Modulation of Adenoviral Infections by CD44

描述（由申请人提供）：在世界范围内，人类腺病毒（Ad）导致8%的病毒感染，导致一系列疾病，如呼吸道、胃肠道、泌尿生殖道感染和角膜结膜炎。流行病影响学校出勤率和工作效率。在高危人群中，如儿童和成人骨髓移植（BMT）患者中有危及生命的播散性感染的报道。根据我们的观察，玻璃质/透明质酸（HA）不仅以剂量依赖的方式增强腺病毒（Ad）载体的转导，而且以CD44依赖的方式增强，我们假设调节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-secretase/PS1复合物对CD44的降解在调节蛋白质转录中起重要作用，我们也假设这种增强与CD44的降解有关，从而导致CD44依赖的信号通路发生变化。为了验证这些假设，提出了以下具体目标：目标1)确定CD44在增强转基因表达中的作用。这将通过a)阐明CD44降解的作用，通过使用PCR删除基质金属蛋白酶（MMP）和y分泌酶切割位点，并通过过表达CD44的降解产物（CD44胞内结构域）来确定其在HA诱导的Ad转导增强中的作用；b)利用PCR诱变技术阐明CD44磷酸化的作用，改变CD44细胞质域的关键磷酸化位点，并研究其对Ad5转导的影响。与CD44相关的下游信号分子抑制剂也将被研究；c)确定Ad5是否可以通过饱和浓度的RGD肽和低孵育温度与HA受体CD44结合以阻断病毒内吞作用。使用不表达CAR或CD46腺病毒受体但表达CD44的CHO细胞，将在HA存在和不存在的情况下确定结合。消化的HA，抗CD44和可溶性CD44将用于研究Ad5与CHO细胞的结合。目的2)利用不同血清型Ad感染人结膜外植体，通过Q-PCR检测HA存在或不存在时释放的病毒颗粒，确定不同血清型野生Ad感染是否会受到CD44信号通路调控的影响。CD44相关信号分子抑制剂的作用也将被测试。揭示CD44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控制这些感染的新药，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.