Viral Populations and Pathogenesis Unit, CNRS, UMR 3569, Institut Pasteur, CEDEX 15, Paris, France.
Chemogenomic and Biological Screening Core Facility, C2RT, Departments of Cell Biology & Infection and of Structural Biology & Chemistry, Institut Pasteur, CEDEX 15, Paris, France.
Nat Commun. 2021 Sep 21;12(1):5553. doi: 10.1038/s41467-021-25796-w.
SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.
SARS-CoV-2 是导致 COVID-19 大流行的病原体，在全球范围内导致超过 1.7 亿例感染和超过 370 万人死亡。为了检测、治疗和接种这种病原体，我们需要更好地了解 SARS-CoV-2 的基本生物学特性。病毒和细胞蛋白酶在 SARS-CoV-2 的复制中都起着至关重要的作用。在这里，我们使用质谱法研究了两种 SARS-CoV-2 复制细胞系模型中病毒和细胞蛋白的蛋白水解切割，以鉴定通过蛋白酶活性产生的蛋白新 N-末端。我们在多种病毒蛋白中发现了以前未知的切割位点，包括主要抗原 S 和 N：这是疫苗和抗体检测工作的主要目标。我们发现与 SARS-CoV-2 主要蛋白酶切割一致的细胞切割事件显著增加，并鉴定出主蛋白酶和木瓜蛋白酶样蛋白酶的 14 个潜在高可信度底物。我们表明，这些细胞蛋白的 siRNA 耗竭会抑制 SARS-CoV-2 的复制，并且靶向其中两种蛋白（酪氨酸激酶 SRC 和丝氨酸/苏氨酸激酶 MYLK）的药物会导致 SARS-CoV-2 滴度呈剂量依赖性降低。总的来说，我们的研究为了解病毒感染背景下的蛋白水解提供了有力的资源，并为开发针对 SARS-CoV-2 的抑制和治疗 COVID-19 的靶向策略提供了信息。
