The SARS-COV2 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-COV2 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-COV2 Spike glycoprotein for the generation of pseudotyped HIV-1, MLV, and 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 of Spike. Infection of 293FT target cells was only possible if the cells were engineered to stably express the human ACE-2 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 5-10 fold. Substitution of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modification of a single amino acid in the furin cleavage site of Spike (R682Q) enhanced infectious particle production another 10-fold. With all enhancing elements combined, the titer of pseudotyped particles reached almost 10⁶ infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV2 Spike was successfully used to detect neutralizing antibodies in plasma from COVID-19 patients, but not plasma from uninfected individuals.

中文翻译：

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SARS-COV2穗糖蛋白的优化假型条件

SARS-COV2 Spike糖蛋白仅负责与宿主细胞受体结合并促进病毒膜与宿主膜之间的融合。产生用SARS-COV2 Spike假型化的病毒颗粒的能力可用于许多类型的研究，例如中和抗体的表征或融合抑制小分子的开发。在这里，我们表征了使用密码子优化的SARS-COV2 Spike糖蛋白来产生假型的HIV-1，MLV和VSV颗粒。全长Spike蛋白在所有三个系统中均无法有效发挥功能，但通过删除Spike胞质尾部的最后19个氨基酸，将其增强了10倍以上。仅当将293FT目标细胞改造为稳定表达人ACE-2受体后，才有可能感染293FT目标细胞 但是稳定地引入该受体的其他拷贝并不能进一步增强敏感性。Spike活化蛋白酶TMPRSS2的稳定引入使感染敏感性提高了5-10倍。用最佳信号肽取代Spike蛋白的信号肽不会增强或减少感染性颗粒的产生。但是，对Spike（R682Q）的弗林蛋白酶切割位点中的单个氨基酸进行修饰可将感染性颗粒的产生提高10倍。综合所有增强元素，假型颗粒的效价几乎达到10 用最佳信号肽取代Spike蛋白的信号肽不会增强或减少感染性颗粒的产生。但是，对Spike（R682Q）的弗林蛋白酶切割位点中的单个氨基酸进行修饰可将感染性颗粒的产生提高10倍。综合所有增强元素，假型颗粒的效价几乎达到10 用最佳信号肽取代Spike蛋白的信号肽不会增强或减少感染性颗粒的产生。但是，对Spike（R682Q）的弗林蛋白酶切割位点中的单个氨基酸进行修饰可将感染性颗粒的产生提高10倍。综合所有增强元素，假型颗粒的效价几乎达到10^6个感染颗粒/毫升。最后，用SARS-COV2 Spike假型化的HIV-1颗粒已成功用于检测COVID-19患者血浆中的中和抗体，但未检测未感染个体血浆中的抗体。