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

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.

Within the eukaryotic cell there is a continual requirement for macromolecules to move between the nucleus and cytoplasm, enabling a range of cellular processes to be efficiently regulated. Many viruses exploit nucleocytoplasmic transport in their replication strategies, and the virus proteins human immunodeficiency virus Rev and herpes simplex virus (HSV) ICP27 were some of the first to be shown to undergo a specialised form of rapid nucleocytoplasmic transport known as shuttling. These proteins were also used to demonstrate a major role for such shuttling proteins in RNA export. Proteins move between the nucleus and cytoplasm by binding specific receptors that recognise discrete peptide signals for either import (NLS) or export (NES). We have recently shown that specific members of a new group of viral shuttling proteins of unknown function, encoded by several alphaherpesvirus UL47 genes, contain variously an NLS that functions as an RNA binding domain, and a novel NES that has a sequence unlike any other NES so far defined. The aims of this proposal are to identify the cellular receptors involved in the nucleocytoplasmic transport of these UL47 proteins, in particular the export receptor for the novel NES. We also aim to address the mechanism of RNA binding by UL47 and determine its role in virus infection. To achieve this we will purify recombinant UL47 protein and its specific transport domains from baculovirus or E.coli expression systems. This will be used to fish out cellular proteins from soluble extracts that bind to either the import or export signals. The cellular binding partner of the novel NES will be fully characterised with regard to its role within the cell. Recombinant protein will also be used to fish out infected cell mRNAs that bind to UL47. Once identified, we will generate recombinant viruses with specific mutations in UL47 to establish the role played by UL47 RNA binding during infection.