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来获得自己的优势，结果是病毒的生命周期和细胞的生理联系在一起。复杂的细胞，如在人体中发现的那些，是高度分隔的，每个分隔被自己的膜所包围。细胞中的主要隔间是细胞核和细胞质，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分隔这两个隔室的核膜上的小孔进行的。在复制期间，病毒必须找到方法将它们各自的构建块传递到细胞中的正确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路径。因此，病毒是解开细胞运输复杂性的极好工具，到目前为止，对病毒的研究一直是确定如何跨越核膜发生运动的基础。这类研究揭示了一组细胞蛋白的存在，它们附着在特定的病毒载体上，并促进它们在毛孔中的移动。虽然最初在病毒感染的细胞中发现了这些蛋白，但很明显，这些细胞蛋白是细胞核和细胞质之间所有运动的基本成分。这一建议涉及一组被称为UL47蛋白的疱疹病毒蛋白，其中几种已知可以在细胞核和细胞质之间有效地移动。两种最具特点的UL47是来自人类单纯疱疹病毒1型和牛疱疹病毒1型的UL47，虽然这些蛋白在病毒生长中所起的作用尚未确定，但它们都是导致感染的病毒颗粒的主要成分。由于这两种病毒都会在宿主动物中引起疾病，确定这些病毒如何与宿主相关并利用它们的宿主，对于抗击疾病至关重要。我们以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的表征，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展示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蛋白来筛选出与UL47已知参与运输的区域结合的细胞成分。然后，我们将把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，我们将致力于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外，我们还希望了解它们为什么表现出这种运输。有初步证据表明，UL47可能参与了另一类病毒产物的结合，信使RNA分子最终被细胞用来制造蛋白质。由于RNA从细胞核到细胞质的运输是病毒生长所必需的，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移动是否是病毒RNA运输的先决条件。有一大批科学家对往返原子核的基本运输领域感兴趣，因此，预计拟议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的细胞成分将引起细胞生物学家的兴趣，并可能揭示特定细胞蛋白质运动的新途径。此外，确定UL47蛋白为什么以这种方式移动将是疱疹病毒学家感兴趣的，并将使我们更接近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.