Molecular interactions between soluable host factors and a gene delivery vector

描述（由申请方提供）：人腺病毒（HAdV）是感染多种细胞类型的无包膜dsDNA病毒。种C HAdV 5是临床上用于基因治疗和疫苗应用的最常用载体。最近，已经表明A和C种HAdV与几种凝血因子（例如，因子X，IX）静脉内递送后允许这些病毒进入上皮细胞和肝脏 肝细胞。这种凝血因子介导的途径导致C型HAdV意外重靶向肝细胞。值得注意的是，这种重定向覆盖了病毒纤维蛋白的天然受体（CAR）的选择性。通过药理学干预或HAd 5六邻体蛋白的突变阻断六邻体-FX相互作用，在体内消除肝脏转导。虽然低分辨率的结构信息HAdV-FX协会可从cryoEM研究，详细的知识之间的分子相互作用的GLA结构域的FX与六邻体的HAd衣壳仍然缺乏。我们最近通过X射线衍射以近原子分辨率确定了基于HAdV 5的载体（命名为Ad 35 F）的晶体结构。尽管有这些新的结构信息，我们仍然缺乏关于病毒衣壳如何影响体内组织嗜性的重要知识。HAdV-FX相互作用的精确模型的缺乏阻碍了抗病毒药物和基因治疗载体的开发。用化学合成的GLA（cs-GLA）肽浸泡的Ad 35 F晶体的初步衍射实验已经相当肯定。最初的Fo-Fc图谱在6？分辨率下表明，六邻体亚基上的GLA结构域结合位点的足迹与先前建议的位置不同。该提议试图通过以下方式极大地提高我们对腺病毒宿主细胞体内向性的理解：1）通过采用X射线衍射方法以近原子分辨率确定与FX-GLA结构域复合的HAdV的结构。这些分析将建立在我们在解决3.5 ½分辨率的Ad 35 F晶体结构和2）分析六邻体亚基中的单点突变体（E451 Q）的结构中获得的广泛知识和专业知识的基础上，该结构大大降低了FX与HAdV的结合，这表明含有该突变的六邻体区域对凝血因子缔合至关重要。我们预计，这些调查将提供更多的了解机制和潜在的分子相互作用，参与腺病毒转导肝细胞。

DESCRIPTION (provided by applicant): Human adenoviruses (HAdV) are nonenveloped dsDNA viruses that infect a variety of cell types. Species C HAdV5 is the most commonly used vector for gene therapy and vaccine applications in the clinic. Recently, it has been shown that association of species A and C HAdVs with several blood coagulation factors (e.g., Factors X, IX) following intravenous delivery allow these viruses to gain access to epithelial cells and liver hepatocytes in vivo, respectively. This clotting factor-mediated pathway results in the unintended retargeting of C-type HAdVs to hepatocytes. Remarkably, this retargeting overrides the selectivity of natural receptor (CAR) for the virus fiber protein. Blocking of the hexon-FX interaction, either by pharmacological intervention or mutation of HAd5 hexon protein, abolishes liver transduction in vivo. Although low-resolution structural information on HAdV-FX association is available from cryoEM studies, detailed knowledge on the molecular interactions between the GLA-domain of FX with the hexon on the HAd capsid is still lacking. We recently determined the crystal structure for a HAdV5 based vector, designated Ad35F, at near atomic resolution by X- ray diffraction. Despite this new structural information, we still lack important knowledge of how the virus capsid influences tissue tropism in vivo. The absence of an accurate model of HAdV-FX interactions hampers the development of antivirals and gene therapy vectors. Preliminary diffraction experiments on Ad35F crystals soaked with chemically synthesized GLA (cs-GLA) peptide have been quite positive. Initial Fo-Fc maps at 6¿ resolution indicate that footprint of th GLA domain binding site on the hexon subunits is different from the location previously suggested. This proposal seeks to greatly improve our understanding of adenovirus host cell tropism in vivo by 1) determining the structure of HAdV in complex with the FX-GLA domain at near atomic resolution by employing X-ray diffraction methods. These analyses will build on the extensive knowledge and expertise that we gained in solving the crystal structure of Ad35F at 3.5 ¿ resolution and 2) analyzing the structure of single point mutant (E451Q) in the hexon subunit that drastically reduces FX binding to HAdV suggesting that the region of the hexon containing this mutation is crucial for clotting factor association. We anticipate that these investigations will provide greater understanding of mechanism and underlying molecular interactions involved in adenovirus transduction of liver hepatocytes.