Modulation of Adenoviral Infections by CD44

描述（由申请人提供）：在全球范围内，人腺病毒（Ad）引起8%的病毒感染，这些病毒感染可引起一系列疾病，如呼吸道、胃肠道和泌尿生殖道感染以及角膜结膜炎。流行病影响到学校出勤率和工作场所的生产力。在高危人群中，如儿童和成人骨髓移植（BMT）患者中，已报告了危及生命的播散性感染。基于我们观察到玻璃体/透明质酸（HA）不仅以剂量依赖性方式而且以CD 44依赖性方式增强腺病毒（Ad）载体转导，我们假设调节CD 44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复合物对CD 44的降解在调节蛋白质转录中起着重要作用，我们还假设这种增强与CD 44依赖性信号通路的降解相关。为了验证这些假设，提出了以下具体目的：目的1）确定CD 44在转基因表达增强中的作用。这将通过a）通过使用PCR删除基质金属蛋白酶（MMP）和γ-分泌酶切割位点并通过过表达CD 44的降解产物来阐明CD 44降解的作用（CD 44胞内结构域）以确定其在HA诱导的Ad转导增强中的作用; B）使用PCR诱变以改变CD 44的胞质结构域中的关键磷酸化位点来阐明CD 44磷酸化的作用，并研究它们对Ad 5转导的影响。还将研究与CD 44相关的下游信号传导分子的抑制剂;和c）使用饱和浓度的RGD肽和低孵育温度以阻断病毒内吞作用来确定Ad 5是否可以结合HA受体CD 44。在存在和不存在HA的情况下，使用不表达CAR或CD 46腺病毒受体但表达CD 44的CHO细胞测定结合。消化的HA、抗CD 44和可溶性CD 44将用于研究Ad 5与CHO细胞的结合。目的2）确定不同血清型的野生型Ad的感染是否可以通过使用不同血清型的Ad感染人结膜外植体并用Q-PCR测量在存在或不存在HA的情况下释放的病毒颗粒来调节CD 44信号传导途径来影响。还将测试CD 44相关信号传导分子抑制剂的作用。揭示CD 44在腺病毒感染中的作用可能会发现潜在的靶点，这些靶点可用于设计新的药物来控制这些感染，从而不仅影响高危患者的生命，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.