Investigation of cellular nucleocytoplasmic transport pathways using the alphaherpesvirus UL47 group of proteins.

所有病毒的一个基本特征是，它们利用正常的细胞过程为自己谋利，其结果是，病毒的生命周期和细胞的生理是密不可分的。复杂的细胞，比如在人体中发现的细胞，是高度区隔的，每个区隔都被自己的膜包围着。细胞中的主要隔室是细胞核和细胞质，细胞核是发现DNA的地方，细胞质是制造蛋白质的地方。大分子在细胞核和细胞质之间的运输是通过核膜上的孔进行的，核膜上的孔将细胞核和细胞质分开。在复制过程中，病毒必须找到方法将它们的单个构建块运送到细胞中正确的位置进行进一步处理，为了做到这一点，它们劫持了这些运输途径。因此，病毒是揭示细胞运输复杂性的绝佳工具，迄今为止，对病毒的研究在确定运动如何跨越核膜方面已经取得了根本性的进展。这些研究揭示了一组细胞蛋白的存在，它们附着在特定的病毒货物上，并促进它们通过毛孔的运动。虽然最初是在病毒感染的细胞中发现的，但已经很清楚，这些细胞蛋白是细胞核和细胞质之间所有运动的基本组成部分。这一建议与一组被称为UL47蛋白的疱疹病毒蛋白有关，其中一些已知能在细胞核和细胞质之间有效移动。两种最具特征的ul47是来自人类单纯疱疹病毒1型和牛疱疹病毒1型的ul47，尽管这些蛋白质在病毒生长中所起的作用尚未确定，但它们都是病毒颗粒在细胞间移动引起感染的主要成分。由于这两种病毒都能在其宿主动物中引起疾病，因此确定这些病毒如何与宿主发生关系并利用宿主对防治疾病具有重要意义。我们之前已经对这些蛋白质如何在细胞核和细胞质之间移动进行了基本表征，并表明所涉及的途径可能是一个尚未表征的系统。我们现在希望积极地证明这些病毒蛋白是如何进入和离开细胞核的，并确定它们的细胞运输伙伴。为了做到这一点，我们将使用大量纯化的UL47蛋白来捞出与已知参与运输的UL47区域结合的细胞成分。然后，我们将结合的蛋白质与人类蛋白质数据库进行比较，并确定它们的身份。如果我们发现新的蛋白质，我们将着手确定这些蛋白质在细胞内的作用。除了了解ul47如何在细胞核和细胞质之间移动之外，我们还希望了解为什么它们表现出这种运输。有初步证据表明UL47可能参与结合另一类病毒产物，信使RNA分子，最终被细胞用来制造蛋白质。由于RNA从细胞核转运到细胞质对病毒生长至关重要，我们希望表征这种RNA结合，并确定UL47在细胞核和细胞质之间的运动是否是病毒RNA转运的先决条件。有一大群科学家对进出原子核的传输这一基本领域感兴趣，因此可以预期，拟议的研究产生的数据可能会有广泛的受众。鉴定细胞核和细胞质之间运动的新细胞成分将是细胞生物学家感兴趣的，并可能揭示特定细胞蛋白质运动的新途径。此外，确定UL47蛋白以这种方式移动的原因将引起疱疹病毒学家的兴趣，并将使我们更接近于理解为什么这些病毒将这些运输蛋白包装到它们的结构中。

A fundamental feature of all viruses is that they exploit normal cellular processes to their own advantage, with the result that the virus life-cycle and the physiology of the cell are inextricably linked. Complex cells such as those found in the human body are highly compartmentalised, with each compartment being surrounded by its own membrane. The major compartments in the cell are the nucleus, where DNA is found, and the cytoplasm, where proteins are made. The transport of large molecules between the nucleus and the cytoplasm takes place through pores in the nuclear membrane that separates these two compartments. During replication, viruses must find ways to deliver their individual building blocks to the correct sites in the cell for further processing, and in order to do this they hijack these transport pathways. As a result, viruses are excellent tools for unravelling the complexities of cellular transport, and to date studies on viruses have been fundamental in determining how movement occurs across the nuclear membrane. Such studies have revealed the presence of a group of cellular proteins that attach to specific viral cargoes and facilitate their movement through the pores. Although initially identified in virus infected cells, it has become clear that these cellular proteins are the basic components of all movement between the nucleus and cytoplasm. This proposal is concerned with a group of herpesvirus proteins called the UL47 proteins, several of which are known to move efficiently between the nucleus and cytoplasm. The two best-characterised UL47s are those from the human herpes simplex virus type 1 and bovine herpesvirus type 1, and although the role that these proteins play in virus growth has not yet been defined, both of them are major components of the virus particles that move from cell to cell causing infection. As both these viruses cause disease in their host animals, determining how these viruses relate to and exploit their host is of fundamental importance in combatting disease. We have previously carried out a basic characterisation of how these proteins move between the nucleus and cytoplasm, and have shown that the pathway involved may be an as yet uncharacterised system. We now wish to positively demonstrate how these viral proteins get into and out of the nucleus, and identify their cellular transport partners. To do this we will use large amounts of purified UL47 proteins to fish out cellular components that bind to the regions of UL47 known to be involved in transport. We will then compare the bound proteins to a database of human proteins and determine their identity. If we find new proteins we will undertake to establish the role of such proteins within the cell. In addition to understanding how the UL47s move between the nucleus and cytoplasm, we also wish to understand why they exhibit such transport. There is preliminary evidence to suggest that UL47 may be involved in binding another class of viral product, the messenger RNA molecules that are eventually used by the cell to make proteins. As the transport of RNA from the nucleus to the cytoplasm is essential for virus growth, we wish to characterise this RNA binding and determine if the movement of UL47 between the nucleus and cytoplasm is a prerequisite for transport of viral RNA. There is a large community of scientists interested in the basic field of transport to and from the nucleus, and therefore it is anticipated that the data generated by the proposed study could have a wide audience. Identification of new cellular components for movement between the nucleus and cytoplasm will be of interest to cell biologists, and may reveal new pathways for the movement of specific cellular proteins. Furthermore, determining why the UL47 proteins move in this way will be of interest to herpesvirologists and will take us a step closer to understanding why these viruses package these transport proteins into their structure.