Junctional adhesion molecule-A is required for hematogenous dissemination of reovirus.

Diverse families of viruses bind immunoglobulin superfamily (IgSF) proteins located in tight junctions (TJs) and adherens junctions of epithelium and endothelium. However, little is known about the roles of these receptors in the pathogenesis of viral disease. Junctional adhesion molecule-A (JAM-A) is an IgSF protein that localizes to TJs and serves as a receptor for mammalian reovirus. We inoculated wild-type (WT) and isogenic JAM-A(-/-) mice perorally with reovirus and found that JAM-A is dispensable for viral replication in the intestine but required for systemic dissemination. Reovirus replication in the brain and tropism for discrete neural regions are equivalent in WT and JAM-A(-/-) mice following intracranial inoculation, suggesting a function for JAM-A in reovirus spread to extraintestinal sites. JAM-A promotes reovirus infection of endothelial cells, providing a conduit for the virus into the bloodstream. These findings indicate that a broadly expressed IgSF viral receptor specifically mediates hematogenous dissemination in the host.

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.

JAM-A regulates permeability and inflammation in the intestine in vivo.

Recent evidence has linked intestinal permeability to mucosal inflammation, but molecular studies are lacking. Candidate regulatory molecules localized within the tight junction (TJ) include Junctional Adhesion Molecule (JAM-A), which has been implicated in the regulation of barrier function and leukocyte migration. Thus, we analyzed the intestinal mucosa of JAM-A-deficient (JAM-A(-/-)) mice for evidence of enhanced permeability and inflammation. Colonic mucosa from JAM-A(-/-) mice had normal epithelial architecture but increased polymorphonuclear leukocyte infiltration and large lymphoid aggregates not seen in wild-type controls. Barrier function experiments revealed increased mucosal permeability, as indicated by enhanced dextran flux, and decreased transepithelial electrical resistance in JAM-A(-/-) mice. The in vivo observations were epithelial specific, because monolayers of JAM-A(-/-) epithelial cells also demonstrated increased permeability. Analyses of other TJ components revealed increased expression of claudin-10 and -15 in the colonic mucosa of JAM-A(-/-) mice and in JAM-A small interfering RNA-treated epithelial cells. Given the observed increase in colonic inflammation and permeability, we assessed the susceptibility of JAM-A(-/-) mice to the induction of colitis with dextran sulfate sodium (DSS). Although DSS-treated JAM-A(-/-) animals had increased clinical disease compared with controls, colonic mucosa showed less injury and increased epithelial proliferation. These findings demonstrate a complex role of JAM-A in intestinal homeostasis by regulating epithelial permeability, inflammation, and proliferation.

IkappaB kinase subunits alpha and gamma are required for activation of NF-kappaB and induction of apoptosis by mammalian reovirus.

Reoviruses induce apoptosis both in cultured cells and in vivo. Apoptosis plays a major role in the pathogenesis of reovirus encephalitis and myocarditis in infected mice. Reovirus-induced apoptosis is dependent on the activation of transcription factor NF-kappaB and downstream cellular genes. To better understand the mechanism of NF-kappaB activation by reovirus, NF-kappaB signaling intermediates under reovirus control were investigated at the level of Rel, IkappaB, and IkappaB kinase (IKK) proteins. We found that reovirus infection leads initially to nuclear translocation of p50 and RelA, followed by delayed mobilization of c-Rel and p52. This biphasic pattern of Rel protein activation is associated with the degradation of the NF-kappaB inhibitor IkappaBalpha but not the structurally related inhibitors IkappaBbeta or IkappaBepsilon. Using IKK subunit-specific small interfering RNAs and cells deficient in individual IKK subunits, we demonstrate that IKKalpha but not IKKbeta is required for reovirus-induced NF-kappaB activation and apoptosis. Despite the preferential usage of IKKalpha, both NF-kappaB activation and apoptosis were attenuated in cells lacking IKKgamma/Nemo, an essential regulatory subunit of IKKbeta. Moreover, deletion of the gene encoding NF-kappaB-inducing kinase, which is known to modulate IKKalpha function, had no inhibitory effect on either response in reovirus-infected cells. Collectively, these findings indicate a novel pathway of NF-kappaB/Rel activation involving IKKalpha and IKKgamma/Nemo, which together mediate the expression of downstream proapoptotic genes in reovirus-infected cells.

JAM-A-independent, antibody-mediated uptake of reovirus into cells leads to apoptosis.

Apoptosis plays a major role in the cytopathic effect induced by reovirus following infection of cultured cells and newborn mice. Strain-specific differences in the capacity of reovirus to induce apoptosis segregate with the S1 and M2 gene segments, which encode attachment protein sigma1 and membrane penetration protein mu1, respectively. Virus strains that bind to both junctional adhesion molecule-A (JAM-A) and sialic acid are the most potent inducers of apoptosis. In addition to receptor binding, events in reovirus replication that occur during or after viral disassembly but prior to initiation of viral RNA synthesis also are required for reovirus-induced apoptosis. To determine whether reovirus infection initiated in the absence of JAM-A and sialic acid results in apoptosis, Chinese hamster ovary (CHO) cells engineered to express Fc receptors were infected with reovirus using antibodies directed against viral outer-capsid proteins. Fc-mediated infection of CHO cells induced apoptosis in a sigma1-independent manner. Apoptosis following this uptake mechanism requires acid-dependent proteolytic disassembly, since treatment of cells with the weak base ammonium chloride diminished the apoptotic response. Analysis of T1L x T3D reassortant viruses revealed that the mu1-encoding M2 gene segment is the only viral determinant of the apoptosis-inducing capacity of reovirus when infection is initiated via Fc receptors. Additionally, a temperature-sensitive, membrane penetration-defective M2 mutant, tsA279.64, is an inefficient inducer of apoptosis. These data suggest that signaling pathways activated by binding of sigma1 to JAM-A and sialic acid are dispensable for reovirus-mediated apoptosis and that the mu1 protein plays an essential role in stimulating proapoptotic signaling.

Junctional adhesion molecule a serves as a receptor for prototype and field-isolate strains of mammalian reovirus.

Reovirus infections are initiated by the binding of viral attachment protein sigma1 to receptors on the surface of host cells. The sigma1 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 sigma1 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 sigma1-encoding S1 gene segment of the strains chosen for study revealed little conservation in the deduced sigma1 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 sigma1 identified regions of conservation and variability, suggesting a likely mode of JAM-A binding via a conserved surface at the base of the sigma1 head domain.

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.

A chimeric adenovirus vector encoding reovirus attachment protein sigma1 targets cells expressing junctional adhesion molecule 1.

The utility of adenovirus (Ad) vectors for gene transduction can be limited by receptor specificity. We developed a gene-delivery vehicle in which the potent Ad5 vector was genetically reengineered to display the mucosal-targeting sigma1 protein of reovirus type 3 Dearing (T3D). A sigma1 construct containing all but a small virion-anchoring domain was fused to the N-terminal 44 aa of Ad5 fiber. This chimeric attachment protein Fibtail-T3Dsigma1 forms trimers and assembles onto Ad virions. Fibtail-T3Dsigma1 was recombined into the Ad5 genome, replacing sequences encoding wild-type fiber. The resulting vector, Ad5-T3Dsigma1, expresses Fibtail-T3Dsigma1 and infects Chinese hamster ovary cells transfected with human or mouse homologs of the reovirus receptor, junctional adhesion molecule 1 (JAM1), but not the coxsackievirus and Ad receptor. Treatment of Caco-2 intestinal epithelial cells with either JAM1-specific antibody or neuraminidase reduced transduction by Ad5-T3Dsigma1, and their combined effect decreased transduction by 95%. Ad5-T3Dsigma1 transduces primary cultures of human dendritic cells substantially more efficiently than does Ad5, and this transduction depends on expression of JAM1. These data provide strong evidence that Ad5-T3Dsigma1 can be redirected to cells expressing JAM1 and sialic acid for application as a vaccine vector.

Structure-function analysis of reovirus binding to junctional adhesion molecule 1. Implications for the mechanism of reovirus attachment.

Mammalian reoviruses are nonenveloped viruses with a long, filamentous attachment protein that dictates disease phenotypes following infection of newborn mice and is a structural homologue of the adenovirus attachment protein. Reoviruses use junctional adhesion molecule 1 (JAM1) as a serotype-independent cellular receptor. JAM1 is a broadly expressed immunoglobulin superfamily protein that forms stable homodimers and regulates tight-junction permeability and lymphocyte trafficking. We employed a series of structure-guided binding and infection experiments to define residues in human JAM1 (hJAM1) important for reovirus-receptor interactions and to gain insight into mechanisms of reovirus attachment. Binding and infection experiments using chimeric and domain deletion mutant receptor molecules indicate that the amino-terminal D1 domain of hJAM1 is required for reovirus attachment, infection, and replication. Reovirus binding to hJAM1 occurs more rapidly than homotypic hJAM1 association and is competed by excess hJAM1 in vitro and on cells. Cross-linking hJAM1 diminishes the capacity of reovirus to bind hJAM1 in vitro and on cells and negates the competitive effects of soluble hJAM1 on reovirus attachment. Finally, mutagenesis studies demonstrate that residues intimately associated with the hJAM1 dimer interface are critical for reovirus interactions with hJAM1. These results suggest that reovirus attachment disrupts hJAM1 dimers and highlight similarities between the attachment strategies of reovirus and adenovirus.

Crystal structure of human junctional adhesion molecule 1: implications for reovirus binding.

Reovirus attachment to cells is mediated by the binding of viral attachment protein sigma 1 to junctional adhesion molecule 1 (JAM1). The crystal structure of the extracellular region of human JAM1 (hJAM1) reveals two concatenated Ig-type domains with a pronounced bend at the domain interface. Two hJAM1 molecules form a dimer that is stabilized by extensive ionic and hydrophobic contacts between the N-terminal domains. This dimeric arrangement is similar to that observed previously in the murine homolog of JAM1, indicating physiologic relevance. However, differences in the dimeric structures of hJAM1 and murine JAM1 suggest that the interface is dynamic, perhaps as a result of its ionic nature. We demonstrate that hJAM1, but not the related proteins hJAM2 and hJAM3, serves as a reovirus receptor, which provides insight into sites in hJAM1 that likely interact with sigma 1. In addition, we present evidence that the previously reported structural homology between sigma 1 and the adenovirus attachment protein, fiber, also extends to their respective receptors, which form similar dimeric structures. Because both receptors are located at regions of cell-cell contact, this similarity suggests that reovirus and adenovirus use conserved mechanisms of entry and pathways of infection.