Respiratory Virus Ion Channels

描述(申请人提供)：流感极大地导致了全球的发病率和死亡率，据估计，下一次流感大流行将导致美国超过8万人死亡，30万人住院，1800万人次就诊。甲型流感病毒的M2离子通道蛋白是抗病毒药物金刚乙胺的靶标，但其用途受到治疗几天内形成的金刚乙胺抗药性逃逸突变的限制。为了开发更有效的抗病毒药物，需要更多的关于M2蛋白的信息，特别是它的跨膜孔和细胞质结构域的结构以及它被激活的机制。以下个人目标旨在阐明M2蛋白的结构和功能的重要特征。1.跨膜孔中的色氨酸41对M2离子通道蛋白的激活是必不可少的；我们将进行功能和结构实验，以确定色氨酸41在M2蛋白激活中的作用。2.M2蛋白的功能依赖于其胞质尾部的完整性。将进行荧光和电子顺磁共振(EPR)光谱实验，以确定M2蛋白的细胞质尾巴是形成位于膜表面的玫瑰花结，还是形成延伸到细胞质的螺旋。3.利用电子顺磁共振(EPR)谱确定M2离子通道的孔道和胞质尾部的主要结构特征。4.为了更好地了解耐药机制，并确定通道的哪些功能特性对其在病毒生命周期中的作用至关重要，将对金刚烷胺耐药突变M2蛋白的功能特性进行表征。5.在另一组实验中，我们将使用一种敏感的方法来测试B型流感病毒的Nb蛋白的离子通道活性。

DESCRIPTION (provided by applicant): Influenza contributes substantially to worldwide morbidity and mortality, and it has been estimated that the next influenza pandemic will result in over 80,000 deaths, 300,000 hospitalizations and 18 million outpatient visits in the United States. The M2 ion channel protein of influenza A virus is the target of the antiviral drug rimantadine, but its usefulness is limited by the formation of rimantadine-resistant escape mutations within a few days of treatment. In order to develop more effective antiviral drugs, more information is needed about the M2 protein, especially the structure of its transmembrane pore and cytoplasmic domains and the mechanism by which it is activated. The following individual aims are designed to elucidate important features of the structure and function of the M2 protein. 1. Tryptophan 41 in the transmembrane pore is essential for activation of the M2 ion channel protein; we will perform functional and structural experiments to ascertain the role of tryptophan 41 in activation of the M2 protein. 2. The function of the M2 protein depends on the integrity of its cytoplasmic tail. Fluorescence and electroparamagnetic resonance (EPR) spectroscopy experiments will be performed to determine whether the cytoplasmic tail of the M2 protein forms a rosette lying on the membrane surface or forms a helix extending into the cytoplasm. 3. Key features of the structure of the pore and the cytoplasmic tail of the M2 ion channel will be determined using (EPR) spectroscopy. 4. In order to understand better the mechanism for resistance and to identify which functional properties of the channel are essential for its role in the virus life cycle, the functional properties of amantadine-resistant mutant M2 proteins will be characterized. 5. In a separate set of experiments we will use a sensitive method to test the NB protein of influenza B virus for ion channel activity.