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

疱疹病毒出口和外皮组装的三维单粒子追踪

• 批准号: 317830695
• 负责人: Dr. Katharina Maria Scherer
• 金额: --
• 依托单位: Department of Chemical Engineering and Biotechnology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/317830695?language=en
• 关键词: Three; dimensional; single; particle; tracking

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.

疱疹病毒是最复杂和最大的临床相关病毒之一。疱疹病毒在脊椎动物和人类中极为广泛，并在其宿主中建立终身感染。九种已知的人类疱疹病毒感染与许多严重疾病有关。虽然存在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的排出-知之甚少。支持疱疹病毒组装和逃逸的公认模型主要是由电子显微照片提供的。电子显微镜的一个问题是对固定样品和非动态状态的限制。该项目的目标是获得新的见解和完善的疱疹病毒组装和出口的模型，通过使用三维单粒子跟踪荧光标记的病毒在活细胞内的真实的时间。单粒子跟踪是研究细胞等异质系统内部动力学的强大工具，因为该技术以亚衍射分辨率定位每个粒子并测量其个体动力学，而不是整体平均值。与之前报道的疱疹病毒在细胞中的轨迹相比，使用主动3D跟踪所增加的观察时间将为我们提供更长的轨迹，并使我们首次能够在数十秒甚至几分钟的时间尺度上跟踪病毒颗粒的路径。这一新的信息将用于统计分析复杂的流动性模式的基础上个别病毒，在流动性状态的停留时间的识别和比较个别粒子的行为的整体平均值。在细胞区室的背景下，这种技术将使我们能够观察和阐明病毒-细胞相互作用的细节，例如在核膜处的核出口，在细胞质中的主动运输和在trans-Golgi膜处的次级粘附，在真实的时间具有高的时间和空间分辨率。最后，我们将结合联合收割机三维单粒子跟踪与双色成像，以揭示病毒衣壳和病毒外壳蛋白在病毒外出和组装过程中的相互作用。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，并开辟一种全新的方式来研究感染细胞内疱疹病毒蛋白质的相互作用。正确的被膜组装对于功能性病毒颗粒的形成是必要的，并且因此对于病毒颗粒在细胞之间和新宿主中的成功传播是必要的。被膜组装的活细胞数据缺失，进一步了解具有生物医学意义，因为它将为疱疹病毒复制提供新的见解，这在寻找新的抗病毒疗法中很重要。