Respiratory Virus Ion Channels

描述（由申请人提供）：流感对全球发病率和死亡率有很大贡献，据估计，下一次流感大流行将导致美国8万多人死亡，30万人住院，1800万人次门诊。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，但由于在治疗几天内形成金刚乙胺抗性逃逸突变，其有效性受到限制。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，特别是其跨膜孔和细胞质结构域的结构及其激活机制。以下个人目标旨在阐明M2蛋白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至关重要；我们将进行功能和结构实验，以确定色氨酸41在M2蛋白活化中的作用。2. M2蛋白的功能取决于其细胞质尾部的完整性。将进行荧光和电顺磁共振（EPR）光谱实验，以确定M2蛋白的细胞质尾部是形成位于膜表面的玫瑰花结还是形成延伸到细胞质中的螺旋。3. 孔隙结构的关键特征和M2离子通道的细胞质尾部将使用（EPR）光谱来确定。4. 为了更好地了解抗性机制，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，将对金刚烷胺抗性M2突变蛋白的功能特性进行表征。5. 在另一组实验中，我们将使用一种灵敏的方法来测试乙型流感病毒的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.