Recognition of decay accelerating factor and alpha(v)beta(3) by inactivated hantaviruses: Toward the development of high-throughput screening flow cytometry assays.

Hantaviruses cause two severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The lack of vaccines or specific drugs to prevent or treat HFRS and HCPS and the requirement for conducting experiments in a biosafety level 3 laboratory (BSL-3) limit the ability to probe the mechanism of infection and disease pathogenesis. In this study, we developed a generalizable spectroscopic assay to quantify saturable fluorophore sites solubilized in envelope membranes of Sin Nombre virus (SNV) particles. We then used flow cytometry and live cell confocal fluorescence microscopy imaging to show that ultraviolet (UV)-killed SNV particles bind to the cognate receptors of live virions, namely, decay accelerating factor (DAF/CD55) expressed on Tanoue B cells and alpha(v)beta(3) integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity (K(d) approximately 26pM). Self-exchange competition binding assays between fluorescently labeled SNV and unlabeled SNV are used to evaluate an infectious unit-to-particle ratio of approximately 1:14,000. We configured the assay for measuring the binding of fluorescently labeled SNV to Tanoue B suspension cells using a high-throughput flow cytometer. In this way, we established a proof-of-principle high-throughput screening (HTS) assay for binding inhibition. This is a first step toward developing HTS format assays for small molecule inhibitors of viral-cell interactions as well as dissecting the mechanism of infection in a BSL-2 environment.