Ebola Virus Secretory Glycoprotein (sGP) Diminishes FcγRIIIB-to-CR3 Proximity on Neutrophils

Abstract

Previous studies have shown that Ebola virus’ secretory glycoprotein (sGP) binds to FcγRIIIB (CD16b) and inhibits L-selectin shedding. In this study, we test the hypothesis that sGP interferes with the physical linkage between CR3 and FcγRIIIB. Neutrophils were stained with rhodamine-conjugated anti-CD16b mAb (which does not inhibit sGP binding) and fluorescein-conjugated anti-CR3 mAb reagents and then incubated in media with or without sGP. Physical proximity between fluorochrome-labeled CR3 and FcγRIIIB on individual cells was measured by resonance energy transfer (RET) imaging, quantitative RET microfluorometry, and single-cell imaging spectrophotometry. Cells incubated with control supernatants displayed a significant RET signal, indicative of physical proximity (<7 nm) between CR3 and FcγRIIIB. In contrast, cells exposed to sGP showed a significant reduction in the CR3-FcγRIIIB RET signal using these methods. Interestingly, colocalization and cocapping of CR3 and FcγRIIIB were not affected, suggesting that the proximity of these two receptors is reduced without triggering dissociation. Thus, sGP alters the physical linkage between FcγRIIIB and CR3.