Recognition of decay accelerating factor and alpha(v)beta(3) by inactivated hantaviruses: Toward the development of high-throughput screening flow cytometry assays.
Recognition of decay accelerating factor and alpha(v)beta(3) by inactivated hantaviruses: Toward the development of high-throughput screening flow cytometry assays.
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DOI：
10.1016/j.ab.2010.03.016
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发表时间：
2010-07-15
影响因子：
2.9
通讯作者：
Hjelle B
中科院分区：
文献类型：
--
作者：
Buranda T;Wu Y;Perez D;Jett SD;BonduHawkins V;Ye C;Edwards B;Hall P;Larson RS;Lopez GP;Sklar LA;Hjelle B
汉坦病毒在人类中引起两种严重疾病：肾综合征出血热（HFRS）或汉坦病毒心肺综合征（HCPS）。由于缺乏预防或治疗HFRS和HCPS的疫苗或特异性药物，以及在生物安全3级实验室（BSL-3）进行实验的要求，限制了探索感染机制和疾病发病机制的能力。在这项研究中，我们开发了一种可推广的光谱分析方法来定量SNV病毒（SNV）颗粒包膜中溶解的饱和荧光团位点。然后，我们使用流式细胞术和活细胞共聚焦荧光显微镜成像发现，紫外线杀死的SNV与活病毒粒子的同源受体结合，即Tanoue B细胞上表达的衰变加速因子（CD55/DAF）和Vero E6细胞上表达的αvβ3整合素。SNV与DAF的结合具有多价性和高亲和力（Kd≈26pM）。使用荧光标记SNV和未标记SNV之间的自交换竞争结合测定来评估感染单位-颗粒比约1:14000。我们已经配置了使用高通量流式细胞仪测量荧光标记SNV与Tanoue B悬浮细胞结合的实验。通过这种方法，我们建立了一种高通量的结合抑制筛选方法。这是开发病毒-细胞相互作用小分子抑制剂HTS格式检测的第一步，也是剖析BSL-2环境中感染机制的第一步。
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 αvβ3 integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity (Kd ≈ 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.
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影响因子：
3.4
作者：
Chigaev, A;Buranda, T;Sklar, LA
通讯作者：
Sklar, LA
影响因子：
5.5
作者：
Bharadwaj, M;Lyons, CR;Hjelle, B
通讯作者：
Hjelle, B
DOI：
10.1016/0005-2736(72)90030-2
发表时间：
1972-01-01
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
DEMEL, RA;VANDEENE.LL;BRUCKDORFER, KR
通讯作者：
BRUCKDORFER, KR
影响因子：
2.7
作者：
Goldsmith, CS;Elliott, LH;Zaki, SR
通讯作者：
Zaki, SR
影响因子：
3.6
作者：
Edwards, BS;Bologa, C;Oprea, TI
通讯作者：
Oprea, TI