Vizgirda Greta, Underwood Alexander P, Fahnøe Ulrik, Weis Nina, Ramirez Santseharay, Bukh Jens
Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark.
Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen Hepatitis C Program (CO-HEP), Copenhagen, Denmark.
PLoS One. 2025 Aug 1;20(8):e0326419. doi: 10.1371/journal.pone.0326419. eCollection 2025.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has evolved into variants with multiple spike protein coding mutations that affect its transmissibility, infectivity, and immune evasion, in particular from neutralizing antibodies. Several of these amino acid changes have been associated with reduced dependency on the principal angiotensin converting enzyme-2 (ACE2) receptor for cell entry. The present study investigates the role of spike protein changes observed in a cell-culture adapted SARS-CoV-2 isolate (DK-AHH1) in modulating entry, ACE2 dependency, and neutralization across different cells, including human liver and lung cell lines. Using a pseudoparticle system, spike proteins with substitutions E484D, P812R, Q954H, and deletion Δ68-76 were evaluated in Vero E6 and Huh7.5, as well as in A549 cells with and without ACE2 overexpression. Pseudoparticles carrying E484D or P812R individually permitted entry in Huh7.5 cells, and their combination further enhanced this capacity. ACE2 blocking experiments revealed the differential roles of these mutations in mediating entry across cell lines. In Vero E6 cells, P812R was the primary driver for ACE2-independent entry, while E484D facilitated ACE2-independent entry in Huh7.5 cells. In A549 cells, all three substitutions (E484D+P812R + Q954H) were required for ACE2-independent entry. Addition of the Δ68-76 deletion did not increase infectivity in any cell line. Notably, pseudoparticles carrying these mutations, maintained susceptibility to neutralization by convalescent plasma from subjects with COVID-19, regardless of the cell line used. These findings highlight the adaptability of SARS-CoV-2 in utilizing alternative entry mechanisms across various cell types, with E484D and P812R playing critical roles in ACE2-independent entry in cell culture. Overall, this study provides valuable insights into how SARS-CoV-2 can alter its receptor usage to ensure robust infectivity of human cell lines while preserving neutralization sensitivity, contributing to our understanding of viral evolution, and informing potential therapeutic strategies targeting viral entry.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）可引发2019冠状病毒病（COVID-19），它已演变成具有多个刺突蛋白编码突变的变体，这些突变影响其传播性、感染性和免疫逃逸能力，尤其是对中和抗体的免疫逃逸能力。其中一些氨基酸变化与病毒进入细胞时对主要血管紧张素转换酶2（ACE2）受体的依赖性降低有关。本研究调查了在一种细胞培养适应性SARS-CoV-2分离株（DK-AHH1）中观察到的刺突蛋白变化在调节不同细胞（包括人肝和肺细胞系）的病毒进入、ACE2依赖性和中和作用方面的作用。使用假病毒颗粒系统，在Vero E6细胞、Huh7.5细胞以及过表达和未过表达ACE2的A549细胞中评估了具有E484D、P812R、Q954H替换以及Δ68-76缺失的刺突蛋白。单独携带E484D或P812R的假病毒颗粒能够进入Huh7.5细胞，二者组合可进一步增强这种能力。ACE2阻断实验揭示了这些突变在介导不同细胞系中病毒进入过程中的不同作用。在Vero E6细胞中，P812R是不依赖ACE2进入细胞的主要驱动因素，而E484D则促进病毒在Huh7.5细胞中不依赖ACE2进入。在A549细胞中，不依赖ACE2进入细胞需要所有三个替换（E484D + P812R + Q954H）。Δ68-76缺失的加入并未增加任何细胞系中的感染性。值得注意的是，携带这些突变的假病毒颗粒，无论使用何种细胞系，对COVID-19康复者的恢复期血浆中和作用均保持敏感。这些发现突出了SARS-CoV-2在利用各种细胞类型的替代进入机制方面的适应性，其中E484D和P812R在细胞培养中不依赖ACE2进入过程中发挥关键作用。总体而言，本研究为SARS-CoV-2如何改变其受体使用方式以确保在保持中和敏感性的同时实现对人细胞系的强大感染性提供了有价值的见解，有助于我们理解病毒进化，并为针对病毒进入的潜在治疗策略提供依据。
