- Buranda, T. and Wu, Y. and Perez, D. and Jett, S. D. and BonduHawkins, V. and Ye, C. Y. and Edwards, B. and Hall, P. and Larson, R. S. and Lopez, G. P. and Sklar, L. A. and Hjelle, B., Recognition of decay accelerating factor and alpha(v)beta(3) by inactivated hantaviruses: Toward the development of high-throughput screening flow cytometry assays,
Analytical Biochemistry, vol. 402 no. 2
pp. 151--160 [doi] .
(last updated on 2013/06/21)
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 HERS 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 a(v)beta(3) integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity (K-d similar to 26 pM). 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. (C) 2010 Elsevier Inc. All rights reserved.