Figure 7. Reovirus virions but not ISVPs bind to soluble NgR1.

Soluble Fc-tagged NgR1, JAM-A, or CAR was immobilized onto protein G beads. Receptor-conjugated beads were incubated with 10¹¹ particles of either reovirus T3SA+ (A) virions or (B) ISVPs, washed extensively, resuspended and boiled in protein-dissociation buffer to release bound material. Precipitated proteins were resolved by SDS-polyacrylamide gel electrophoresis and visualized by colloidal blue staining. (C) Reovirus λ band intensity was quantified by the Odyssey imaging system for three independent experiments. Results are expressed as the mean optical density relative to that of NgR1-precipated λ bands. Error bars indicate SD. (D, E) Plates were coated with soluble Fc-tagged NgR1, JAM-A, or CAR and adsorbed with serial dilutions of reovirus T3SA+ virions (D) or ISVPs (E) in a FLISA binding assay. Wells were washed and incubated with reovirus-specific antibodies and fluorescent secondary antibodies to detect virions or ISVPs bound to receptor protein. Signal intensity was quantified using the Odyssey imaging system. Results are shown as the mean relative FLISA signal for three independent experiments. Error bars indicate SD. (F) Both reovirus virions and ISVPs efficiently bind JAM-A. However, ISVPs are incapable of engaging NgR1, suggesting that the reovirus ligand for NgR1 is either σ3 or the virion-associated conformer of σ1.