Distinctive Roles of Furin and TMPRSS2 in SARS-CoV-2 Infectivity.
Distinctive Roles of Furin and TMPRSS2 in SARS-CoV-2 Infectivity.
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FURIN和TMPRSS2在SARS-COV-2感染性中的独特作用。
DOI：
10.1128/jvi.00128-22
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发表时间：
2022-04-27
影响因子：
5.4
通讯作者：
中科院分区：
文献类型：
--
作者：
严重急性呼吸综合征冠状病毒(SARS-CoV-2)的刺突蛋白(S)与血管紧张素转换酶2(ACE2)受体结合后，可引导肺部和其他组织的感染。为了有效感染，S蛋白在两个位置上被切割：S1/S2和S2‘。已证明，在细胞和小动物模型中，表面S蛋白S1/S2(PRRAR685↓)[下划线的碱性氨基酸是指识别呋喃所需的关键残基]的“启动”对于SARS-CoV-2在细胞和小动物模型中的感染性是重要的。在本研究中，我们首次通过蛋白质组学明确地将融合激活位点S2‘确定为KPSKR815↓(下划线的碱性氨基酸是指识别Furin所需的关键残基)，并证明了ACE2与S蛋白的结合显著增强了这种切割。新型药物呋喃西林抑制剂(BoS抑制剂)有效地阻断了HeLa细胞内源性S蛋白的加工，而肺源性CALU-3细胞的SARS-CoV-2感染完全被呋喃沙林(BoS)和II型跨膜丝氨酸蛋白酶2(TMPRSS2)的联合抑制剂(Camostat)所阻止。细胞间融合和S蛋白加工的定量分析表明，在没有S1/S2启动的情况下，TMPRSS2介导的融合增强需要TMPRSS2脱落ACE2。我们进一步证明了ACE2的集合素二聚化结构域在TMPRSS2对细胞间融合的影响中是必不可少的。总体而言，我们的结果表明，Furin和TMPRSS2在病毒进入和传染性方面发挥了协同作用，支持Furin和TMPRSS2抑制剂作为有效的抗SARS-CoV-2药物。新冠肺炎的病原体SARS-CoV-2迄今已导致全球610万人死亡。病毒的刺突蛋白(S)通过与血管紧张素转换酶2(ACE2)受体结合，引导肺部和其他组织的感染。为了有效感染，S蛋白在两个位置上被切割：S1/S2和S2‘。S_1/S_2的切割导致构象改变，有利于血管紧张素转换酶2识别S蛋白。S2的裂解对于触发膜融合和病毒进入宿主细胞是至关重要的。我们的研究突出了在SARS-CoV-2进入过程中S蛋白ACE2与宿主蛋白酶Furin和TMPRSS2之间相互作用的复杂动力学，并表明无毒的Furin抑制剂和TMPRSS2抑制剂的组合显著地减少了病毒进入肺细胞，∼平均协同作用使病毒感染减少了95%。这代表了一种强大的新的抗病毒方法，以减少病毒在感染SARS-CoV-2或未来相关冠状病毒的个人中的传播。
The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2′. The “priming” of the surface S protein at S1/S2 (PRRAR685↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2′ as KPSKR815↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2′. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2′ cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.
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影响因子：
5.4
作者：
Heurich, Adeline;Hofmann-Winkler, Heike;Poehlmann, Stefan
通讯作者：
Poehlmann, Stefan
影响因子：
64.8
作者：
Hoffmann, Markus;Moesbauer, Kirstin;Poehlmann, Stefan
通讯作者：
Poehlmann, Stefan
影响因子：
5.4
作者：
Glowacka, Ilona;Bertram, Stephanie;Poehlmann, Stefan
通讯作者：
Poehlmann, Stefan
影响因子：
5.6
作者：
Forget, J;Yao, XJ;Cohen, EA
通讯作者：
Cohen, EA
影响因子：
15.1
作者：
Gunst JD;Staerke NB;Pahus MH;Kristensen LH;Bodilsen J;Lohse N;Dalgaard LS;Brønnum D;Fröbert O;Hønge B;Johansen IS;Monrad I;Erikstrup C;Rosendal R;Vilstrup E;Mariager T;Bove DG;Offersen R;Shakar S;Cajander S;Jørgensen NP;Sritharan SS;Breining P;Jespersen S;Mortensen KL;Jensen ML;Kolte L;Frattari GS;Larsen CS;Storgaard M;Nielsen LP;Tolstrup M;Sædder EA;Østergaard LJ;Ngo HTT;Jensen MH;Højen JF;Kjolby M;Søgaard OS
通讯作者：
Søgaard OS