Structural analysis of herpesvirus assembly by cryo electron tomography

将电子冷冻断层扫描应用于病毒感染的细胞，目的是阐明病毒和宿主细胞的大分子复合体在接近自然状态的原位参与和相互作用。与Beate Sodeik(汉诺威)和Hans-Georg Kräusslich(Heidelberg)团队合作，我们将分别研究疱疹病毒组装和人类免疫缺陷病毒在质膜上萌发的方面。该项目是基于我们早先对分离的病毒颗粒的总体结构的研究，以及将该技术扩展到病毒感染的宿主细胞的目标。从未感染细胞获得的初步数据显示，电子束可以充分穿透外围细胞区域，以便在低剂量条件下获取断层扫描数据集。此外，我们将建立冷冻水合状态下宿主细胞的切片，以克服细胞标本厚度对电子冷冻断层扫描的固有限制。应用荧光显微镜和电子冷冻显微镜的混合方法，我们将能够专注于组装过程的特定状态。所选择的技术允许将现有的生化研究结果与亚细胞水平的自然结构信息联系起来。

Applying electron cryo tomography to virus infected cells we aim to elucidate the involvement and interaction of macromolecular complexes from both, the virus and the host cell, in situ in a near native state. Collaborating with the groups of Beate Sodeik (Hannover) and Hans-Georg Kräusslich (Heidelberg) we will investigate aspects of herpesvirus assembly and human immunodeficiency virus budding at the plasma membrane, respectively. The project is based on our earlier studies on the overall architecture of isolated virus particles for both viruses and targets on extending the technique to virus infected host cells. Preliminary data obtained from uninfected cells show that peripheral cell regions can be sufficiently penetrated by the electron beam to allow acquisition of tomographic data sets under low dose conditions. Additionally, we will establish the sectioning of host cells in the frozen hydrated state to overcome the inherent limitations for electron cryo-tomography imposed by the thickness of the cellular specimens. Applying a hybrid approach of fluorescence microscopy and electron cryo-microscopy we will be able to focus on defined states of the assembly process. The chosen technique allows linking existing results from biochemical studies to native structural information at the sub-cellular level.