Pathways and kinetics of foamy virus uptake and its glycoprotein-mediated fusion process

脂膜包裹的病毒已经进化出各种巧妙的策略来将它们的遗传信息传递到敏感的宿主，它们依赖于敏感的宿主进行复制。首先要克服的障碍是细胞膜，无论是在细胞表面还是在内部细胞器。被包膜的病毒使用嵌入在其病毒膜上的糖蛋白与细胞膜融合，从而提供其包含病毒基因组的内部蛋白质壳(衣壳)进入宿主细胞细胞质的途径。这项建议的总体目标是以泡沫病毒(FVS)为模型系统，深入了解包膜病毒特别是逆转录病毒的入侵机制。FV是最古老的逆转录病毒之一，与它们的自然宿主共同进化，是非人类灵长类动物、猫、牛和马的地方病。FV在基因治疗策略中很有希望成为基因转移载体，其特点是在自然宿主和人畜共感染的人类中表现出明显的无致病性。我们已经开发了带有荧光标记的具有感染性的FV颗粒，使我们能够研究病毒复制，并通过延时显微镜技术在活细胞中追踪单个病毒颗粒的命运。利用这一新工具，我们可以证明FV主要通过内吞机制进入宿主细胞，以严格依赖于细胞周期的方式将它们的遗传信息传递到宿主细胞核，并需要细胞有丝分裂才能将病毒基因组整合到宿主细胞染色质中。此外，我们还开发了一种在活细胞中追踪衣壳和糖蛋白标记的荧光抗体的单一病毒颗粒的方法。使用这种方法，我们经常观察到导致病毒衣壳释放到细胞质中的生产性融合事件。最引人注目的是，我们检测到了一种以前未知的中间进入步骤(系留状态)，其特征是病毒衣壳和糖蛋白信号之间的距离增加，随后这两种结构都会在细胞内独立运动。这项建议的重点是阐明FV摄取的初始步骤的细节，并特别关注结合使用遗传、生化和成像技术的融合过程。我们将开发新的病毒工具，用于定量和可视化以前描述的FV融合的其他中间步骤，如病毒和细胞脂的混合或病毒和宿主细胞细胞质之间的小分子交换，然后释放病毒衣壳。结合这些新工具和已建立的荧光标记FV颗粒的单病毒颗粒跟踪方法，我们将表征FV的摄取途径，包括鉴定所利用的内吞途径的类型，确定单个摄取步骤的动力学和顺序，并通过分析包含不同逆转录病毒种类成分的嵌合病毒来研究它们如何依赖于不同的病毒成分。

Lipid membrane enveloped viruses have evolved a variety of elegant strategies to deliver their genetic information into susceptible host, on which they depend for replication. The first barriers to overcome are cellular membranes, either at the cell surface or internal organelles. Enveloped viruses use glycoproteins embedded in their viral membrane for fusion with cellular membranes, thereby providing access of their inner protein shell (capsid) containing the viral genome to the host cell cytoplasm. The overall aim of this proposal is to gain insight into the entry mechanisms of enveloped viruses and retroviruses in particular, using foamy viruses (FVs) as a model system. FVs are one of the most ancient retroviruses having co-evolved with their natural hosts and are endemic to non-human primates, cats, cattle and horses. A hallmark of FVs, which are promising candidates as gene transfer vehicles in gene therapeutic strategies, is their apparent apathogenicity in natural hosts and also in zoonotically infected humans. We have developed fluorescently tagged, infectious FV particles allowing us to study viral replication and tracing the fate of single viral particles by time-lapse microscopy techniques in the living cells. Using this new tool, we could demonstrate that FVs enter host cells predominantly by endocytic mechanisms, deliver their genetic information to the host cell nucleus in a strictly cell cycle dependent manner and require cellular mitosis for viral genome integration into host cell chromatin. Furthermore, we developed a method for single viral particle tracing of capsid and glycoprotein tagged fluorescent FVs in living cells. Using this method, we frequently observed productive fusion events resulting in release of the viral capsid into the cytoplasm. Most strikingly, we detected a previously unknown intermediated entry step (tethering state), characterized by an increased distance between viral capsid and glycoprotein signals that precedes subsequent independent intracellular movement of both structures. The focus of this proposal is to elucidate the details of initial steps of FV uptake with a special focus on the fusion process using a combination of genetic, biochemical and imaging techniques. We will develop new viral tools for quantitation and visualization of other previously characterized intermediate steps of FV fusion such as mixing of viral and cellular lipids or exchange of small molecules between virus and host cell cytoplasm prior to release of the viral capsid. Combining these new tools and the established single viral particle tracing methodologies of fluorescently tagged FV particles, we will characterize the uptake pathways of FVs including identification of the types of endocytic pathways exploited, determine the kinetics and the order of the individual uptake steps and investigate how they depend on different viral components by analyzing chimeric viruses containing components of different retrovirus species.