Structure and inhibition of the SARS coronavirus envelope protein ion channel.
Structure and inhibition of the SARS coronavirus envelope protein ion channel.
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DOI：
10.1371/journal.ppat.1000511
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发表时间：
2009-07
期刊：
影响因子：
6.7
通讯作者：
Torres J
中科院分区：
文献类型：
--
作者：
Pervushin K;Tan E;Parthasarathy K;Lin X;Jiang FL;Yu D;Vararattanavech A;Soong TW;Liu DX;Torres J
冠状病毒的包膜(E)蛋白是一种含有至少一个α-螺旋跨膜结构域的小多肽。由于病毒粒子形态或趋向性的改变，E蛋白的缺失或失活导致病毒减毒。除了其形态发生特性外，蛋白E还具有膜渗透活性。此外，药物六亚甲基阿米洛利（HMA），而不是阿米洛利，可以抑制一些合成冠状病毒E蛋白的体外离子通道活性，也可以抑制病毒复制。我们之前已经证明，对于导致严重急性呼吸综合征（SARS-CoV）的冠状病毒物种，E蛋白的跨膜结构域（ETM）形成五聚体α-螺旋束，这可能是观察到的通道活性的原因。利用十二烷基磷脂酰胆碱胶束的溶液核磁共振和能量最小化，我们得到了该通道的一个模型，该模型具有规则的α-螺旋，形成一个五聚体左手平行束。药物HMA被发现结合在通道的腔内，在c端和n端开口，并且，与amiloride相反，诱导额外的ETM化学位移。在全细胞膜片钳装置中，全长SARS-CoV E在人胚胎肾293 （HEK-293）细胞中短暂表达时显示通道活性。六亚甲基氨酰胺（HMA）显著降低了该活性，而氨酰胺没有显著降低。本文提出的通道结构提供了一种可能的抑制原理，并为未来基于结构的药物设计这种潜在的药理靶点提供了平台。冠状病毒是一种病毒性病原体，会导致鸟类和哺乳动物患上多种致命疾病，并导致人类患上普通感冒。然而，在2003年，一种动物冠状病毒能够感染人类并产生严重急性呼吸系统综合症（SARS），导致一场近乎大流行。此类事件在未来很可能再次发生，因此有必要采取新的抗病毒策略。一种名为“包膜”的冠状病毒小蛋白对发病机制很重要，影响病毒包膜的形成和病毒在体内的分布。体外研究表明，合成冠状病毒包膜蛋白具有通道活性，在某些情况下，这种活性被药物六亚甲基阿米洛利抑制，但不被阿米洛利抑制。在本文中，我们描述了负责该通道活动的结构。我们还确定了药物六亚甲基阿米洛利在通道中的结合位点，并表明阿米洛利对蛋白质的核磁共振信号只有轻微的影响。这些结果的有效性得到了表达全长SARS-CoV E的哺乳动物细胞的支持，其中通道活性被六亚甲基阿米洛利抑制，但阿米洛利仅轻度抑制。该通道的结构模型为了解冠状病毒包膜蛋白离子通道活性提供了有价值的见解，并可作为开发新型抗病毒药物的平台。
The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one α-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric α-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular α-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target. Coronaviruses are viral pathogens that cause a variety of lethal diseases in birds and mammals, and common colds in humans. In 2003, however, an animal coronavirus was able to infect humans and produced severe acute respiratory syndrome (SARS), causing a near pandemic. Such events are likely to reoccur in the future, and new antiviral strategies are necessary. A small coronavirus protein called ‘envelope’ is important for pathogenesis, affecting the formation of the viral envelope and the distribution of the virus in the body. In vitro studies have shown that synthetic coronavirus envelope proteins have channel activity that in some cases has been inhibited by the drug hexamethylene amiloride, but not by amiloride. In the present paper, we have characterized the structure responsible for this channel activity. We have also determined the binding site of the drug hexamethylene amiloride in the channel, and shown that amiloride has only a mild effect on the NMR signals from the protein. The validity of these results is supported using mammalian cells expressing full length SARS-CoV E, where channel activity was inhibited by hexamethylene amiloride, but only mildly by amiloride. The structural model described for this channel provides a valuable insight into coronavirus envelope protein ion channel activity, and could serve as a platform for the development of novel anti-viral drugs.
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影响因子：
2.7
作者：
González JM;Gomez-Puertas P;Cavanagh D;Gorbalenya AE;Enjuanes L
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Enjuanes L
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