A monoclonal antibody specific for reovirus outer-capsid protein sigma 3 inhibits sigma 1-mediated hemagglutination by steric hindrance

Reovirus virions are nonenveloped icosahedral particles consisting of two c oncentric protein shells, termed outer capsid and core. Outer capsid protei n sigma1 is the viral attachment protein and binds carbohydrate molecules o n the surface of host cells. Monoclonal antibody (MAb) 4F2, which is specif ic for outer-capsid protein sigma3, blocks the binding of sigma1 protein to sialic acid and inhibits reovirus-induced hemagglutination (HA). To determ ine whether MAb 4F2 inhibits HA by altering sigma1-sigma3 interactions or b y steric hindrance, we analyzed the effect of 4F2 immunoglobulin G (IgG) an d 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 mug per ml, whereas that of Fabs was > 200 mug per ml. Dynamic light sca ttering analysis showed that at the concentration of IgG sufficient to inhi bit HA, virion-antibody complexes were monodispersed and not aggregated. Th e affinity of 4F2 Fabs for T3D virions was only threefold less than that of intact IgG, which suggests that differences in HA inhibition titer exhibit ed by 4F2 IgG and Fabs are not attributable to differences in the affinity of these molecules for T3D virions. We used cryoelectron microscopy and thr ee-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 co nformational: changes in outer-capsid proteins sigma3 and mu1. These result s suggest that MAb 4F2 inhibits reovirus binding to sialic acid by steric h indrance and provide insight into the conformational flexibility of reoviru s outer capsid proteins.