کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10532864 | 961756 | 2010 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Recognition of decay accelerating factor and αvβ3 by inactivated hantaviruses: Toward the development of high-throughput screening flow cytometry assays
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
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چکیده انگلیسی
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 αvβ3 integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity (Kd â¼Â 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 402, Issue 2, 15 July 2010, Pages 151-160
Journal: Analytical Biochemistry - Volume 402, Issue 2, 15 July 2010, Pages 151-160
نویسندگان
Tione Buranda, Yang Wu, Dominique Perez, Stephen D. Jett, Virginie BonduHawkins, Chunyan Ye, Bruce Edwards, Pamela Hall, Richard S. Larson, Gabriel P. Lopez, Larry A. Sklar, Brian Hjelle,