Recognition of DAF and α v β 3 by inactivated Hantaviruses, towards the development of HTS flow cytometry assays

Hantaviruses cause two severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardio-pulmonary 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 have developed a generalizable spectroscopic assay to quantify saturable fluorophore sites solubilized in envelope membranes of Sin Nombre virus (SNV) particles. We then use flow cytometry and live cell confocal fluorescence microscopy imaging to show that UV-killed SNV bind to the cognate receptors of live virions, namely, decay accelerating factor (CD55/DAF) expressed on Tanoue B cells and α v β 3 integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity ( K d ≈ 26pM). Self-exchange competition binding assays between fluorescently labeled SNV and unlabeled SNV are used to evaluate an infectious unit-to-particle ratio of ∼ 1:14000. We have 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 establish a proof of principle high throughput screening assay for binding inhibition. This is a first step towards the development of HTS format assays for small molecule inhibitors of viral-cell interactions, as well as dissecting the mechanism of infection in a BSL-2 environment. after After lipid used measurements degree of We show that and in