Pfx DNA polymerase (Invitrogen) and One Shot MAX Efficiency DH5␣-T1R competent cells (Invitrogen).
Viral replication assays. Viral plaque assays were performed using L cells (61). Viral yield was calculated using the following formula: log 10 yield tx ϭ log 10 (PFU/ml) tx Ϫ log 10 (PFU/ml)t 0 , where t is the time postinoculation.
Conformation-specific antibody neutralization. Virus strains were incubated with 10 g/ml of MAb 5C6 or a mouse IgG2␣ isotype control at room temperature for 1 h. Cells seeded in confluent monolayers in 24-well plates (Corning) were inoculated with the virus-antibody mixture at room temperature for 1 h. Cells were washed twice with PBS, and fresh medium was added. After incubation at 37°C for 20 h, cells were fixed, and infectivity was determined by flow cytometry using Alexa-647-labeled reovirus polyclonal antiserum.
Hemagglutination assay. Purified reovirus virions (10 11 particles) were distributed into 96-well U-bottom microtiter plates (Costar) and serially diluted 2-fold in 0.05 ml of PBS. Human type O erythrocytes (Vanderbilt University Blood Bank) were washed twice with PBS and resuspended at a concentration of 1% (vol/vol). Erythrocytes (0.05 ml) were added to wells containing virus particles and incubated at 4°C for 3 h. A partial or complete shield of erythrocytes on the well bottom was interpreted as a positive hemagglutination result; a smooth, round button of erythrocytes was interpreted as a negative result. Hemagglutination (HA) titer is expressed as 10 11 particles divided by the number of particles/HA unit. One HA unit equals the number of particles sufficient to produce hemagglutination.
Hemagglutination inhibition assay. Human erythrocytes were treated with A. ureafaciens neuraminidase (M.P. Biomedicals, LLC) at room temperature for 1 h, incubated with 4 HA units of reovirus, incubated at 4°C for 3 h, and scored for agglutination.
STD-NMR spectroscopy. NMR spectra were recorded at 283 K using 3-mm tubes and a Bruker AVIII-600 spectrometer equipped with a room temperature probe head and processed with TOPSPIN 3.0 (Bruker). Samples contained 2 mM GM2 glycan (Elicityl), 20 mM potassium phosphate (pH 7.4), and 150 mM NaCl with and without 16.8 M T1L 1 or the mutant protein. Samples were prepared in D 2 O, and no additional water suppression was used to preserve the anomeric proton signals. A sample without protein also was used for spectral assignment. The off-and onresonance irradiation frequencies were set to Ϫ30 ppm and 7.3 ppm, respectively. A selective pulse irradiation power of 57 Hz and saturation time of 2 s were used, and the total relaxation delay was 3 s. A 50-ms continuous-wave spin-lock pulse with a strength of 3.2 kHz was employed to suppress residual protein signals. Spectra were referenced using HDO as an internal standard (64) and multiplied with a Gaussian window function prior to Fourier transformation.
Virus attachment by flow cytometry. Cells were adsorbed with reovirus strains at 4°C for 1 h to prevent internalization, washed twice with PBS, and stained with Alexa-647 or Alexa-488 labeled reovirus polyclonal antiserum. Labeling was performed using the AlexaFluor antibody labeling kit (Molecular Probes) according to the manufacturer's instructions. The percentage of cells bound by virus was quantified using an LSR-II flow cytometer (BD Biosciences). The data were analyzed with FlowJo software (Tree Star).
Infection of mice. C57BL/6 mice were obtained from the Jackson Laboratory to establish a breeding colony at Vanderbilt University. GM2 Ϫ/Ϫ mice lacking functional copies of galgt1 and galgt2 on a C57BL/6 background (30) were generated by The Center for Functional Glycomics (The Scripps Research Institute) and provided by Dapeng Zhou (MD Anderson Cancer Center).
Two-to three-day-old mice were inoculated intracranially in the right cerebral hemisphere with reovirus diluted in 5 l PBS using a Hamilton syringe and 30-gauge needle. For analysis of viral replication, mice were euthanized at various intervals postinoculation, and organs were excised, collected in 1 ml of PBS, and frozen and thawed twice prior to homogenization using a TissueLyser (Qiagen). Viral titer was quantified by plaque assay using L cells. For histopathological and immunohistochemical analyses, mice were euthanized at various intervals following inoculation, and organs were excised and fixed overnight in 10% formalin. Fixed organs were paraffin embedded, and 6-m sections were prepared. Sections were evaluated for histological damage following hematoxylin-and-eosin (H&E) staining. Reovirus proteins were detected using reovirus polyclonal antiserum. T cells and macrophages were detected using CD3-and F4/80-specific antibodies, respectively.
All animal husbandry and experimental procedures were performed in accordance with U.S. Public Health Service policy and approved by the Vanderbilt University School of Medicine Institutional Animal Care and Use Committee, All animal work was performed under Vanderbilt animal protocol M/05/198. Magnetic resonance imaging. Mice were inoculated with 10 8 PFU of reovirus. Twenty-one days postinoculation, mice were anesthetized via inhalation of 2% isoflurane and 98% oxygen. Animals were secured in a prone position with the head in a 25-mm-inner-diameter radiofrequency (RF) coil and placed in a Varian 7T horizontal-bore magnetic resonance imaging system (Varian Inc.) to collect imaging data. Respiration rate and internal body temperature were continuously monitored. A constant body temperature of 37°C was maintained using heated airflow.
For each animal, multislice scout images were collected in all three imaging planes (axial, sagittal, and coronal) using a gradient echo sequence with a repetition time (TR) of 75 ms, an echo time (TE) of 5 ms, a slice thickness of 2 mm, and a flip angle of 35°. An average of four acquisitions were obtained for each animal. Additional parameters include a 50-mm by 50-mm field of view (FOV) and a 128-by-128 data matrix.
Following localization of the brain, T2-weighted fast-spin echo images were collected over 12 to 20 imaging slices for all three imaging planes (axial, coronal, and sagittal), with a 20-mm by 20-mm FOV, a slice thickness of 0.75 mm, and a 128-by-128 data matrix. Additional parameters include a TR of 5 s, an echo train length of 16, echo spacing of 8 ms, and a TE of 64 ms. Ventricular quantification. Ventricular volume was quantified using MatLab 2013a (The MathWorks, Inc.). A region of interest (ROI) encompassing the entire brain was manually drawn for each slice, and a signal intensity threshold 1.25 times the mean signal intensity in a manually draw region of cortical gray matter was used to segment voxels within the ROI corresponding to a ventricle. The total ventricular volume was cal-culated as the sum of the number of voxels within the segmented ventricular region multiplied by the volume of each voxel. The appearance of both eyes in the coronal plane of analysis was used as a marker to standardize regions for ventricular volume quantification. Eight consecutive images following the landmark image were used for volumetric determination.
Statistical analysis. Statistical analyses were performed using Prism (GraphPad). P values of less than 0.05 were considered to be statistically significant. Descriptions of the specific tests are found in the figure legends. Viral replication assays, hemagglutination assays, infectivity assays comprising more than two virus strains, and animal studies were analyzed by analysis of variance (ANOVA) followed by Bonferroni's correction for multiple tests. Student's t tests were used for analysis of infectivity and binding experiments in which only two strains were compared.

ACKNOWLEDGMENTS
We are grateful to Melanie Dietrich and Alexandra Thor (Interfaculty Institute of Biochemistry, University of Tübingen) for assistance with the expression and purification of the 1 proteins. We acknowledge the staff in the Vanderbilt University Center for Small Animal Imaging, Flow Cytometry Shared Resource, and Translational Pathology Shared Resource for technical support. We thank William S. Bush (Case Western University) for help with statistical analysis and Remco Sprangers (Max Planck Institut for Developmental Biology, Tübingen) for assistance in recording the NMR data. We thank James Paulson (The Scripps Research Institute) and Dapeng Zhou (MD Anderson Cancer Center) for establishing and providing the GM2 Ϫ/Ϫ mice, respectively. This work was supported by U.S. Public Health Service award R01 AI076983 and the Elizabeth B. Lamb Center for Pediatric Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.