Influenza PDZ Ligand Directed Pathogenesis

描述（由申请人提供）：人类大流行毒株的毒力增加与禽流感病毒（AIV）基因引入人类病毒有关，高致病性流感毒株感染与急性肺水肿有关，这增强了病毒的发病机制。感染期间直接肺泡液积聚增加的分子机制目前尚不清楚，确定高致病性人流感病毒的毒力因素对于开发抑制未来大流行严重性的治疗方法至关重要。我们已经证明，只有AIV NS1 PLs特异性结合Kv1.4的PDZ结构域，Kv1.4是一种调节肺泡液积聚的钾通道，Scribble是一种调节蛋白，指导通道通往根尖膜并介导紧密连接（TJ）的形成。Kv1.4钾通道包含一个n端PDZ结构域和一个c端PL基序，PL相互作用调节Kv1.4顶端膜的定位和功能。Kv1.4通过将钾泵入肺泡空间并同时指导肺泡对液体的吸收来调节肺泡液的积聚。据报道，阻止钾通道功能会导致肺泡液积聚和肺水肿（）。Kv1.4的PL基序与AIV ns1中的PL基序几乎相同，并且是Scribble 2个PDZ结构域的共识PDZ配体。Scribble是一种含有4个PDZ结构域的支架蛋白，它调节整体膜蛋白的顶膜定位和紧密连接的Rho定向通透性。我们假设高致病性流感病毒的NS1 PLs竞争Kv1.4和Scribble上的PL结合位点，阻断Kv1.4的肺泡液吸收功能，并解除紧密连接组装，从而导致肺泡内液体积聚增强。目的：在本研究中，我们将确定来自高致病性AIV菌株的PL是否阻断Kv1.4功能并改变紧密连接蛋白组装，从而增加肺部液体积聚。这些研究解决了流感病毒在肺泡内直接液体积聚和增强大流行毒株发病机制的具体手段。拟议的研究将确定潜在的流感毒力决定因素，确定在治疗上调节未来大流行性流感病毒发病机制的靶点，并为使用现有的钾通道激活剂减少感染期间肺部液体积聚提供依据。人类大流行毒株的毒力增强与禽流感病毒（AIV）基因进入人类病毒有关，高致病性流感毒株的感染与肺部积液有关，这加剧了疾病和感染的致命后果。拟议的研究涉及流感病毒指导肺内液体积聚并增加由大流行性流感病毒引起的疾病严重程度的特定手段。研究结果将为在未来流感大流行期间使用药物调节流感疾病严重程度提供一种手段，并为评估现有药物（激活钾通道）在流感病毒感染期间减少肺部液体积聚的能力提供依据。

DESCRIPTION (provided by applicant): Increased virulence of human pandemic strains is associated with the introduction of avian influenza virus (AIV) genes into human viruses, and infection by highly pathogenic influenza strains is associated with acute pulmonary edema which enhances viral pathogenesis. The molecular mechanisms that direct increased alveolar fluid accumulation during infection are currently unknown and defining virulence factors in highly pathogenic human influenza viruses is crucial for developing therapeutics that stems the severity of future pandemics. We have shown that only AIV NS1 PLs bind specifically to the PDZ domain of the Kv1.4, a potassium channel that regulates alveolar fluid accumulation, and to Scribble, a regulatory protein that directs channels to apical membranes and mediates tight junction (TJ) formation. The Kv1.4 potassium channel contains both an N-terminal PDZ domain and a C-terminal PL motif and PL interactions regulate Kv1.4 apical membrane localization and function. Kv1.4 regulates alveolar fluid accumulation by pumping potassium into the alveolar space and concomitantly directing fluid resorption from the alveolus. Preventing potassium channel function reportedly causes the accumulation of alveolar fluid and pulmonary edema ( ). The PL motif of Kv1.4 is nearly identical to PLs present in AIV NS1s and is a consensus PDZ ligand for 2 PDZ domains of Scribble. Scribble is a scaffolding protein containing 4 PDZ domains which regulates apical membrane localization of integral membrane proteins and Rho directed permeability of tight junctions. We hypothesize that the NS1 PLs of highly pathogenic influenza viruses compete for PL binding sites on Kv1.4 and Scribble, blocking alveolar fluid resorptive functions of Kv1.4 and uncoupling tight junction assembly which results in enhanced accumulation of fluid in alveoli. Objective: In this proposal we will determine whether the PL from highly pathogenic AIV strains block Kv1.4 function and alter tight junction protein assembly which increases pulmonary fluid accumulation. These studies address a specific means for influenza viruses to direct fluid accumulation within alveoli and enhance the pathogenesis of pandemic strains. Proposed studies will define a potential influenza virulence determinant, identify targets for therapeutically regulating the pathogenesis of future pandemic influenza viruses and provide a rationale for using existing potassium channel activators to reduce fluid accumulation in the lung during infection. Increased virulence of human pandemic strains is associated with the introduction of avian influenza virus (AIV) genes into human viruses, and infection by highly pathogenic influenza strains is associated with fluid accumulation in the lung which enhances disease and the lethal outcome of infection. Proposed studies address a specific means for influenza viruses to direct fluid accumulation within the lung and increase the severity of disease caused by pandemic influenza viruses. Results will provide a means for using drugs to regulate influenza disease severity during future pandemics and rationales for evaluating existing drugs (that activate potassium channels) for their ability to reduce fluid accumulation in the lung during influenza virus infection.