Studies on Influenza B Virus BM2 Protein Ion Channel

说明(由申请人提供)：流感在很大程度上导致了全球的发病率和死亡率。甲型流感病毒引起了所有的人类大流行，但乙型流感病毒感染每年都会发生，近年来它们占流感病毒感染总数的50%。使用反向遗传学程序改变甲型和乙型流感病毒的基因组，故意将致病决定因素引入病毒基因组，再加上流感病毒的气溶胶传播，这一相对简单的方法使这些病毒成为生物恐怖分子制造流行病和破坏的理想目标。最近，我们发现B型流感病毒的BM2蛋白是一种具有离子通道活性的寡聚体完整膜蛋白。BM2蛋白导致病毒内部的酸化，这是病毒在内小体中脱壳所必需的。BM2蛋白的功能与甲型流感病毒M2离子通道蛋白有关，后者是抗病毒药物金刚烷胺的靶点：该药物不抑制B型流感病毒复制或BM2离子通道活性。拟议的实验旨在阐明BM2蛋白的结构和功能的重要特征及其在B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。将确定BM2蛋白的磷酸化状态，并将使用反向遗传学程序确定影响BM2功能的特定突变的效果，以将这些突变引入B型流感病毒。(2)BM2蛋白活性形式的低聚状态将使用具有不同性质的BM2亚基的混合物来确定。(3)将通过半胱氨酸扫描突变鉴定BM2通道的孔衬残基，然后进行生化和功能研究。(4)对BM2蛋白活性的调控机制进行研究。(5)我们将开发体外检测重组BM2蛋白活性的系统，为制药行业筛选针对BM2的抗病毒化合物并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.