Cleavage of the SARS coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro.
Cleavage of the SARS coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro.
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DOI：
10.1371/journal.pone.0007870
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发表时间：
2009-11-17
期刊：
影响因子：
3.7
通讯作者：
Altmeyer R
中科院分区：
文献类型：
--
作者：
Kam YW;Okumura Y;Kido H;Ng LF;Bruzzone R;Altmeyer R
被包膜的病毒进入宿主细胞需要被细胞内或细胞外的酶通过裂解激活病毒包膜糖蛋白。为了深入了解蛋白水解酶切割的分子基础及其对病毒侵入效率的影响，我们研究了SARS冠状病毒重组天然全长S蛋白三聚体(TriSpike)对多种呼吸道蛋白酶切割的敏感性。纯化的TriSpike蛋白在体外很容易被胰酶、纤溶酶和TMPRSS11a三种不同的呼吸道蛋白酶切割。高效液相色谱法和氨基酸序列分析确定两个精氨酸残基(R667和R797)为潜在的蛋白酶切割位点(S)。用ACE2表达人支气管上皮细胞16HBE，证实了蛋白酶依赖的增强SARS-CoV感染的作用。呼吸道蛋白水解酶以依赖于先前受体结合的方式调节SARS-CoV的感染性。突变结构(R667A、R797A或R797AR667A)进一步显示了精氨酸残基的作用。R667或R797突变不影响S蛋白的表达，但导致对SARS-CoVpp伪分型的疗效不同。R667A SARS-CoVpp突变体在蛋白酶处理后缺乏病毒进入增强作用。这些结果表明，SARS S蛋白对呼吸道蛋白水解酶的切割很敏感，而且，蛋白酶介导的病毒侵袭的增强依赖于SARS S蛋白与ACE2结合时的特定构象。这些数据对SARS-CoV沿呼吸系统的细胞进入机制具有直接的意义，并进一步扩大了寻找潜在的治疗SARS-CoV药物的可能性。
Entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike) of the severe acute respiratory syndrome coronavirus (SARS-CoV) to cleavage by various airway proteases. Purified triSpike proteins were readily cleaved in vitro by three different airway proteases: trypsin, plasmin and TMPRSS11a. High Performance Liquid Chromatography (HPLC) and amino acid sequencing analyses identified two arginine residues (R667 and R797) as potential protease cleavage site(s). The effect of protease-dependent enhancement of SARS-CoV infection was demonstrated with ACE2 expressing human bronchial epithelial cells 16HBE. Airway proteases regulate the infectivity of SARS-CoV in a fashion dependent on previous receptor binding. The role of arginine residues was further shown with mutant constructs (R667A, R797A or R797AR667A). Mutation of R667 or R797 did not affect the expression of S-protein but resulted in a differential efficacy of pseudotyping into SARS-CoVpp. The R667A SARS-CoVpp mutant exhibited a lack of virus entry enhancement following protease treatment. These results suggest that SARS S-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of SARS S-protein upon ACE2 binding. These data have direct implications for the cell entry mechanism of SARS-CoV along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against SARS-CoV.
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影响因子：
64.8
作者：
Deng, HK;Liu, R;Landau, NR
通讯作者：
Landau, NR
影响因子：
5.4
作者：
Bosch, Berend Jan;Bartelink, Willem;Rottier, Peter J. M.
通讯作者：
Rottier, Peter J. M.
DOI：
10.1046/j.1432-1327.2000.01346.x
发表时间：
2000-06-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Chen, Y;Shiota, M;Kido, H
通讯作者：
Kido, H
受体结合后SARS冠状病毒尖峰的构象重组：对膜融合的影响。
影响因子：
3.7
作者：
Beniac DR;deVarennes SL;Andonov A;He R;Booth TF
通讯作者：
Booth TF
影响因子：
5.4
作者：
Jia, HP;Look, DC;McCray, PB
通讯作者：
McCray, PB