Studies on Influenza B Virus BM2 Protein Ion Channel

描述（由申请方提供）：流感是全球发病率和死亡率的主要原因。甲型流感病毒引起了所有人类大流行，但B流感病毒感染每年发生，近年来它们已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B型流感病毒的基因组以故意将致病性决定簇引入病毒基因组中的相对简单性，加上流感病毒的气溶胶传播，使得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。最近，我们发现流感B病毒的BM 2蛋白是具有离子通道活性的寡聚整合膜蛋白。BM2蛋白引起病毒粒子内部的酸化，这是病毒在内体中脱壳所需的。BM 2蛋白以与甲型流感病毒M2离子通道蛋白相关的方式发挥功能，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BM 2离子通道活性。所提出的实验旨在阐明BM 2蛋白的结构和功能的重要特征及其在流感B病毒生命周期中的作用。(1)我们将使用生物化学、诱变和反向遗传学来表征BM2蛋白。将确定BM 2蛋白的磷酸化状态，并使用反向遗传学程序将影响BM 2功能的特定突变引入流感B病毒中，以确定这些突变的影响。(2)将使用具有可区分性质的BM 2亚基的混合物来确定BM 2蛋白的活性形式的寡聚状态。(3)将使用半胱氨酸扫描诱变，然后进行生物化学和功能研究，鉴定BM 2通道的孔衬残基。(4)将研究调节BM 2蛋白活性的机制。(5)我们将开发用于测量重组BM 2蛋白的体外活性的系统，以便为制药工业提供有用的手段来筛选针对BM 2的抗病毒化合物并用于生物物理实验。

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.