Studies on Influenza B Virus BM2 Protein Ion Channel

描述（由申请人提供）：流感是全球发病率和死亡率的重要因素。甲型流感病毒引起了所有人类大流行，但乙型流感病毒感染每年都会发生，最近几年，乙型流感病毒感染占流感病毒感染总数的50%。使用反向遗传学程序来改变甲型和乙型流感病毒的基因组，故意将致病性决定因素引入病毒基因组，再加上流感病毒的气溶胶传播，相对简单，这使得这些病毒成为生物恐怖分子用来制造流行病和破坏的理想候选者。近年来，我们发现乙型流感病毒BM2蛋白是一种具有离子通道活性的寡聚整体膜蛋白。BM2蛋白引起病毒粒子内部酸化，这是病毒在核内体中脱壳所必需的。BM2蛋白的功能与甲型流感病毒M2离子通道蛋白的功能相关，后者是抗病毒药物金刚烷胺的靶标：该药物不抑制乙型流感病毒的复制或BM2离子通道活性。本实验旨在阐明BM2蛋白结构和功能的重要特征及其在乙型流感病毒生命周期中的作用。(1)我们将利用生物化学、诱变和反向遗传学对BM2蛋白进行表征。BM2蛋白的磷酸化状态将被确定，影响BM2功能的特定突变的影响将通过将这些突变引入B型流感病毒的反向遗传学程序来确定。(2) BM2蛋白活性形式的寡聚状态将使用具有可区分性质的BM2亚基的混合物来确定。(3) BM2通道的孔壁残基将通过半胱氨酸扫描诱变进行鉴定，然后进行生化和功能研究。(4) BM2蛋白活性的调节机制将被研究。(5)我们将开发用于体外测量重组BM2蛋白活性的系统，为制药行业筛选BM2特异性抗病毒化合物和用于生物物理实验提供有用的手段。

DESCRIPTION (provided by the applicant): Influenza contributes substantially to worldwide morbidity and mortality. Influenza A virus has caused all human pandemics but influenza B virus infections occur annually and in some recent years they have constituted 50% of total influenza virus infections. The relative simplicity of using reverse genetics procedures to alter the genome of influenza A and B viruses to deliberately introduce pathogenicity determinants into the viral genome coupled with the aerosol transmission of influenza viruses, makes the viruses ideal candidates for use by bioterrorists to create epidemics and havoc. Recently, we discovered that the BM2 protein of influenza B virus is an oligomeric integral membrane protein with an ion channel activity. BM2 protein causes acidification of the virion interior that is required for virus uncoating in endosomes. The BM2 protein functions in a manner related to that of the influenza A virus M2 ion channel protein, the latter being the target of the anti-viral drug amantadine: the drug does not inhibit influenza B virus replication or the BM2 ion channel activity. The proposed experiments are designed to elucidate the important features of the structure and function of the BM2 protein and its role in the influenza B virus lifecycle. (1) We will characterize the BM2 protein using biochemistry, mutagenesis and reverse genetics. The phosphorylation state of the BM2 protein will be determined and the effect of specific mutations that affect BM2 function will be determined using reverse genetics procedures to introduce these mutations into influenza B virus. (2) The oligomeric state of the active form of the BM2 protein will be determined using mixtures of BM2 subunits with distinguishable properties. (3) The pore-lining residues of the BM2 channel will be identified using cysteine-scanning mutagenesis followed by biochemical and functional studies. (4) The mechanisms by which the activity of the BM2 protein is modulated will be studied. (5) We will develop systems for measuring the activity of recombinant BM2 protein in vitro in order to provide a useful means for the pharmaceutical industry to screen for antiviral compounds specific for BM2 and to use in biophysical experiments.