Isoform-Specific Regulation and Localization of the Coxsackie and Adenovirus Rece

描述（由申请人提供）：腺病毒和腺病毒受体与气道上皮生物学和疾病的发展密切相关。腺病毒于1953年首次从扁桃体中发现。1968年，美国武装部队急性呼吸道疾病委员会发现腺病毒感染是新兵急性发热性呼吸道疾病的主要原因之一。在过去的50年里，腺病毒研究已经产生了关于病毒性肺炎的发病机制、疫苗接种以及分子病毒学和细胞生物学的基本方面的令人印象深刻的知识。然而，腺病毒仍然是一个重大的民用和军事威胁。腺病毒受体（柯萨奇病毒和腺病毒受体; CAR）的发现和最近的发现，CAR在气道上皮细胞-细胞粘附中起着核心作用，产生了新的机制，病毒逃逸上皮表面。一个尚未解决的主要问题是，当受体安全地分离在基底外侧膜上时，这些致病病毒如何启动感染。已经描述了CAR的几种蛋白质同种型，包括两种跨膜形式（CARex 7和CARex 8）。这两种异构体仅在C-末端不同，这表明它们的一些相互作用以及因此的定位和调节可能不同。在极化上皮细胞中，CARex 7位于基底外侧表面上，因此与顶表面上的潜在病毒相互作用隔离。相比之下，虽然CARex 8是一种丰度较低的同种型，但我们最近的工作揭示CARex 8位于顶端，在那里它可以介导腺病毒感染从顶端表面开始。我们假设受体丰度和顶端定位是由PDZ与膜相关鸟苷酸激酶反向1，亚型B（MAGI-1 B）的相互作用调节的。我们的目标是通过PDZ结构域分离、突变、结合和竞争，最终在极化气道细胞中了解这种相互作用的分子基础。此外，我们假设与MAGI-1b的相互作用是在顶端运输途径内与含PDZ结构域的蛋白质竞争。我们将采用候选蛋白的方法，并通过免疫细胞化学和免疫共沉淀-Western印迹分析研究新的相互作用。了解根尖定位背后的分子机制具有临床意义，原因有几个。目前还没有针对柯萨奇病毒或腺病毒感染的特异性治疗方法;因此，在病毒爆发时阻断病毒顶端结合的能力将是一个重大的治疗进展。另一方面，增强受体顶端表达的能力对于有效的腺病毒介导的基因治疗具有高度相关性。此外，这项工作将使一个研究生和本科生团队在病毒学和医学的界面上进行重要的研究。 公共卫生相关性：病毒引起的急性呼吸道疾病每年造成大量人类疾病和死亡。我们最近发现一种结合柯萨奇病毒和腺病毒的受体存在于气道上皮细胞暴露于空气的表面。了解是什么原因导致这种表面位置以及如何调节将提供对病毒感染易感性的深入了解，并导致预防病毒感染和促进腺病毒介导的基因治疗用于治疗遗传性和获得性呼吸道疾病的策略。

DESCRIPTION (provided by applicant): Adenovirus and the adenovirus receptor are intricately linked to airway epithelia biology, and to the development of disease. Adenoviruses were first identified from tonsils in 1953. In 1968 the Commission of Acute Respiratory Diseases of the U.S. Armed Forces found that adenovirus infection was one of the major causes of acute febrile respiratory illness in recruits. Over the past 5 decades adenovirus research has yielded impressive knowledge about the pathogenesis of viral pneumonias, vaccination, and basic aspects of molecular virology and cellular biology. However, adenovirus remains a significant civilian and military threat. The discovery of the receptor for adenovirus (Coxsackievirus and adenovirus receptor; CAR) and the recent finding that CAR plays a central role in airway epithelial cell-cell adhesion have yielded novel mechanisms for viral escape from epithelial surfaces. A major unanswered question that remains is how these pathogenic viruses initiate infection when the receptor is safely segregated on the basolateral membrane. Several protein isoforms have been described for CAR, including two transmembrane forms (CAREx7 and CAREx8). These two isoforms differ only in their C-termini, suggesting that some of their interactions, and hence localization and regulation, may differ. In polarized epithelial cells, CAREx7 resides on the basolateral surface and is thus sequestered away from potential viral interactions on the apical surface. In contrast, although CAREx8 is a less abundant isoform, our recent work reveals that CAREx8 is localized apically where it can mediate initiation of adenovirus infection from the apical surface. We hypothesize that receptor abundance and apical localization are regulated by a PDZ-based interaction with membrane-associated guanylate kinase inverted 1, isoform b (MAGI-1b). We aim to understand the molecular basis of this interaction through PDZ domain isolation, mutation, binding and competition, ultimately in polarized airway cells. Moreover, we hypothesize that the interaction with MAGI-1b is in competition with PDZ-domain-containing proteins within the apical trafficking pathway. We will take a candidate protein approach and investigate novel interactions by immunocytochemistry and co-immunoprecipitation-Western blot analysis. Understanding the molecular mechanisms behind apical localization is clinically significant for several reasons. Currently there is no specific treatment for coxsackievirus or adenovirus infection; thus, the ability to block apical binding of the virus in the face of viral outbreaks would be a significant therapeutic advance. On the other hand, the ability to augment apical expression of the receptor would have high relevance for efficient adenoviral-mediated gene therapy. Moreover, this work will expose a team of graduate and undergraduate students to vital research at the interface of virology and medicine. PUBLIC HEALTH RELEVANCE: Viral-induced acute respiratory disease causes a significant amount of human illness and death each year. We have recently discovered one form of the receptor that binds both coxsackievirus and adenovirus is present on the air exposed surface of the airway epithelium. Understanding what causes this surface location and how this is regulated will provide insight into susceptibility to viral infections, and lead to strategies both for prevention of virus infection and facilitation of adenovirus-mediated gene therapy for the treatment of inherited and acquired respiratory diseases.