Production of a molecular network pathway for herpesviruses and the nucleolus.

The mammalian cell nucleus is a membrane bound organelle. Within the nucleus several nuclear bodies or subnuclear structures exist. The largest of these is the nucleolus. For many years the role of the nucleolus was thought to be the site of ribosome biogenesis. However, recent analysis suggests that the nucleolus may have important roles in many aspects of cell biology. A novel way to understand these novel non-classical roles of the nucleolus is to analyse the relationship between viruses and the nucleolus. Many important virally encoded proteins localise to the nucleolus and this localisation is thought to be essential for the correct functioning of these viral proteins and efficient replication of the virus. Therefore, if we can understand why the viral proteins target the nucleolus, and how the nucleolus helps these viral proteins function it may help in understanding the wider non-classical roles of the nucleolus in general. Therefore the aim of this fellowship is to analyse the changes within the nucleolus during a herpesvirus infection. To do this I will determine the way cellular proteins are either lost or moved into the nucleolus during an infection. Moreover, we will identify which viral proteins are responsible for these changes. Once identified, we will then assess what happens to the replication of the herpesvirus when we inhibit the nucleolar proteins from being either lost or moved into the nucleolus. Finally, once the interaction between the herpesvirus proteins and the nucleolus have been identified, I will cross reference this data with existing databases which have mapped protein-protein interactions between herpesvirus proteins and also databases which have mapped protein-protein interactions between cellular proteins. This data will then be assembled to build up a molecular interaction network, showing which molecular pathways in the host cell each viral protein subverts or utilises. This can then be traced back to the nucleolar proteins which will help in understanding of how the nucleolar proteins function.

The nucleolus is now thought to be plurifunctional in nature. In addition, to its classical role in ribosomal biogenesis, it is now believed to have many non-classical roles in cell biology including cell cycle regulation, viral replication, tumourigenesis and cellular stress responses. Interestingly, an increasing number of viral proteins encoded by RNA and DNA viruses have been shown to localise to the nucleolus. For example, a genome-wide screen of 3 distinct herpesviruses, HSV-1, CMV and EBV, has shown that a least 12 herpesvirus-encoded proteins specifically localise to the nucleolus. This highlights that nucleolar interactions have important implications in the life cycle of herpesvirus. However, no information is available regarding how a virus affects the nucleolus during the course of an infection. Moreover, little is known about how these changes in the nucleolus aid virus replication. Therefore the overall aim of this fellowship is to build a molecular network which highlights the inter-relationship between herpesviruses and the nucleolus. To assemble this molecular network I aim to firstly to collate the nucleolar proteome changes during three distinct herpesvirus infections. This will be achieved using a SILAC-based quantitative proteomics approach. Once these changes are confirmed, I will assess how these nucleolar proteome changes relate to virus infection. Finally, data from this SILAC screen will be assembled in conjunction with pre-existing virus and human protein-protein interactome databases to build a molecular network highlighting the inter-relationship between herpesviruses and the nucleolus. The work described herein therefore clearly falls within the remit of the Biochemistry and Cell Biology Committee's priority area of the Systems Analysis of Multi-Protein Complexes. Furthermore, the work will lead to fundamental new insights into cell biology processes in general.