Optimized Pseudotyping Conditions for the SARS-COV-2 Spike Glycoprotein.
Optimized Pseudotyping Conditions for the SARS-COV-2 Spike Glycoprotein.
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DOI：
10.1128/jvi.01062-20
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发表时间：
2020-10-14
影响因子：
5.4
通讯作者：
Ritter DG
中科院分区：
文献类型：
--
作者：
Johnson MC;Lyddon TD;Suarez R;Salcedo B;LePique M;Graham M;Ricana C;Robinson C;Ritter DG
在处理致病性病毒的工作中，对病毒传染性进行快速定量检测是有用的，这种检测可以在没有严格生物控制限制的情况下进行。实现这一目标的一种常见方法是产生含有病原病毒表面糖蛋白的病毒假颗粒，该病毒假颗粒与含有敏感报告系统的复制缺陷病毒颗粒结合。这些假颗粒利用来自致病病毒的糖蛋白进入细胞，导致感染的读数。阻断致病性病毒进入的条件，如中和抗体，也会阻断病毒假颗粒的进入。然而，病毒糖蛋白通常不适于产生假颗粒。在这里，我们描述了一系列修饰，导致产生相对高滴度的SARS-COV-2假颗粒，适用于检测来自COVID-19患者的中和抗体。严重急性呼吸综合征冠状病毒2 （SARS-COV-2）刺突糖蛋白仅负责与宿主细胞受体结合并促进病毒与宿主膜之间的融合。产生带有SARS-COV-2 Spike的假型病毒颗粒的能力对于许多类型的研究都很有用，例如对中和抗体的表征或融合抑制小分子的开发。在这里，我们描述了使用密码子优化的SARS-COV-2刺突糖蛋白产生假型HIV-1、小鼠白血病病毒（MLV）和水泡性口炎病毒（VSV）颗粒。全长Spike蛋白在这三种系统中都不有效，但通过删除细胞质尾部的最后19个氨基酸，其功能增强了10倍以上。293FT靶细胞只有在稳定表达人血管紧张素转换酶2 （ACE2）受体的情况下才有可能被感染，但稳定地引入该受体的额外拷贝并不能进一步增强易感性。稳定引入刺突激活蛋白酶TMPRSS2进一步提高了5- 10倍的感染易感性。用最佳信号肽替代Spike蛋白的信号肽并不会增加或减少感染性颗粒的产生。然而，D614G和R682Q的修饰进一步增强了感染性颗粒的产生。所有增强因子结合后，假型HIV-1颗粒的滴度几乎达到106个感染性颗粒/ml。最后，将带有SARS-COV-2刺突的HIV-1假型颗粒成功用于检测2019冠状病毒病（COVID-19）患者血浆中的中和抗体，但无法检测未感染者血浆中的中和抗体。在处理致病性病毒的工作中，对病毒传染性进行快速定量检测是有用的，这种检测可以在没有严格生物控制限制的情况下进行。实现这一目标的一种常见方法是产生含有病原病毒表面糖蛋白的病毒假颗粒，该病毒假颗粒与含有敏感报告系统的复制缺陷病毒颗粒结合。这些假颗粒利用来自致病病毒的糖蛋白进入细胞，导致感染的读数。阻断致病性病毒进入的条件，如中和抗体，也会阻断病毒假颗粒的进入。然而，病毒糖蛋白通常不适于产生假颗粒。在这里，我们描述了一系列修饰，导致产生相对高滴度的SARS-COV-2假颗粒，适用于检测来自COVID-19患者的中和抗体。
In work with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus, leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here, we describe a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudoparticles that are suitable for the detection of neutralizing antibodies from COVID-19 patients. The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV-2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here, we characterized the use of a codon-optimized SARS-COV-2 Spike glycoprotein for the generation of pseudotyped HIV-1, murine leukemia virus (MLV), and vesicular stomatitis virus (VSV) particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail. Infection of 293FT target cells was possible only if the cells were engineered to stably express the human angiotensin-converting enzyme 2 (ACE2) receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike-activating protease TMPRSS2 further enhanced susceptibility to infection by 5- to 10-fold. Replacement of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modifications D614G and R682Q further enhanced infectious particle production. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV-2 Spike were successfully used to detect neutralizing antibodies in plasma from coronavirus disease 2019 (COVID-19) patients, but not in plasma from uninfected individuals. IMPORTANCE In work with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus, leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here, we describe a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudoparticles that are suitable for the detection of neutralizing antibodies from COVID-19 patients.