J. Denise Wetzel

Peyer's Patch Dendritic Cells Process Viral Antigen from Apoptotic Epithelial Cells in the Intestine of Reovirus-infected Mice

We explored the role of Peyers patch (PP) dendritic cell (DC) populations in the induction of immune responses to reovirus strain type 1 Lang (T1L). Immunofluorescence staining revealed the presence of T1L structural (σ1) and nonstructural (σNS) proteins in PPs of T1L-infected mice. Cells in the follicle-associated epithelium contained both σ1 and σNS, indicating productive viral replication. In contrast, σ1, but not σNS, was detected in the subepithelial dome (SED) in association with CD11c+/CD8α−/CD11blo DCs, suggesting antigen uptake by these DCs in the absence of infection. Consistent with this possibility, PP DCs purified from infected mice contained σ1, but not σNS, and PP DCs from uninfected mice could not be productively infected in vitro. Furthermore, σ1 protein in the SED was associated with fragmented DNA by terminal deoxy-UTP nick-end labeling staining, activated caspase-3, and the epithelial cell protein cytokeratin, suggesting that DCs capture T1L antigen from infected apoptotic epithelial cells. Finally, PP DCs from infected mice activated T1L-primed CD4+ T cells in vitro. These studies show that CD8α−/CD11blo DCs in the PP SED process T1L antigen from infected apoptotic epithelial cells for presentation to CD4+ T cells, and therefore demonstrate the cross-presentation of virally infected cells by DCs in vivo during a natural viral infection.

Junctional Adhesion Molecule A Serves as a Receptor for Prototype and Field-Isolate Strains of Mammalian Reovirus

ABSTRACT Reovirus infections are initiated by the binding of viral attachment protein σ1 to receptors on the surface of host cells. The σ1 protein is an elongated fiber comprised of an N-terminal tail that inserts into the virion and a C-terminal head that extends from the virion surface. The prototype reovirus strains type 1 Lang/53 (T1L/53) and type 3 Dearing/55 (T3D/55) use junctional adhesion molecule A (JAM-A) as a receptor. The C-terminal half of the T3D/55 σ1 protein interacts directly with JAM-A, but the determinants of receptor-binding specificity have not been identified. In this study, we investigated whether JAM-A also mediates the attachment of the prototype reovirus strain type 2 Jones/55 (T2J/55) and a panel of field-isolate strains representing each of the three serotypes. Antibodies specific for JAM-A were capable of inhibiting infections of HeLa cells by T1L/53, T2J/55, and T3D/55, demonstrating that strains of all three serotypes use JAM-A as a receptor. To corroborate these findings, we introduced JAM-A or the structurally related JAM family members JAM-B and JAM-C into Chinese hamster ovary cells, which are poorly permissive for reovirus infection. Both prototype and field-isolate reovirus strains were capable of infecting cells transfected with JAM-A but not those transfected with JAM-B or JAM-C. A sequence analysis of the σ1-encoding S1 gene segment of the strains chosen for study revealed little conservation in the deduced σ1 amino acid sequences among the three serotypes. This contrasts markedly with the observed sequence variability within each serotype, which is confined to a small number of amino acids. Mapping of these residues onto the crystal structure of σ1 identified regions of conservation and variability, suggesting a likely mode of JAM-A binding via a conserved surface at the base of the σ1 head domain.

Utilization of sialic acid as a coreceptor is required for reovirus-induced biliary disease

Infection of neonatal mice with some reovirus strains produces a disease similar to infantile biliary atresia, but previous attempts to correlate reovirus infection with this disease have yielded conflicting results. We used isogenic reovirus strains T3SA- and T3SA+, which differ solely in the capacity to bind sialic acid as a coreceptor, to define the role of sialic acid in reovirus encephalitis and biliary tract infection in mice. Growth in the intestine was equivalent for both strains following peroral inoculation. However, T3SA+ spread more rapidly from the intestine to distant sites and replicated to higher titers in spleen, liver, and brain. Strikingly, mice infected with T3SA+ but not T3SA- developed steatorrhea and bilirubinemia. Liver tissue from mice infected with T3SA+ demonstrated intense inflammation focused at intrahepatic bile ducts, pathology analogous to that found in biliary atresia in humans, and high levels of T3SA+ antigen in bile duct epithelial cells. T3SA+ bound 100-fold more efficiently than T3SA- to human cholangiocarcinoma cells. These observations suggest that the carbohydrate-binding specificity of a virus can dramatically alter disease in the host and highlight the need for epidemiologic studies focusing on infection by sialic acid-binding reovirus strains as a possible contributor to the pathogenesis of neonatal biliary atresia.

Organ-specific roles for transcription factor NF-κB in reovirus-induced apoptosis and disease

Reovirus induces apoptosis in cultured cells and in vivo. In cell culture models, apoptosis is contingent upon a mechanism involving reovirus-induced activation of transcription factor NF-kappaB complexes containing p50 and p65/RelA subunits. To explore the in vivo role of NF-kappaB in this process, we tested the capacity of reovirus to induce apoptosis in mice lacking a functional nfkb1/p50 gene. The genetic defect had no apparent effect on reovirus replication in the intestine or dissemination to secondary sites of infection. In comparison to what was observed in wild-type controls, apoptosis was significantly diminished in the CNS of p50-null mice following reovirus infection. In sharp contrast, the loss of p50 was associated with massive reovirus-induced apoptosis and uncontrolled reovirus replication in the heart. Levels of IFN-beta mRNA were markedly increased in the hearts of wild-type animals but not p50-null animals infected with reovirus. Treatment of p50-null mice with IFN-beta substantially diminished reovirus replication and apoptosis, which suggests that IFN-beta induction by NF-kappaB protects against reovirus-induced myocarditis. These findings reveal an organ-specific role for NF-kappaB in the regulation of reovirus-induced apoptosis, which modulates encephalitis and myocarditis associated with reovirus infection.

Type I interferons produced by hematopoietic cells protect mice against lethal infection by mammalian reovirus

We defined the function of type I interferons (IFNs) in defense against reovirus strain type 1 Lang (T1L), which is a double-stranded RNA virus that infects Peyers patches (PPs) after peroral inoculation of mice. T1L induced expression of mRNA for IFN-α, IFN-β, and Mx-1 in PPs and caused localized intestinal infection that was cleared in 10 d. In contrast, T1L produced fatal systemic infection in IFNαR1 knockout (KO) mice with extensive cell loss in lymphoid tissues and necrosis of the intestinal mucosa. Studies of bone-marrow chimeric mice indicated an essential role for hematopoietic cells in IFN-dependent viral clearance. Dendritic cells (DCs), including conventional DCs (cDCs), were the major source of type I IFNs in PPs of reovirus-infected mice, whereas all cell types expressed the antiviral protein Mx-1. Neither NK cells nor signaling via Toll-like receptor 3 or MyD88 were essential for viral clearance. These data demonstrate a requirement for type I IFNs in the control of an intestinal viral infection and indicate that cDCs are a significant source of type I IFN production in vivo. Therefore, innate immunity in PPs is an essential component of host defense that limits systemic spread of pathogens that infect the intestinal mucosa.

Isolation and Molecular Characterization of a Novel Type 3 Reovirus from a Child with Meningitis

Mammalian reoviruses are non-enveloped viruses that contain a segmented, double-stranded RNA genome. Reoviruses infect most mammalian species, although infection with these viruses in humans is usually asymptomatic. We report the isolation of a novel reovirus strain from a 6.5-week-old child with meningitis. Hemagglutination and neutralization assays indicated that the isolate is a serotype 3 strain, leading to the designation T3/Human/Colorado/1996 (T3C/96). Sequence analysis of the T3C/96 S1 gene segment, which encodes the viral attachment protein, sigma 1, confirmed the serotype assignment for this strain and indicated that T3C/96 is a novel reovirus isolate. T3C/96 is capable of systemic spread in newborn mice after peroral inoculation and produces lethal encephalitis. These results suggest that serotype 3 reoviruses can cause meningitis in humans.

Detection of Mammalian Reovirus RNA by Using Reverse Transcription-PCR: Sequence Diversity within the λ3-Encoding L1 Gene

ABSTRACT Reoviruses infect virtually all mammalian species, and infection of humans is associated with mild gastrointestinal or upper respiratory illnesses. To improve reovirus detection strategies, we developed a reverse transcription-PCR technique to amplify a fragment of the reovirus L1 gene segment. This assay was capable of detecting 44 of 44 reovirus field isolate strains and was sufficiently sensitive to detect nearly a single viral particle (1.16 ± 0.13) per PCR of prototype strain type 3 Dearing. Pairwise comparisons of the 44 partial L1 gene sequences revealed that nucleotide variability ranged from 0 to 24.7%, with most of the nucleotide polymorphism occurring at synonymous positions. Phylogenetic trees generated from amplified L1 gene sequences suggest that multiple alleles of the L1 gene cocirculate in nature and that genetic diversity of the L1 gene is largely independent of the host species, geographic locale, or date of isolation. Phylogenetic trees constructed from the L1 gene sequences are distinct from those constructed from the four reovirus S-class gene segments, which supports the hypothesis that reovirus gene segments reassort in nature. This study establishes a new sensitive and specific technique for the identification of mammalian reoviruses and enhances our understanding of reovirus evolution.

Reovirus Preferentially Infects the Basolateral Surface and Is Released from the Apical Surface of Polarized Human Respiratory Epithelial Cells

Mammalian reoviruses infect respiratory and gastrointestinal epithelia and cause disease in neonates. Junctional adhesion molecule-A (JAM-A) is a serotype-independent receptor for reovirus. JAM-A localizes to tight junctions and contributes to paracellular permeability in polarized epithelia. To investigate the mechanisms of reovirus infection of polarized epithelial cells, we assessed reovirus replication, release, and spread after apical and basolateral adsorption to primary human airway epithelial cultures. Reovirus infection of human airway epithelia was more efficient after adsorption to the basolateral surface than after adsorption to the apical surface, and it was dependent on JAM-A. Reovirus binding to carbohydrate coreceptor sialic acid inhibited apical infection, which was partially ameliorated by treatment of the cultures with neuraminidase. Despite the preference for basolateral infection, reovirus was released from the apical surface of respiratory epithelia and did not disrupt tight junctions. These results establish the existence of an infectious circuit for reovirus in polarized human respiratory epithelial cells.

Adaptation of Reovirus to Growth in the Presence of Protease Inhibitor E64 Segregates with a Mutation in the Carboxy Terminus of Viral Outer-Capsid Protein ς3

ABSTRACT Reovirus virions are internalized into cells by receptor-mediated endocytosis. Within the endocytic compartment, the viral outer capsid undergoes acid-dependent proteolysis leading to degradation of ς3 protein and proteolytic cleavage of μ1/μ1C protein. E64 is a specific inhibitor of cysteine-containing proteases that blocks disassembly of reovirus virions. To identify domains in reovirus proteins that influence susceptibility to E64-mediated inhibition of disassembly, we selected variant viruses by serial passage of strain type 3 Dearing (T3D) in murine L929 cells treated with E64. E64-adapted variant viruses (D-EA viruses) produced 7- to 17-fold-greater yields than T3D did after infection of cells treated with 100 μM E64. Viral genes that segregate with growth of D-EA viruses in the presence of E64 were identified by using reassortant viruses isolated from independent crosses of E64-sensitive strain type 1 Lang and two prototype D-EA viruses. Growth of reassortant viruses in the presence of E64 segregated with the S4 gene, which encodes outer-capsid protein ς3. Sequence analysis of S4 genes of three D-EA viruses isolated from independent passage series revealed a common tyrosine-to-histidine mutation at amino acid 354 in the deduced amino acid sequence of ς3. Proteolysis of D-EA virions by endocytic protease cathepsin L occurred with faster kinetics than proteolysis of wild-type T3D virions. Treatment of D-EA virions, but not T3D virions, with cathepsin D resulted in proteolysis of ς3, a property that also was found to segregate with the D-EA S4 gene. These results indicate that a region in ς3 protein containing amino acid 354 influences susceptibility of ς3 to proteolysis during reovirus disassembly.

A Single Mutation in the Carboxy Terminus of Reovirus Outer-Capsid Protein σ3 Confers Enhanced Kinetics of σ3 Proteolysis, Resistance to Inhibitors of Viral Disassembly, and Alterations in σ3 Structure

ABSTRACT Mammalian reoviruses undergo acid-dependent proteolytic disassembly within endosomes, resulting in formation of infectious subvirion particles (ISVPs). ISVPs are obligate intermediates in reovirus disassembly that mediate viral penetration into the cytoplasm. The initial biochemical event in the reovirus disassembly pathway is the proteolysis of viral outer-capsid protein σ3. Mutant reoviruses selected during persistent infection of murine L929 cells (PI viruses) demonstrate enhanced kinetics of viral disassembly and resistance to inhibitors of endocytic acidification and proteolysis. To identify sequences in σ3 that modulate acid-dependent and protease-dependent steps in reovirus disassembly, the σ3 proteins of wild-type strain type 3 Dearing; PI viruses L/C, PI 2A1, and PI 3-1; and four novel mutant σ3 proteins were expressed in insect cells and used to recoat ISVPs. Treatment of recoated ISVPs (rISVPs) with either of the endocytic proteases cathepsin L or cathepsin D demonstrated that an isolated tyrosine-to-histidine mutation at amino acid 354 (Y354H) enhanced σ3 proteolysis during viral disassembly. Yields of rISVPs containing Y354H in σ3 were substantially greater than those of rISVPs lacking this mutation after growth in cells treated with either acidification inhibitor ammonium chloride or cysteine protease inhibitor E64. Image reconstructions of electron micrographs of virus particles containing wild-type or mutant σ3 proteins revealed structural alterations in σ3 that correlate with the Y354H mutation. These results indicate that a single mutation in σ3 protein alters its susceptibility to proteolysis and provide a structural framework to understand mechanisms of σ3 cleavage during reovirus disassembly.