Modulation of Adenoviral Infections by CD44

描述(申请人提供)：在世界范围内，人类腺病毒(Ad)引起8%的病毒感染，导致一系列疾病，如呼吸道、胃肠道和泌尿生殖道感染，以及角结膜炎。流行病影响到学校出勤率和工作场所的生产力。据报道，在儿童和成人骨髓移植(BMT)患者等高危人群中存在威胁生命的播散性感染。根据我们的观察，玻璃/透明质酸(HA)不仅以剂量依赖的方式而且以CD44依赖的方式增强腺病毒(Ad)的载体转导，我们推测调节CD44与其配体HA之间的相互作用将抑制几种血清型的腺病毒感染。由于金属蛋白酶和v-分泌酶/PS1复合体对CD44的降解在调节蛋白质转录方面起着重要作用，我们还假设这种增强与CD44的降解以及由此导致的CD44依赖的信号通路的变化有关。为了验证这些假说，提出了以下具体目标：目的1)确定CD44在促进转基因表达中的作用。这将通过a)通过聚合酶链式反应删除基质金属蛋白酶和γ-分泌酶裂解位点来阐明CD44降解的作用，并通过过表达CD44的降解产物(CD44胞内区)来确定其在HA诱导的Ad转导增强中的作用；b)通过PCR突变来阐明CD44磷酸化改变CD44胞浆结构域中关键的磷酸化位点的作用，并研究它们对Ad5转导的影响。还将研究与CD44相关的下游信号分子的抑制剂；以及c)利用饱和浓度的RGD多肽和较低的孵育温度来确定Ad5是否能与HA受体CD44结合以阻断病毒的内吞作用。使用不表达CAR或CD46腺病毒受体但表达CD44的CHO细胞，将在存在和不存在HA的情况下确定结合。用消化后的HA、抗CD44和可溶性CD44研究Ad5与CHO细胞的结合。目的2)用不同血清型的AdS感染人结膜组织，用Q-PCR检测有或没有HA时释放的病毒颗粒，以确定CD44信号通路的调节是否影响不同血清型的野生型Ad的感染。CD44相关信号分子抑制剂的作用也将得到测试。解开CD44在腺病毒感染中的作用，可能会发现潜在的靶点，可以用来设计控制这些感染的新药，从而不仅影响高危患者的生活，还影响普通人群的健康和生产力。

DESCRIPTION (provided by applicant): Worldwide, human adenoviruses (Ad) cause 8% of viral infections responsible for a spectrum of diseases such as respiratory, gastro-intestinal, and urogenital tract infections, and keratoconjunctivitis. Epidemics affect school attendance and work-place productivity. Life-threatening disseminated infections have been reported among high-risk populations such as pediatric and adult bone marrow transplant (BMT) patients. Based on our observation that vitreous/hyaluronan (HA) enhances adenovirus (Ad) vector transduction not only in a dose dependent manner but also in a CD44 dependent manner, we hypothesize that modulating the interaction between CD44 and its ligand HA will inhibit adenoviral infections by several serotypes. Since degradation of CD44 by metalloproteinases and by the v-secretase/PS1 complex plays an important role in regulating protein transcription, we also hypothesize that this enhancement is related to degradation of CD44 with resultant changes in the CD44-dependent signaling pathways. To examine these hypotheses, the following Specific Aims are proposed: Aim 1) Determine the role of CD44 in the enhancement of transgene expression. This will be accomplished by a) elucidating the role of CD44 degradation by using PCR to delete the matrix metalloproteinase (MMP) and y-secretase cleavage sites and by over-expressing the degradation product of CD44 (the CD44 intracellular domain) to determine its role in the HA induced enhancement of Ad transduction; b) elucidating the role of CD44 phosphorylation using PCR mutagenesis to alter critical phosphorylation sites in the cytoplasmic domain of CD44 and studying their effects on Ad5 transduction. Inhibitors of downstream signaling molecules associated with CD44 will also be studied; and c) determining if Ad5 can bind to the HA receptor CD44 using saturating concentrations of RGD peptide and low incubation temperatures to block viral endocytosis. Binding will be determined in the presence and absence of HA using CHO cells that do not express the CAR or CD46 adenoviral receptors but do express CD44. Digested HA, anti-CD44 and soluble CD44 will be used to study binding of Ad5 to CHO cells. Aim 2) Establish if infections by wild type Ad of different serotypes can be influenced by modulation of the CD44 signaling pathway using Ads of different serotypes to infect human conjunctiva explants and measuring the viral particles released in the presence or absence of HA with Q-PCR. The effect of CD44 related signaling molecule inhibitors will also be tested. Unraveling the role that CD44 plays in adenoviral infections may uncover potential targets that can be used to design new drugs to control these infections thus impacting not only the lives of at-risk patients but also the health and productivity of the general population.