Three-dimensional single particle tracking of herpesvirus egress and tegument assembly

疱疹病毒是临床上最复杂和最大的病毒之一。疱疹病毒在脊椎动物和人类中极其广泛，并在宿主中建立终身感染。已知的九种人类疱疹病毒的感染与许多严重疾病有关。尽管有许多关于疱疹病毒复制周期的研究，但对感染周期的后期阶段-病毒颗粒的组装及其从细胞中流出-知之甚少。对公认的疱疹病毒组装和排泄模型的支持主要由电子显微镜提供。电子显微镜的一个问题是对固定样品和非动态样品的限制。本项目的目标是通过使用实时荧光标记的病毒在活细胞内的三维单粒子跟踪来获得新的见解并完善疱疹病毒组装和输出的模型。单粒子跟踪是研究细胞等非均质系统内部动力学的有力工具，因为该技术以亚衍射分辨率定位每个粒子，并测量其单独的动力学，而不是总体平均。与以前报告疱疹病毒在细胞中的轨迹的工作相比，使用主动3D跟踪所带来的观察时间的增加将为我们提供更长的轨迹，并使我们第一次能够在几十秒甚至几分钟的时间尺度上跟踪病毒颗粒的路径。这些新的信息将被用于基于单个病毒的复杂移动模式的统计分析，在移动状态下的停留时间的识别，以及单个粒子行为与总体平均行为的比较。在细胞室的背景下，这项技术将使我们能够以高时间和空间分辨率实时观察和阐明病毒与细胞相互作用的细节，如核膜上的核外泄、细胞质中的主动运输和跨高尔基膜的二次包膜。最后，我们将结合三维单颗粒跟踪和双色成像来揭示病毒外泄和组装过程中病毒衣壳和病毒被蛋白之间的相互作用。通过实施双色、三维单粒子跟踪，我们将把技术能力发挥到极限，为研究感染细胞内疱疹病毒蛋白的相互作用开辟一条全新的途径。正确的被膜组装对于功能性病毒颗粒的形成是必要的，因此对于病毒颗粒在细胞之间和新宿主之间的成功传播是必要的。由于缺少被膜组装的活细胞数据，进一步了解它具有生物医学意义，因为它将为疱疹病毒复制提供新的见解，这对寻找新的抗病毒疗法是重要的。

Herpesviruses are among the most complex and largest of the clinically relevant viruses. Herpesviruses are extremely widespread in vertebrates and humans and establish life-long infections in their hosts. Infections by the nine known human herpesviruses are associated with many serious diseases. Although many studies exist about the replication cycle of herpesviruses, not much is known about the later stages of the infection cycle - the assembly of virus particles and their egress from the cell. Support for the accepted model of herpesvirus assembly and egress was provided mostly by electron micrographs. A problem of electron microscopy is the limitation to fixed specimens and non-dynamic states. The goal of this project is to gain new insight and refine the model of herpesvirus assembly and egress by the use of three-dimensional single particle tracking of fluorescently labeled viruses in real time inside living cells. Single-particle tracking is a powerful tool to study the dynamics inside heterogeneous systems like cells because this technique locates each particle with sub-diffraction resolution and measures its individual dynamics, instead of an ensemble average. Compared to previous work reporting on herpesviruses trajectories in cells, the increased observation times afforded by use of active 3D tracking will provide us with much longer trajectories and enable us for the first time to follow the path of the virus particles on timescales of tens of seconds or even minutes. This novel information will be used for the statistical analysis of complex mobility patterns based on individual viruses, identification of dwell times in mobility states and comparison of individual particle behavior to the ensemble average. In the context of cellular compartments, this technique will allow us to observe and elucidate details of viral - cell interactions during processes such as nuclear egress at the nuclear envelope, active transport in the cytoplasm and secondary envelopment at trans-Golgi membranes for example, with high temporal and spatial resolution in real time. Finally, we will combine three-dimensional single particle tracking with dual colour imaging to uncover interactions between virus capsids and viral tegument proteins during virus egress and assembly. By implementation of two-color, three-dimensional single particle tracking we will take the technical capabilities to the limits and open up a completely new way to study herpesvirus protein interactions inside infected cells. Correct tegument assembly is necessary for functional virus particle formation, and thus for successful spreading of virus particles between cells and to new hosts. Live-cell data of tegument assembly is missing and further understanding is of biomedical interest, since it will provide fresh insights into herpesvirus replication, which is important in the search for novel antiviral therapies.