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

Lead Research Organisation: University of Leeds
Department Name: Inst of Molecular & Cellular Biology

Abstract

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.

Technical Summary

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.
 
Description The overall aim of the fellowship was to develop a quantitative proteomic approach to assess the changes with the nucleolar proteome during herpesvirus infection. To this end, I utilised a modified method incorporating Stable Isotope Labeling with Amino acids in Cell culture (SILAC) coupled to LC-MS/MS. Once all the data has been collected for 3 different herpesviruses and importantly the proteome changes have been confirmed, I have assembled the data into interaction networks based upon the proteins observed to change in the SILAC screen and various pre-exisiting virus and human protein-protein interactomes. These data networks have then been used to identify which cellular protein changes into or out of the nucleolus are essential for virus replication.

We have shown dramatic changes in the nucleolar proteome during the course of a herpesvirus infection. Examples include :

(i) Network pathway analysis has identified that a relatively large group of cellular proteins involved in cellular DNA replication are redistributed out of the nucleolus during herpesvirus infection; these include DNA topoisomerase 1 and 2; hMSH2; poly(ADP-ribose) pol I; the complete Mini-chromosome complex (MCM2-7); Upstream binding factor (UBF); Rad50 and structural maintenance of chromosomes (SMC) proteins.

(ii) KSHV ORF57 enters the nucleolus and effects multiple pathways involved in RNA processing, incluing polyadenylation and mRNA export.

(iii) Network data has identified 2 other novel members of the cellular hTREX complex, namely Cip29 and SRAG which are required for ORF-57-mediated viral intronless mRNA nuclear export. Further siRNA-mediated depletion studies have shown that both Cip29 and SRAG are all essential for ORF57-mediated mRNA nuclear export of viral intronless mRNAs.

(iv) The assembly of the KSHV interaction network has also highlighted a dramatic redistribution of DNA repair and recombination enzymes into the nucleolus. This may be of particular significant, as KSHV replication can cause chromosome instability.
Exploitation Route Potential for new antiviral targets for oncogenic viruses


The SILAC-based proteomic analysis has been very successful and highlighted a number of new avenues which we have investigated in detail, this has lead to high impact publications (EMBO and PLoS pathogens). Moreover, it has provided new lines of inquiry which we are now being following up through recently awarded grant applications (BBSRC). Finally, it has highlighted a number of therapeutic avenues which we would like to further explore for the treatment of oncogenic herpesviruses.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description It has provided a greater understanding of how virus interaction with the host cell and provides opportunities to develop novel antiviral agents
First Year Of Impact 2011
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description Sequestering nucleolar-localised cellular proteins enhances KSHV DNA replication
Amount £160,401 (GBP)
Funding ID YCR 
Organisation Yorkshire Cancer Research 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2011 
End 05/2014
 
Description Targeting mRNA export pathways as a novel antiviral strategy for KSHV
Amount £178,323 (GBP)
Funding ID 12-1045 
Organisation Association for International Cancer Research 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2012 
End 09/2015
 
Description Viral control of the m6A methylome
Amount £440,000 (GBP)
Funding ID BB/M006557/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2015 
End 05/2018
 
Description Virus-mediated nucleolar polyadenylation
Amount £373,000 (GBP)
Funding ID BBSRC 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2013 
End 03/2016
 
Description Leeds Discovery Zone 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Discovery zone is large exhibition of biological science related activities to get children interested in sceince. My lab runs a stall on extracting DNA from fruit and explaining what DNA is. School participants are varied from affulent or very poor areas of Leeds. The children really enjoy this activity.
Year(s) Of Engagement Activity 2016,2017,2018,2019,2020,2021
URL http://www.fbs.leeds.ac.uk/outreach/schools/lfos.php
 
Description Presentation at Beaver camp 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk and experiment to extract DNA from fruit as local scout and beavers group in Leeds
Year(s) Of Engagement Activity 2015,2016,2017,2018,2019,2020
 
Description School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I do a talk about DNA and viruses and run an practical (extraction of DNA from fruit) and imaging of cheek cells to KS1 and KS2 (years 3,4,5,6) pupils at Bramhope primary school. Once a year as part of science week
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017,2018,2019,2020