Department of Immunology and Microbiology, Scripps Biomedical Research at the University of Florida, Jupiter, Florida, USA.
Department of Medicinal Chemistry, University of Washington, Seattle, Washington, USA.
J Virol. 2023 Mar 30;97(3):e0165022. doi: 10.1128/jvi.01650-22. Epub 2023 Feb 15.
Truncations of the cytoplasmic tail (CT) of entry proteins of enveloped viruses dramatically increase the infectivity of pseudoviruses (PVs) bearing these proteins. Several mechanisms have been proposed to explain this enhanced entry, including an increase in cell surface expression. However, alternative explanations have also been forwarded, and the underlying mechanisms for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein remain undetermined. Here, we show that the partial or complete deletion of the CT (residues 19 to 35) does not modify SARS-CoV-2 S protein expression on the cell surface when the S2 subunit is measured, whereas it is significantly increased when the S1 subunit is measured. We also show that the higher level of S1 in these CT-truncated S proteins reflects the decreased dissociation of the S1 subunit from the S2 subunit. In addition, we demonstrate that CT truncation further promotes S protein incorporation into PV particles, as indicated by biochemical analyses and cryo-electron microscopy. Thus, our data show that two distinct mechanisms contribute to the markedly increased infectivity of PVs carrying CT-truncated SARS-CoV-2 S proteins and help clarify the interpretation of the results of studies employing such PVs. Various forms of PVs have been used as tools to evaluate vaccine efficacy and study virus entry steps. When PV infectivity is inherently low, such as that of SARS-CoV-2, a CT-truncated version of the viral entry glycoprotein is widely used to enhance PV infectivity, but the mechanism underlying this enhanced PV infectivity has been unclear. Here, our study identified two mechanisms by which the CT truncation of the SARS-CoV-2 S protein dramatically increases PV infectivity: a reduction of S1 shedding and an increase in S protein incorporation into PV particles. An understanding of these mechanisms can clarify the mechanistic bases for the differences observed among various assays employing such PVs.
包膜病毒进入蛋白的细胞质尾部（CT）截断极大地提高了携带这些蛋白的假病毒（PV）的感染性。已经提出了几种机制来解释这种增强的进入，包括细胞表面表达的增加。然而，也提出了替代解释，并且严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）S 蛋白的潜在机制仍未确定。在这里，我们表明，当测量 S2 亚基时，S 蛋白的 CT（残基 19 至 35）的部分或完全缺失不会改变 SARS-CoV-2 S 蛋白在细胞表面的表达，而当测量 S1 亚基时，它会显著增加。我们还表明，在这些 CT 截断的 S 蛋白中，S1 水平的升高反映了 S1 亚基与 S2 亚基的解离减少。此外，我们证明 CT 截断进一步促进了 S 蛋白掺入 PV 颗粒，这可以通过生化分析和冷冻电子显微镜证明。因此，我们的数据表明，两种不同的机制导致携带 CT 截断 SARS-CoV-2 S 蛋白的 PV 的感染性显著增加，并有助于澄清使用此类 PV 进行的研究结果的解释。各种形式的 PV 已被用作评估疫苗功效和研究病毒进入步骤的工具。当 PV 的感染性固有较低时，例如 SARS-CoV-2，病毒进入糖蛋白的 CT 截断形式被广泛用于增强 PV 的感染性，但这种增强的 PV 感染性的机制尚不清楚。在这里，我们的研究确定了 SARS-CoV-2 S 蛋白 CT 截断极大地增加 PV 感染性的两种机制：S1 脱落减少和 S 蛋白掺入 PV 颗粒增加。对这些机制的理解可以澄清使用此类 PV 进行的各种测定中观察到的差异的机制基础。
