Molecular interactions between soluable host factors and a gene delivery vector

描述(申请人提供)：人腺病毒(HAdV)是感染多种细胞类型的无包膜dsDNA病毒。物种C HAdV5是临床上最常用的基因治疗和疫苗应用载体。最近，有研究表明，在静脉注射后，A和C类HAdv与几种凝血因子(如X、IX因子)的结合使这些病毒能够进入上皮细胞和肝脏 活体肝细胞。这种凝血因子介导的途径导致C型HAdv意外地重定向到肝细胞。值得注意的是，这种重定向超越了天然受体(CAR)对病毒纤维蛋白的选择性。通过药物干预或HAd5Hexon蛋白突变来阻断Hexon-FX相互作用，可取消体内的肝转导。尽管HAdV-FX结合的低分辨结构信息可以从低温EM研究中获得，但关于FX的GLA结构域与HAD衣壳上的六邻体之间的分子相互作用仍然缺乏详细的知识。最近，我们用X射线衍射法测定了一种基于HAdV5的载体的晶体结构，命名为Ad35F，具有近原子分辨率。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趋向性的重要知识。缺乏准确的HAdV-FX相互作用模型阻碍了抗病毒药物和基因治疗载体的发展。用化学合成的GLA(cs-GLA)多肽对Ad35F晶体进行了初步的衍射实验，得到了相当好的结果。分辨率为6？的初始Fo-Fc图表明，GLA结构域结合位点在六邻体亚基上的足迹与先前提出的位置不同。这一建议旨在通过以下方法极大地提高我们对腺病毒在体内的宿主细胞趋向性的理解：1)利用X射线衍射方法在近原子分辨率下确定HAdV与FX-GLA结构域的复合体的结构。这些分析将建立在我们在解决Ad35F在3.5？分辨率下的晶体结构方面获得的广泛知识和专业知识的基础上，以及2)分析Hexon亚单位中单点突变(E451Q)的结构，该结构极大地减少了FX与HAdV的结合，这表明包含该突变的Hexon区域对于凝血因子关联至关重要。我们期望这些研究将提供更多对腺病毒转导肝细胞的机制和潜在的分子相互作用的理解。

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.