A second functional furin site in the SARS-CoV-2 spike protein

The ubiquitously-expressed proteolytic enzyme furin is closely related to the pathogenesis of SARS-CoV-2 and therefore represents a key target for antiviral therapy. Based on bioinformatic analysis and pseudovirus tests, we discovered a second functional furin site located in the spike protein. Furin still increased the infectivity of mutated SARS-CoV-2 pseudovirus in 293T-ACE2 cells when the canonical polybasic cleavage site (682–686) was deleted. However, K814A mutation eliminated the enhancing effect of furin on virus infection. Furin inhibitor prevented infection by 682–686-deleted SARS-CoV-2 in 293T-ACE2-furin cells, but not the K814A mutant. K814A mutation did not affect the activity of TMPRSS2 and cathepsin L but did impact the cleavage of S2 into S2′ and cell–cell fusion. Additionally, we showed that this functional furin site exists in RaTG13 from bat and PCoV-GD/GX from pangolin. Therefore, we discovered a new functional furin site that is pivotal in promoting SARS-CoV-2 infection.

普遍表达的蛋白水解酶弗林蛋白酶（furin）与新型冠状病毒（SARS-CoV-2）的致病机制密切相关，因此是抗病毒治疗的关键靶点。本研究通过生物信息学分析与假病毒实验，在刺突蛋白（spike protein）中发现了第二个功能性弗林蛋白酶切割位点。当经典多碱基切割位点（canonical polybasic cleavage site，682–686位）缺失后，弗林蛋白酶仍可增强293T-ACE2细胞中突变型新型冠状病毒假病毒的感染性；但K814A突变可消除弗林蛋白酶对病毒感染的增强效应。弗林蛋白酶抑制剂可抑制682–686位缺失的新型冠状病毒在293T-ACE2-furin细胞中的感染，但对K814A突变株无效。K814A突变不影响跨膜丝氨酸蛋白酶2（TMPRSS2）与组织蛋白酶L（cathepsin L）的活性，但可影响S2蛋白切割为S2′片段以及细胞间融合过程。此外，本研究证实该功能性弗林蛋白酶切割位点同时存在于蝙蝠源RaTG13冠状病毒以及穿山甲源PCoV-GD/GX冠状病毒中。综上，本研究发现了一个新的功能性弗林蛋白酶切割位点，其在促进新型冠状病毒感染过程中发挥关键作用。