Mammalian reovirus nonstructural protein microNS forms large inclusions and colocalizes with reovirus microtubule-associated protein micro2 in transfected cells

Cells infected with mammalian orthoreoviruses contain large cytoplasmic phase-dense inclusions believed to be the sites of viral replication and assembly, but the morphogenesis, structure, and specific functions of these "viral factories" are poorly understood. Using immunofluorescence microscopy, we found that reovirus nonstructural protein microNS expressed in transfected cells forms inclusions that resemble the globular viral factories formed in cells infected with reovirus strain type 3 Dearing from our laboratory (T3D(N)). In the transfected cells, the formation of microNS large globular perinuclear inclusions was dependent on the microtubule network, as demonstrated by the appearance of many smaller microNS globular inclusions dispersed throughout the cytoplasm after treatment with the microtubule-depolymerizing drug nocodazole. Coexpression of microNS and reovirus protein micro2 from a different strain, type 1 Lang (T1L), which forms filamentous viral factories, altered the distributions of both proteins. In cotransfected cells, the two proteins colocalized in thick filamentous structures. After nocodazole treatment, many small dispersed globular inclusions containing microNS and micro2 were seen, demonstrating that the microtubule network is required for the formation of the filamentous structures. When coexpressed, the micro2 protein from T3D(N) also colocalized with microNS, but in globular inclusions rather than filamentous structures. The morphology difference between the globular inclusions containing microNS and micro2 protein from T3D(N) and the filamentous structures containing microNS and micro2 protein from T1L in cotransfected cells mimicked the morphology difference between globular and filamentous factories in reovirus-infected cells, which is determined by the micro2-encoding M1 genome segment. We found that the first 40 amino acids of microNS are required for colocalization with micro2 but not for inclusion formation. Similarly, a fusion of microNS amino acids 1 to 41 to green fluorescent protein was sufficient for colocalization with the micro2 protein from T1L but not for inclusion formation. These observations suggest a functional difference between microNS and microNSC, a smaller form of the protein that is present in infected cells and that is missing amino acids from the amino terminus of microNS. The capacity of microNS to form inclusions and to colocalize with micro2 in transfected cells suggests a key role for microNS in forming viral factories in reovirus-infected cells.     

Increased ubiquitination and other covariant phenotypes attributed to a strain- and temperature-dependent defect of reovirus core protein mu2

Reovirus replication and assembly are thought to occur within cytoplasmic inclusion bodies, which we call viral factories. A strain-dependent difference in the morphology of these structures reflects more effective microtubule association by the mu2 core proteins of some viral strains, which form filamentous factories, than by those of others, which form globular factories. For this report, we identified and characterized another strain-dependent attribute of the factories, namely, the extent to which they colocalized with conjugated ubiquitin (cUb). Among 16 laboratory strains and field isolates, the extent of factory costaining for cUb paralleled factory morphology, with globular strains exhibiting higher levels by far. In reassortant viruses, factory costaining for cUb mapped primarily to the mu2-encoding M1 genome segment, although contributions by the lambda3- and lambda2-encoding L1 and L2 genome segments were also evident. Immunoprecipitations revealed that cells infected with globular strains contained higher levels of ubiquitinated mu2 (Ub-mu2). In M1-transfected cells, cUb commonly colocalized with aggregates formed by mu2 from globular strains but not with microtubules coated by mu2 from filamentous strains, and immunoprecipitations revealed that mu2 from globular strains displayed higher levels of Ub-mu2. Allelic changes at mu2 residue 208 determined these differences. Nocodazole treatment of cells infected with filamentous strains resulted in globular factories that still showed low levels of costaining for cUb, indicating that higher levels of costaining were not a direct result of decreased microtubule association. The factories of globular strains, or their mu2 proteins expressed in transfected cells, were furthermore shown to gain microtubule association and to lose colocalization with cUb when cells were grown at reduced temperature. From the sum of these findings, we propose that mu2 from globular strains is more prone to temperature-dependent misfolding and as a result displays increased aggregation, increased levels of Ub-mu2, and decreased association with microtubules. Because so few of the viral strains formed factories that were regularly associated with ubiquitinated proteins, we conclude that reovirus factories are generally distinct from cellular aggresomes.     

Reovirus sigmaNS protein is required for nucleation of viral assembly complexes and formation of viral inclusions

Progeny virions of mammalian reoviruses are assembled in the cytoplasm of infected cells at discrete sites termed viral inclusions. Studies of temperature-sensitive (ts) mutant viruses indicate that nonstructural protein sigmaNS and core protein mu2 are required for synthesis of double-stranded (ds) RNA, a process that occurs at sites of viral assembly. We used confocal immunofluorescence microscopy and ts mutant reoviruses to define the roles of sigmaNS and mu2 in viral inclusion formation. In cells infected with wild-type (wt) reovirus, sigmaNS and mu2 colocalize to large, perinuclear structures that correspond to viral inclusions. In cells infected at a nonpermissive temperature with sigmaNS-mutant virus tsE320, sigmaNS is distributed diffusely in the cytoplasm and mu2 is contained in small, punctate foci that do not resemble viral inclusions. In cells infected at a nonpermissive temperature with mu2-mutant virus tsH11.2, mu2 is distributed diffusely in the cytoplasm and the nucleus. However, sigmaNS localizes to discrete structures in the cytoplasm that contain other viral proteins and are morphologically indistinguishable from viral inclusions seen in cells infected with wt reovirus. Examination of cells infected with wt reovirus over a time course demonstrates that sigmaNS precedes mu2 in localization to viral inclusions. These findings suggest that viral RNA-protein complexes containing sigmaNS nucleate sites of viral replication to which other viral proteins, including mu2, are recruited to commence dsRNA synthesis.     

Linkage between reovirus-induced apoptosis and inhibition of cellular DNA synthesis: role of the S1 and M2 genes.

The mammalian reoviruses are capable of inhibiting cellular DNA synthesis and inducing apoptosis. Reovirus strains type 3 Abney (T3A) and type 3 Dearing (T3D) inhibit cellular DNA synthesis and induce apoptosis to a substantially greater extent than strain type 1 Lang (T1L). We used T1L x T3A and T1L x T3D reassortant viruses to identify viral genes associated with differences in the capacities of reovirus strains to elicit these cellular responses to viral infection. We found that the S1 and M2 genome segments determine differences in the capacities of both T1L x T3A and T1L x T3D reassortant viruses to inhibit cellular DNA synthesis and to induce apoptosis. These genes encode viral outer-capsid proteins that play important roles in viral attachment and disassembly. To extend these findings, we used field isolate strains of reovirus to determine whether the strain-specific differences in inhibition of cellular DNA synthesis and induction of apoptosis are also associated with viral serotype, a property determined by the S1 gene. In these experiments, type 3 field isolate strains were found to inhibit cellular DNA synthesis and to induce apoptosis to a greater extent than type 1 field isolate strains. Statistical analysis of these data indicate a significant correlation between the capacity of T1L x T3A and T1L x T3D reassortant viruses and field isolate strains to inhibit cellular DNA synthesis and to induce apoptosis. These findings suggest that reovirus-induced inhibition of cellular DNA synthesis and induction of apoptosis are linked and that both phenomena are induced by early steps in the viral replication cycle.     

Reovirus-induced apoptosis of MDCK cells is not linked to viral yield and is blocked by Bcl-2.

In this study, we investigated the relationship between reovirus-induced apoptosis and viral growth. Madin-Darby canine kidney (MDCK) epithelial cells infected with prototype reovirus strains type 1 Lang (T1L) or type 3 Dearing (T3D) were found to undergo apoptosis, and T3D induced apoptosis of MDCK cells to a substantially greater extent than T1L. By using T1L x T3D reassortant viruses, we found that differences in the capacities of these strains to induce apoptosis are determined by the viral S1 and M2 gene segments. These genes encode viral outer-capsid proteins that play important roles in viral entry into cells. T1L grew significantly better in MDCK cells than T3D, and these differences in growth segregated with the viral L1 and M1 gene segments. The L1 and M1 genes encode viral core proteins involved in viral RNA synthesis. Bcl-2 overexpression in MDCK cells inhibited reovirus-induced apoptosis but did not substantially affect reovirus growth. These findings indicate that differences in the capacities of reovirus strains to induce apoptosis and grow in MDCK cells are determined by different viral genes and that premature cell death by apoptosis does not limit reovirus growth in MDCK cells.     

Reovirus Replication Protein μ2 Influences Cell Tropism by Promoting Particle Assembly within Viral Inclusions

The double-stranded RNA virus mammalian reovirus displays broad cell, tissue, and host tropism. A critical checkpoint in the reovirus replication cycle resides within viral cytoplasmic inclusions, which are biosynthetic centers of genome multiplication and new-particle assembly. Replication of strain type 3 Dearing (T3) is arrested in Madin-Darby canine kidney (MDCK) cells at a step subsequent to inclusion development and prior to formation of genomic double-stranded RNA. This phenotype is primarily regulated by viral replication protein μ2. To understand how reovirus inclusions differ in productively and abortively infected MDCK cells, we used confocal immunofluorescence and thin-section transmission electron microscopy (TEM) to probe inclusion organization and particle morphogenesis. Although no abnormalities in inclusion morphology or viral protein localization were observed in T3-infected MDCK cells using confocal microscopy, TEM revealed markedly diminished production of mature progeny virions. T3 inclusions were less frequent and smaller than those formed by T3-T1M1, a productively replicating reovirus strain, and contained decreased numbers of complete particles. T3 replication was enhanced when cells were cultivated at 31°C, and inclusion ultrastructure at low-temperature infection more closely resembled that of a productive infection. These results indicate that particle assembly in T3-infected MDCK cells is defective, possibly due to a temperature-sensitive structural or functional property of μ2. Thus, reovirus cell tropism can be governed by interactions between viral replication proteins and the unique cell environment that modulate efficiency of particle assembly.     

Differences in the capacity of reovirus strains to induce apoptosis are determined by the viral attachment protein sigma 1.

Reoviruses are important models for studies of viral pathogenesis; however, the mechanisms by which these viruses produce cytopathic effects in infected cells have not been defined. In this report, we show that murine L929 (L) cells infected with prototype reovirus strains type 1 Lang (TIL) and type 3 Dearing (T3D) undergo apoptosis and that T3D induces apoptosis to a substantially greater extent than T1L. Using T1L x T3D reassortant viruses, we found that differences in the capacity of T1L and T3D to induce apoptosis are determined by the viral S1 gene segment, which encodes the viral attachment protein sigma 1 and the non-virion-associated protein sigma 1s. Apoptosis was induced by UV-inactivated, replication-incompetent reovirus virions, which do not contain sigma 1s and do not mediate its synthesis in infected cells. Additionally, T3D-induced apoptosis was inhibited by anti-reovirus monoclonal antibodies that inhibit T3D cell attachment and disassembly. These results indicate that sigma 1, rather than sigma 1s, is required for induction of apoptosis by the reovirus and suggest that interaction of virions with cell surface receptors is an essential step in this mechanism of cell killing.     

Reovirus sigma NS and mu NS proteins form cytoplasmic inclusion structures in the absence of viral infection

Reovirus replication occurs in the cytoplasm of infected cells and culminates in the formation of crystalline arrays of progeny virions within viral inclusions. Two viral nonstructural proteins, sigma NS and micro NS, and structural protein sigma 3 form protein-RNA complexes early in reovirus infection. To better understand the minimal requirements of viral inclusion formation, we expressed sigma NS, mu NS, and sigma 3 alone and in combination in the absence of viral infection. In contrast to its concentration in inclusion structures during reovirus replication, sigma NS expressed in cells in the absence of infection is distributed diffusely throughout the cytoplasm and does not form structures that resemble viral inclusions. Expressed sigma NS is functional as it complements the defect in temperature-sensitive, sigma NS-mutant virus tsE320. In both transfected and infected cells, mu NS is found in punctate cytoplasmic structures and sigma 3 is distributed diffusely in the cytoplasm and the nucleus. The subcellular localization of mu NS and sigma 3 is not altered when the proteins are expressed together or with sigma NS. However, when expressed with micro NS, sigma NS colocalizes with mu NS to punctate structures similar in morphology to inclusion structures observed early in viral replication. During reovirus infection, both sigma NS and mu NS are detectable 4 h after adsorption and colocalize to punctate structures throughout the viral life cycle. In concordance with these results, sigma NS interacts with mu NS in a yeast two-hybrid assay and by coimmunoprecipitation analysis. These data suggest that sigma NS and mu NS are the minimal viral components required to form inclusions, which then recruit other reovirus proteins and RNA to initiate viral genome replication.     

Genetic mapping of reovirus virulence and organ tropism in severe combined immunodeficient mice: organ-specific virulence genes.

We used reovirus reassortant genetics and severe combined immunodeficient (SCID) mice to define viral genes important for organ tropism and virulence in the absence of antigen-specific immunity. Adult SCID mice infected with reovirus serotype 1 strain Lang (T1L) died after 20 +/- 6 days, while infection with serotype 3 strain Dearing (T3D) was lethal after 77 +/- 22 days. One hundred forty-five adult SCID mice were infected with T1L, T3D, and 25 different T1L x T3D reassortant reoviruses, and gene segments associated with the increased virulence of T1L were identified. Gene segments S1, L2, M1, and L1 accounted for > 90% of the genetically determined increase in T1L virulence. Gene segment M1 was independently important for virulence, with S1, L2, and L1 alone or in combination also playing a role. T1L grew to higher titers in multiple organs and caused more severe hepatitis than T3D. Seventy adult SCID mice, T1L, T3D, and 15 T1L x T3D reassortant viruses were used to map genetic determinants of viral titers in the brain, intestines, and liver, as well as the severity of hepatitis. Different sets of gene segments were important for determining viral titers in different organs. Gene segments L1 (encoding a core protein) and L2 (encoding the core spike of the virion) were important in all of the organs analyzed. The M1 gene segment (encoding a core protein), but not the S1 gene segment, was a critical determinant of reovirus titer in the liver and severity of hepatitis. The S1 gene segment (encoding the viral cell attachment protein and a nonstructural protein), but not the M1 gene segment, was a critical determinant of titers in intestines and brains. These studies demonstrate that viral growth in different organs is dependent on different subsets of the genes important for virulence. The virion-associated protein products of the four gene segments (L1, L2, M1, and S1) important for virulence and organ tropism in SCID mice likely form a structural unit, the reovirus vertex. Organs (the brain and intestines versus the liver) differ in properties that determine which virulence genes, and thus which parts of this structural unit, are important.     

Mutations in reovirus outer-capsid protein sigma3 selected during persistent infections of L cells confer resistance to protease inhibitor E64.

Mutations selected in reoviruses isolated from persistently infected cultures (PI viruses) affect viral entry into cells. Unlike wild-type (wt) viruses, PI viruses can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins in cellular endosomes during viral entry. In this study, we show that E64, an inhibitor of cysteine proteases such as those present in the endocytic compartment, blocks growth of wt reovirus by inhibiting viral disassembly. To determine whether PI viruses can grow in the presence of an inhibitor of endocytic proteases, we compared yields of wt and PI viruses in cells treated with E64. Prototype PI viruses L/C, PI 2A1, and PI 3-1 produced substantially greater yields than wt viruses type 1 Lang (T1L) and type 3 Dearing (T3D) in E64-treated cells. To identify viral genes that segregate with growth of PI viruses in the presence of E64, we tested reassortant viruses isolated from independent crosses of T1L and each of the prototype PI viruses for growth in cells treated with E64. Growth of reassortant viruses in the presence of E64 segregated exclusively with the S4 gene, which encodes viral outer-capsid protein sigma3. These results suggest that mutations in sigma3 protein selected during persistent infection alter its susceptibility to cleavage during viral disassembly. To determine the temporal relationship of acid-dependent and protease-dependent steps in reovirus disassembly, cells were infected with wt strain T1L or T3D, and medium containing either ammonium chloride or E64d, a membrane-permeable form of E64, was added at various times after adsorption. Susceptibility to inhibition by both ammonium chloride and E64 was abolished when either inhibitor was added at times greater than 60 min after adsorption. These findings indicate that acid-dependent and protease-dependent disassembly events occur with similar kinetics early in reovirus replication, which suggests that these events take place within the same compartment of the endocytic pathway.     

Core protein mu2 is a second determinant of nucleoside triphosphatase activities by reovirus cores.

NTPase activities in mammalian reovirus cores were examined under various conditions that permitted several new differences to be identified between strains type 1 Lang (T1L) and type 3 Dearing (T3D). One difference concerned the ratio (at pH 8.5) of ATP hydrolysis at 50 degrees C to that at 35 degrees C. A genetic analysis using T1L x T3D reassortant viruses implicated the L3 and M1 gene segments in this difference, with M1 influencing ATPase activity most strongly at high temperatures. L3 and M1 encode the core proteins lambda1 and mu2, respectively. Another difference concerned the absolute levels of GTP hydrolysis by cores at 45 degrees C and pH 6.5. A genetic analysis using T1L x T3D reassortants implicated the M1 gene as the sole determinant of this difference. The results of these experiments, coupled with previous findings (S. Noble and M. L. Nibert, J. Virol. 71:2182-2191, 1997), suggest either that a single type of NTPase in cores is strongly influenced by two different core proteins--lambda1 and mu2--or that cores contain two different types of NTPase influenced by the two proteins. The findings appear relevant for understanding the complex functions of reovirus cores in RNA synthesis and capping.     

Reovirus-induced G(2)/M cell cycle arrest requires sigma1s and occurs in the absence of apoptosis

Serotype-specific differences in the capacity of reovirus strains to inhibit proliferation of murine L929 cells correlate with the capacity to induce apoptosis. The prototype serotype 3 reovirus strains Abney (T3A) and Dearing (T3D) inhibit cellular proliferation and induce apoptosis to a greater extent than the prototype serotype 1 reovirus strain Lang (T1L). We now show that reovirus-induced inhibition of cellular proliferation results from a G(2)/M cell cycle arrest. Using T1L x T3D reassortant viruses, we found that strain-specific differences in the capacity to induce G(2)/M arrest, like the differences in the capacity to induce apoptosis, are determined by the viral S1 gene. The S1 gene is bicistronic, encoding the viral attachment protein sigma1 and the nonstructural protein sigma1s. A sigma1s-deficient reovirus strain, T3C84-MA, fails to induce G(2)/M arrest, yet retains the capacity to induce apoptosis, indicating that sigma1s is required for reovirus-induced G(2)/M arrest. Expression of sigma1s in C127 cells increases the percentage of cells in the G(2)/M phase of the cell cycle, supporting a role for this protein in reovirus-induced G(2)/M arrest. Inhibition of reovirus-induced apoptosis failed to prevent virus-induced G(2)/M arrest, indicating that G(2)/M arrest is not the result of apoptosis related DNA damage and suggests that these two processes occur through distinct pathways.     

Reovirus M2 gene is associated with chromium release from mouse L cells.

In this study, we investigated the interaction of reovirus particles with cell membranes by using a 51Cr release assay. We confirmed prior observations (J. Borsa, B. D. Morash, M. D. Sargent, T. P. Copps, P. A. Lievaart, and J. G. Szekely, J. Gen. Virol. 45:161-170, 1979) that intermediate subviral particles (ISVPs) of reovirus type 3 strain Abney (T3A) induced the release of 51Cr from preloaded L cells and showed that the intact virion and core forms did not. Reovirus type 1 strain Lang (T1L) ISVPs were found to be less efficient at 51Cr release than T3A ISVPs. Reassortants between these strains indicated that the 51Cr release phenotype segregates with the M2 gene segment. Biochemical studies indicated that the ISVPs' acquisition of the capacity to induce 51Cr release followed the cleavage of the viral M2 gene product mu 1/mu 1C to fragments delta and phi during virion conversion to ISVP but did not directly correlate with this cleavage. These studies suggest that the reovirus M2 gene product (in its cleaved form) plays a role in interacting with cell membranes.     

A single-amino-acid polymorphism in reovirus protein μ2 determines repression of interferon signaling and modulates myocarditis

Myocarditis is indicated as the second leading cause of sudden death in young adults. Reovirus induces myocarditis in neonatal mice, providing a tractable model system for investigation of this important disease. Alpha/beta-interferon (IFN-α/β) treatment improves cardiac function and inhibits viral replication in patients with chronic myocarditis, and the host IFN-α/β response is a determinant of reovirus strain-specific differences in induction of myocarditis. Virus-induced IFN-β stimulates a signaling cascade that establishes an antiviral state and further induces IFN-α/β through an amplification loop. Reovirus strain-specific differences in induction of and sensitivity to IFN-α/β are associated with the viral M1, L2, and S2 genes. The reovirus M1 gene-encoded μ2 protein is a strain-specific repressor of IFN-β signaling, providing one possible mechanism for the variation in resistance to IFN and induction of myocarditis between different reovirus strains. We report here that μ2 amino acid 208 determines repression of IFN-β signaling and modulates reovirus induction of IFN-β in cardiac myocytes. Moreover, μ2 amino acid 208 determines reovirus replication, both in initially infected cardiac myocytes and after viral spread, by regulating the IFN-β response. Amino acid 208 of μ2 also influences the cytopathic effect in cardiac myocytes after spread. Finally, μ2 amino acid 208 modulates myocarditis in neonatal mice. Thus, repression of IFN-β signaling mediated by reovirus μ2 amino acid 208 is a determinant of the IFN-β response, viral replication and damage in cardiac myocytes, and myocarditis. These results demonstrate that a single amino acid difference between viruses can dictate virus strain-specific differences in suppression of the host IFN-β response and, consequently, damage to the heart.     

Reovirus variants selected during persistent infections of L cells contain mutations in the viral S1 and S4 genes and are altered in viral disassembly.

Reoviruses isolated from persistently infected cultures (PI viruses) can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins during viral entry into cells. We used reassortant viruses isolated from crosses of wild-type (wt) reovirus strain, type 1 Lang, and three independent PI viruses, L/C, PI 2A1, and PI 3-1, to identify viral genes that segregate with the capacity of PI viruses to grow in cells treated with ammonium chloride. Growth of reassortant viruses in ammonium chloride-treated cells segregated with the S1 gene of L/C and the S4 gene of PI 2A1 and PI 3-1. The S1 gene encodes viral attachment protein sigma1, and the S4 gene encodes outer-capsid protein sigma3. To identify mutations in sigma3 selected during persistent reovirus infection, we determined the S4 gene nucleotide sequences of L/C, PI 2A1, PI 3-1, and four additional PI viruses. The deduced amino acid sequences of sigma3 protein of six of these PI viruses contained a tyrosine-to-histidine substitution at residue 354. To determine whether mutations selected during persistent infection alter cleavage of the viral outer capsid, the fate of viral structural proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment of virions of wt and PI viruses with chymotrypsin in vitro. Proteolysis of PI virus outer-capsid proteins sigma3 and mu1C occurred with faster kinetics than proteolysis of wt virus outer-capsid proteins. These results demonstrate that mutations in either the S1 or S4 gene alter acid-dependent disassembly of the reovirus outer capsid and suggest that increased efficiency of proteolysis of viral outer-capsid proteins is important for maintenance of persistent reovirus infections of cultured cells.     

The viral sigma1 protein and glycoconjugates containing alpha2-3-linked sialic acid are involved in type 1 reovirus adherence to M cell apical surfaces

Type 1 reoviruses invade the intestinal mucosa of mice by adhering selectively to M cells in the follicle-associated epithelium and then exploiting M cell transport activity. The purpose of this study was to identify the apical cell membrane component and viral protein that mediate the M cell adherence of these viruses. Virions and infectious subviral particles of reovirus type 1 Lang (T1L) adhered to rabbit M cells in Peyer's patch mucosal explants and to tissue sections in an overlay assay. Viral adherence was abolished by pretreatment of sections with periodate and in the presence of excess sialic acid or lectins MAL-I and MAL-II (which recognize complex oligosaccharides containing sialic acid linked alpha2-3 to galactose). The binding of T1L particles to polarized human intestinal (Caco-2(BBe)) cell monolayers was correlated with the presence of MAL-I and MAL-II binding sites, blocked by excess MAL-I and -II, and abolished by neuraminidase treatment. Other type 1 reovirus isolates exhibited MAL-II-sensitive binding to rabbit M cells and polarized Caco-2(BBe) cells, but type 2 or type 3 isolates including type 3 Dearing (T3D) did not. In assays using T1L-T3D reassortants and recoated viral cores containing T1L, T3D, or no sigma1 protein, MAL-II-sensitive binding to rabbit M cells and polarized Caco-2(BBe) cells was consistently associated with the T1L sigma1. MAL-II-recognized oligosaccharide epitopes are not restricted to M cells in vivo, but MAL-II immobilized on virus-sized microparticles bound only to the follicle-associated epithelium and M cells. The results suggest that selective binding of type 1 reoviruses to M cells in vivo involves interaction of the type 1 sigma1 protein with glycoconjugates containing alpha2-3-linked sialic acid that are accessible to viral particles only on M cell apical surfaces.