A monoclonal antibody specific for reovirus outer-capsid protein sigma3 inhibits sigma1-mediated hemagglutination by steric hindrance.

Reovirus virions are nonenveloped icosahedral particles consisting of two concentric protein shells, termed outer capsid and core. Outer-capsid protein sigma1 is the viral attachment protein and binds carbohydrate molecules on the surface of host cells. Monoclonal antibody (MAb) 4F2, which is specific for outer-capsid protein sigma3, blocks the binding of sigma1 protein to sialic acid and inhibits reovirus-induced hemagglutination (HA). To determine whether MAb 4F2 inhibits HA by altering sigma1-sigma3 interactions or by steric hindrance, we analyzed the effect of 4F2 immunoglobulin G (IgG) and Fab fragments (Fabs) on HA induced by reovirus strain type 3 Dearing (T3D). The concentration of 4F2 IgG sufficient to inhibit T3D-induced HA was 12.5 microg per ml, whereas that of Fabs was >200 microg per ml. Dynamic light scattering analysis showed that at the concentration of IgG sufficient to inhibit HA, virion-antibody complexes were monodispersed and not aggregated. The affinity of 4F2 Fabs for T3D virions was only threefold less than that of intact IgG, which suggests that differences in HA inhibition titer exhibited by 4F2 IgG and Fabs are not attributable to differences in the affinity of these molecules for T3D virions. We used cryoelectron microscopy and three-dimensional image analysis to visualize T3D virions alone and in complex with either IgG or Fabs of MAb 4F2. IgG and Fabs bind the same site at the distal portion of sigma3, and binding of IgG and Fabs induces identical conformational changes in outer-capsid proteins sigma3 and mu1. These results suggest that MAb 4F2 inhibits reovirus binding to sialic acid by steric hindrance and provide insight into the conformational flexibility of reovirus outer-capsid proteins.

Reovirus binding to cell surface sialic acid potentiates virus-induced apoptosis.

Reovirus induces apoptosis in cultured cells and in vivo. Genetic studies indicate that the efficiency with which reovirus strains induce apoptosis is determined by the viral S1 gene, which encodes attachment protein sigma1. However, the biochemical properties of sigma1 that influence apoptosis induction are unknown. To determine whether the capacity of sigma1 to bind cell surface sialic acid determines the magnitude of the apoptotic response, we used isogenic reovirus mutants that differ in the capacity to engage sialic acid. We found that T3SA+, a virus capable of binding sialic acid, induces high levels of apoptosis in both HeLa cells and L cells. In contrast, non-sialic-acid-binding strain T3SA- induces little or no apoptosis in these cell types. Differences in the capacity of T3SA- and T3SA+ to induce apoptosis are not due to differences in viral protein synthesis or production of viral progeny. Removal of cell surface sialic acid with neuraminidase abolishes the capacity of T3SA+ to induce apoptosis. Similarly, incubation of T3SA+ with sialyllactose, a trisaccharide comprised of lactose and sialic acid, blocks apoptosis. These findings demonstrate that reovirus binding to cell surface sialic acid is a critical requirement for the efficient induction of apoptosis and suggest that virus receptor utilization plays an important role in regulating cell death.

Junction adhesion molecule is a receptor for reovirus.

Virus attachment to cells plays an essential role in viral tropism and disease. Reovirus serotypes 1 and 3 differ in the capacity to target distinct cell types in the murine nervous system and in the efficiency to induce apoptosis. The binding of viral attachment protein sigma1 to unidentified receptors controls these phenotypes. We used expression cloning to identify junction adhesion molecule (JAM), an integral tight junction protein, as a reovirus receptor. JAM binds directly to sigma1 and permits reovirus infection of nonpermissive cells. Ligation of JAM is required for reovirus-induced activation of NF-kappaB and apoptosis. Thus, reovirus interaction with cell-surface receptors is a critical determinant of both cell-type specific tropism and virus-induced intracellular signaling events that culminate in cell death.

Utilization of sialic acid as a coreceptor enhances reovirus attachment by multistep adhesion strengthening.

Many serotype 3 reoviruses bind to two different host cell molecules, sialic acid and an unidentified protein, using discrete receptor-binding domains in viral attachment protein, final sigma1. To determine mechanisms by which these receptor-binding events cooperate to mediate cell attachment, we generated isogenic reovirus strains that differ in the capacity to bind sialic acid. Strain SA+, but not SA-, bound specifically to sialic acid on a biosensor chip with nanomolar avidity. SA+ displayed 5-fold higher avidity for HeLa cells when compared with SA-, although both strains recognized the same proteinaceous receptor. Increased avidity of SA+ binding was mediated by increased k(on). Neuraminidase treatment to remove cell-surface sialic acid decreased the k(on) of SA+ to that of SA-. Increased k(on) of SA+ enhanced an infectious attachment process, since SA+ was 50-100-fold more efficient than SA- at infecting HeLa cells in a kinetic fluorescent focus assay. Sialic acid binding was operant early during SA+ attachment, since the capacity of soluble sialyllactose to inhibit infection decreased rapidly during the first 20 min of adsorption. These results indicate that reovirus binding to sialic acid enhances virus infection through adhesion of virus to the cell surface where access to a proteinaceous receptor is thermodynamically favored.

Cleavage susceptibility of reovirus attachment protein sigma1 during proteolytic disassembly of virions is determined by a sequence polymorphism in the sigma1 neck.

A requisite step in reovirus infection of the murine intestine is proteolysis of outer-capsid proteins to yield infectious subvirion particles (ISVPs). When converted to ISVPs by intestinal proteases, virions of reovirus strain type 3 Dearing (T3D) lose 90% of their original infectivity due to cleavage of viral attachment protein sigma1. In an analysis of eight field isolate strains of type 3 reovirus, we identified one additional strain, type 3 clone 31 (T3C31), that loses infectivity and undergoes sigma1 cleavage upon conversion of virions to ISVPs. We examined the sigma1 deduced amino acid sequences of T3D and the eight field isolate strains for a correlation between sequence variability and sigma1 cleavage. The sigma1 proteins of T3D and T3C31 contain a threonine at amino acid position 249, whereas an isoleucine occurs at this position in the sigma1 proteins of the remaining strains. Thr249 occupies the d position of a heptad repeat motif predicted to stabilize sigma1 oligomers through alpha-helical coiled-coil interactions. This region of sequence comprises a portion of the fibrous tail domain of sigma1 known as the neck. Substitution of Thr249 with isoleucine or leucine resulted in resistance to cleavage by trypsin, whereas replacement with asparagine did not affect cleavage susceptibility. These results demonstrate that amino acid position 249 is an independent determinant of T3D sigma1 cleavage susceptibility and that an intact heptad repeat is required to confer cleavage resistance. We performed amino-terminal sequence analysis on the sigma1 cleavage product released during trypsin treatment of T3D virions to generate ISVPs and found that trypsin cleaves sigma1 after Arg245. Thus, the sequence polymorphism at position 249 controls cleavage at a nearby site in the neck region. The relevance of these results to reovirus infection in vivo was assessed by treating virions with the contents of a murine intestinal wash under conditions that result in generation of ISVPs. The pattern of sigma1 cleavage susceptibility generated by using purified protease was reproduced in assays using the intestinal wash. These results provide a mechanistic explanation for sigma1 cleavage during exposure of virions to intestinal proteases and may account for certain strain-dependent patterns of reovirus pathogenesis.

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.

Delusional misidentification and platelet monoamine oxidase.

Recently, reduced platelet monoamine oxidase (MAO) activity was demonstrated in two cases of paranoid schizophrenia exhibiting Capgras' syndrome. The present authors report platelet MAO studies in an additional patient with the Delusion of Doubles. In this case, one of delusional hyperidentification (false recognition), decreased platelet MAO activity compared with normal controls was detected. The patient, while not schizophrenic, showed platelet MAO activity comparable with that of a chronic schizophrenic control group. The possible significance of the findings are discussed.

The problem of generalization in the operant conditioning of social speech in the severely subnormal. Use of reversal to establish generalizability.

Many severely subnormal patients talk little to each other. In this experiment, three pairs of subjects were reinforced for talking to each other, and learned to do so quite quickly. Whether social speech would continue to occur without the benefit of external reinforcement was examined by observing the subjects through a one-way mirror in a bare interview room adjacent to the teaching room immediately after each training session. On some occasions untrained subjects were observed in the bare room with the trained subjects. The reinforcement of social speech was demonstrated to be effective by the use of a reversal design (baseline, reinforcement, no reinforcement, reinforcement), where the rate of speech increased considerably when reinforcement was available but decreased when it was discontinued. Generalization of the increased social speech, however, was very poor and only significantly above baseline levels with the pair who seemed responsive to social as well as material reinforcement. The implications of this for training programmes are discussed.

Improvement of retardates' mealtime behaviors by timeout procedures using multiple baseline techniques.

Undesirable mealtime behaviors of a hospital cottage of retardates were reduced by contingent timeout procedures applied by ward personnel successively to one undesirable behavior after another, in a multiple baseline design. In some cases the timeout procedure was to remove the subject from the room until the meal was finished; in other cases (depending on the health of the child and the initial rate of the behavior to be reduced), timeout consisted of a 15-sec removal of the child's meal tray. Undesirable behaviors were defined as stealing, using fingers inappropriately, messy use of utensils, and pigging (eating directly with mouth or eating spilled food). Timeout was applied to these behaviors in that order, and in each case led to a marked and useful reduction in the behavior throughout the group. As these undesirable behaviors were reduced, more appropriate mealtime behaviors emerged: as inappropriate use of fingers declined (under contingent timeout), messy utensil behavior increased; later, as messy utensil behavior declined (under contingent timeout), a defined category of neat utensil behavior increased. Weights of the subjects were monitored steadily throughout the study and showed essentially no change.

Inappropriate speech in a severely retarded child: a case study in language conditioning and generalization.

The modification of inappropriate speech, a class of behaviors rather than a limited number of specific examples, is little known in the severely retarded. In this study, operant techniques were used to modify the strikingly bizarre and inappropriate speech of a severely retarded boy. The boy's appropriate verbal responses to questions about magazine pictures were reinforced with candy. When he responded inappropriately, the magazine was withdrawn, and social interaction was discontinued for a 10-sec timeout period. Negative responses were ignored, the next picture displayed, and the next question asked immediately. In 10 sessions, appropriate responses increased from 26% to 86% of all responses. A reversal of reinforcement was then introduced, in which inappropriate responses were reinforced, appropriate responses resulted in timeout, and negative responses were treated as before. This reduced the percentage of appropriate responses to 24%. Subsequent sessions of reinforcement for appropriate responses increased appropriate responses to 96% of all responses. At significant stages in the experiment, a measure of possible generalization was attempted. Although some generalization was recorded, it was minimal: some explanations are discussed.