Abstract：
Autophagy and lysosomal pathways are involved in the cell entry of SARS-CoV-2 virus. To infect the host cell, the spike protein of SARS-CoV-2 binds to the cell surface receptor angiotensin-converting enzyme 2 (ACE2). To allow the fusion of the viral envelope with the host cell membrane, the spike protein has to be cleaved. One possible mechanism is the endocytosis of the SARS-CoV-2-ACE2 complex and subsequent cleavage of the spike protein, mainly by the lysosomal protease cathepsin L. However, detailed molecular and dynamic insights into the role of cathepsin L in viral cell entry remain elusive. To address this, HeLa cells and iPSC-derived alveolarspheres were treated with recombinant SARS-CoV-2 spike protein, and the changes in mRNA and protein levels of cathepsins L, B, and D were monitored. Additionally, we studied the effect of cathepsin L deficiency on spike protein internalization and investigated the influence of the spike protein on cathepsin L promoters in vitro. Furthermore, we analyzed variants in the genes coding for cathepsin L, B, D, and ACE2 possibly associated with disease progression using data from Regeneron\'s COVID Results Browser and our own cohort of 173 patients with COVID-19, exhibiting a variant of ACE2 showing significant association with COVID-19 disease progression. Our in vitro studies revealed a significant increase in cathepsin L mRNA and protein levels following exposure to the SARS-CoV-2 spike protein in HeLa cells, accompanied by elevated mRNA levels of cathepsin B and D in alveolarspheres. Moreover, an increase in cathepsin L promoter activity was detected in vitro upon spike protein treatment. Notably, the knockout of cathepsin L resulted in reduced internalization of the spike protein. The study highlights the importance of cathepsin L and lysosomal proteases in the SARS-CoV-2 spike protein internalization and suggests the potential of lysosomal proteases as possible therapeutic targets against COVID-19 and other viral infections.
摘要：
自噬和溶酶体途径参与SARS-CoV-2病毒的细胞进入。感染宿主细胞，SARS-CoV-2的刺突蛋白与细胞表面受体血管紧张素转换酶2（ACE2）结合。为了使病毒包膜与宿主细胞膜融合，刺突蛋白必须被切割。一种可能的机制是SARS-CoV-2-ACE2复合物的内吞作用以及随后的刺突蛋白的裂解，主要由溶酶体蛋白酶组织蛋白酶L.详细的分子和动态的见解，在病毒细胞进入组织蛋白酶L的作用仍然难以捉摸。为了解决这个问题，用重组SARS-CoV-2刺突蛋白处理HeLa细胞和iPSC来源的肺泡球，以及组织蛋白酶L的mRNA和蛋白质水平的变化，B,和D被监测。此外,我们研究了组织蛋白酶L缺乏对刺突蛋白内化的影响，并在体外研究了刺突蛋白对组织蛋白酶L启动子的影响。此外，我们分析了编码组织蛋白酶L的基因变异，B,D,使用Regeneron的COVID结果浏览器和我们自己的173例COVID-19患者队列的数据，ACE2可能与疾病进展相关，显示出ACE2变体，显示出与COVID-19疾病进展显著相关。我们的体外研究表明，在HeLa细胞中暴露于SARS-CoV-2刺突蛋白后，组织蛋白酶LmRNA和蛋白质水平显着增加，伴有肺泡球中组织蛋白酶B和D的mRNA水平升高。此外，在刺突蛋白处理后，在体外检测到组织蛋白酶L启动子活性的增加。值得注意的是,组织蛋白酶L的敲除导致刺突蛋白的内化减少。该研究强调了组织蛋白酶L和溶酶体蛋白酶在SARS-CoV-2刺突蛋白内化中的重要性，并暗示了溶酶体蛋白酶作为对抗COVID-19和其他病毒感染的可能治疗靶标的潜力。
