CRYO-EM STRUCTURAL STUDIES OF ADENOVIRUS CELL ENTRY

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。冷冻电子显微镜（cryo-EM）结合三维图像重建技术和新开发的时间分辨方法，提供了一种强有力的方法来检查病毒-宿主细胞相互作用以及病毒进入细胞过程中发生的动态结构变化。事实上，这些方法通常代表了大（1亿道尔顿）大分子复合物和具有高度移动的受体结合位点的病毒/受体复合物的唯一可行的结构方法，如对腺病毒所假设的。这些研究将阐明腺病毒/受体在细胞表面结合、内化成包被的小凹以及病毒颗粒从内体逃逸过程中的结构事件。具体目的包括成像与单克隆抗体的Fab片段复合的腺病毒颗粒，所述单克隆抗体阻断细胞进入以定位五邻体基础病毒组分上的Arg-Gly-Asp整联蛋白结合位点。此外，将解析与可溶形式的内化受体整合素avb 3和avb 5形成的病毒复合物艾德的结构，以评价受体结合后可能发生的结构变化。我们还将研究病毒和病毒/受体复合物的构象变化，作为改变pH值以模拟细胞外（pH 8）和早期内体（pH 6.0 - 6.5）环境的结果。低pH处理后快速冷冻（毫秒内）允许捕获结构中间体。另一个目的涉及计算腺病毒温度敏感突变体tsl的图像重建，tsl对于细胞进入是有缺陷的。将分析tsl突变体和野生型之间的结构差异以分离生产性腺病毒细胞进入所必需的元件。这些cryo-EM结构的研究将提供一个更好的了解腺病毒，一个有前途的载体在体内基因治疗的细胞进入过程。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cryo-electron microscopy (cryo-EM) combined with three-dimensional image reconstruction techniques and newly developed time-resolved methods provide a powerful way to examine virus-host cell interactions as well as the dynamic structural changes that occur during viral cell entry. In fact these methods often represent the only feasible structural approach for large (100 million Dalton) macromolecular complexes and virus/receptor complexes with highly mobile receptor binding sites, as has been postulated for adenovirus. These proposed studies will elucidate the structural events during adenovirus/receptor binding at thle cell surface, intemalization into coatedpits, and escape ofthe virus particle from the endosome. Specific aims include imaging the adenovirus particle complexed with a Fab fragment of a monoclonal antibody that blocks cell entry in order to localize the Arg-Gly-Asp integrin binding site on the penton base viral component. In addition, the structure of the virus complex ed with a soluble form of the internalization receptor, the integrins avb3 and avb5, will be solved in order to evaluate possible structural changes that occur upon receptor binding. We will also examine conformational changes in the virus and virus/receptor complexes as a consequence of varying the pH value to mimic both the extracellular (pH 8) and early endosomal (pH 6.0 - 6.5) environments. Rapid freezing (within milliseconds) after low pH treatment allows the trapping of structural intermediates. A further objective involves calculating an image reconstruction of the adenovirus temperature sensitive mutant, tsl, which is defective for cell entry. The structural differences between the tsl mutant and wild type will be analyzed to isolate elements essential for productive adenoviral cell entry. These cryo-EM structural studies will provide a greater understanding of the cell entry process of adenovirus, a promising vector for in vivo gene therapy.