Lee Jin-Gu, Huang Weiliang, Lee Hangnoh, van de Leemput Joyce, Kane Maureen A, Han Zhe
Center for Precision Disease Modeling, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA.
Division of Endocrinology, Diabetes and Nutrition, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA.
Cell Biosci. 2021 Mar 25;11(1):58. doi: 10.1186/s13578-021-00568-7.
SARS-CoV-2 causes COVID-19 which has a widely diverse disease profile. The mechanisms underlying its pathogenicity remain unclear. We set out to identify the SARS-CoV-2 pathogenic proteins that through host interactions cause the cellular damages underlying COVID-19 symptomatology.
We examined each of the individual SARS-CoV-2 proteins for their cytotoxicity in HEK 293 T cells and their subcellular localization in COS-7 cells. We also used Mass-Spec Affinity purification to identify the host proteins interacting with SARS-CoV-2 Orf6 protein and tested a drug that could inhibit a specific Orf6 and host protein interaction.
We found that Orf6, Nsp6 and Orf7a induced the highest toxicity when over-expressed in human 293 T cells. All three proteins showed membrane localization in COS-7 cells. We focused on Orf6, which was most cytotoxic and localized to the endoplasmic reticulum, autophagosome and lysosomal membranes. Proteomics revealed Orf6 interacts with nucleopore proteins (RAE1, XPO1, RANBP2 and nucleoporins). Treatment with Selinexor, an FDA-approved inhibitor for XPO1, attenuated Orf6-induced cellular toxicity in human 293 T cells.
Our study revealed Orf6 as a highly pathogenic protein from the SARS-CoV-2 genome, identified its key host interacting proteins, and Selinexor as a drug candidate for directly targeting Orf6 host protein interaction that leads to cytotoxicity.
严重急性呼吸综合征冠状病毒2(SARS-CoV-2)引发的2019冠状病毒病(COVID-19)具有广泛多样的疾病特征。其致病机制仍不清楚。我们着手鉴定SARS-CoV-2致病蛋白,这些蛋白通过与宿主相互作用导致COVID-19症状背后的细胞损伤。
我们检测了SARS-CoV-2的每一种蛋白在人胚肾293T细胞中的细胞毒性及其在非洲绿猴肾COS-7细胞中的亚细胞定位。我们还利用质谱亲和纯化技术鉴定与SARS-CoV-2 Orf6蛋白相互作用的宿主蛋白,并测试了一种可抑制特定Orf6与宿主蛋白相互作用的药物。
我们发现,在人293T细胞中过表达时,Orf6、非结构蛋白6(Nsp6)和Orf7a诱导的毒性最高。这三种蛋白在COS-7细胞中均显示膜定位。我们聚焦于细胞毒性最强且定位于内质网、自噬体和溶酶体膜的Orf6。蛋白质组学显示Orf6与核孔蛋白( Rae1、输出蛋白1(XPO1)、核转运蛋白2(RANBP2)和核孔蛋白)相互作用。用美国食品药品监督管理局(FDA)批准的XPO1抑制剂塞利尼索治疗,可减轻Orf6在人293T细胞中诱导的细胞毒性。
我们的研究揭示Orf6是SARS-CoV-2基因组中的一种高致病性蛋白,鉴定了其关键的宿主相互作用蛋白,并发现塞利尼索是一种直接靶向导致细胞毒性的Orf6-宿主蛋白相互作用的候选药物。