Quantum Dot-Conjugated SARS-CoV-2 Spike Pseudo-Virions Enable Tracking of Angiotensin Converting Enzyme 2 Binding and Endocytosis
Quantum Dot-Conjugated SARS-CoV-2 Spike Pseudo-Virions Enable Tracking of Angiotensin Converting Enzyme 2 Binding and Endocytosis
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DOI：
10.1021/acsnano.0c05975
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发表时间：
2020-09-22
期刊：
影响因子：
17.1
通讯作者：
Oh, Eunkeu
中科院分区：
文献类型：
--
作者：
Gorshkov, Kirill;Susumu, Kimihiro;Oh, Eunkeu
SARS-CoV-2 感染的第一步是刺突蛋白的受体结合域与宿主细胞质膜上的 ACE2 受体结合。在这里，我们使用与荧光量子点（QD）缀合的重组 Spike 受体结合域生成了一种多功能成像探针。该探针能够与 ACE2 结合的金纳米粒子进行能量转移猝灭，从而能够监测溶液中的结合事件。中和抗体和重组人 ACE2 阻断猝灭，证明了特异性结合相互作用。在用 ACE2-GFP 转染的细胞中，我们观察到探针在细胞表面立即结合，然后发生内吞作用。中和抗体和 ACE2-Fc 以低纳摩尔效力完全阻止结合和内吞作用。重要的是，我们将能够使用这种 QD 纳米颗粒探针来识别和验证人类细胞中 SARS-CoV-2 Spike 和 ACE2 受体结合的抑制剂。这项工作能够轻松、快速、高通量地基于细胞筛选冠状病毒刺突介导的细胞识别和进入的抑制剂。
The first step of SARS-CoV-2 infection is binding of the spike protein's receptor binding domain to the host cell's ACE2 receptor on the plasma membrane. Here, we have generated a versatile imaging probe using recombinant Spike receptor binding domain conjugated to fluorescent quantum dots (QDs). This probe is capable of engaging in energy transfer quenching with ACE2-conjugated gold nanoparticles to enable monitoring of the binding event in solution. Neutralizing antibodies and recombinant human ACE2 blocked quenching, demonstrating a specific binding interaction. In cells transfected with ACE2-GFP, we observed immediate binding of the probe on the cell surface followed by endocytosis. Neutralizing antibodies and ACE2-Fc fully prevented binding and endocytosis with low nanomolar potency. Importantly, we will be able to use this QD nanoparticle probe to identify and validate inhibitors of the SARS-CoV-2 Spike and ACE2 receptor binding in human cells. This work enables facile, rapid, and high-throughput cell-based screening of inhibitors for coronavirus Spike-mediated cell recognition and entry.