Arginine methylation regulates the function of a key herpesvirus nucleocytoplasmic shuttle protein

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

Abstract

mRNA is transcribed in the nucleus and must be exported from the nucleus into the cytoplasm to get translated into a protein. In the nucleus, the RNA has to undergo a series of processing events prior to export and these are performed by a number of multi-protein complexes. However, how these complexes are recruited to the RNA and how each multi-protein complex is regulated to perform its specific function at the correct time is unknown. Herein, we aim to investigate how the function of one of these RNA binding multi-protein complexes is regulated using a viral model. We have previously demonstrated that the herpesvirus ORF57 protein is important for exporting herpesvirus mRNA out of the nucleus to get translated. ORF57 functions by binding to the herpesvirus RNA and specifically recruiting cellular proteins onto the viral RNA, which are essential for its correct processing and export. This system has uniquely identified that a multi-protein complex called hTREX is the only cellular protein complex which is required by ORF57 for herpesvirus mRNA nuclear export. Moreover, we have demonstrated that once hTREX is bound by ORF57 to the herpesvirus mRNA, the whole complex is transported through a domain within the nucleus called the nucleolus, before it is exported from the nucleus into the cytoplasm. Therefore, ORF57 is a multifunctional protein which sequentially, binds the viral RNA, then recruits hTREX to the viral RNA, then transports this complex to the nucleolus, then allows the complex to exit the nucleus. But what tells ORF57 to perform its many functions in the correct manner is unknown. This functional diversity of a protein can be controlled by different chemical modifications to the protein, such as adding or taking away a phosphate, acetate or methyl group on the protein. Interestingly, we have demonstrated that the herpesvirus ORF57 protein undergoes one such modification. We have shown that ORF57 can be methylated at arginine residues. Therefore, we aim to determine if adding or taking away methyl groups affects ORF57's function and in turn regulates the multi-protein complex responsible for exporting viral mRNA from the nucleus. To assess the role of methylation we will first identify which residues of ORF57 are methylated. We will then mutate these, so that ORF57 cannnot undergo methylation. This will then allow us to assess what effect it has upon the functioning of the ORF57 protein. Secondly, we will identify which cellular enzymes are responsible for performing this post-translational modification on the viral ORF57 protein. Finally, we have preliminary data to suggest that taking away the methyl group on ORF57 (or demethylating ORF57), is the trigger to tell ORF57 to exit the nucleus. We believe this happens in the nucleolus and we will identify which enzyme in the nucleolus performs this function. These data will provide a better understanding of how multi-protein complexes function to enable RNAs to exit the nucleus and also provide a better understanding of how viruses function in order to identify new antiviral targets in the longer term.

Technical Summary

Processing of mRNA from transcription to translation is regulated by several multi-protein complexes. These multi-protein complexes are coupled to one another allowing processing events to occur in a strict temporal manner. However, how these complexes are regulated is unknown. We have utilised a viral model to study the function of one of these multi-protein complexes, which is involved in mRNA nuclear export. KSHV ORF57 orchestrates the assembly of an export-competent viral ribonucleoprotein particle (RNP) by recruitment of the hTREX complex to intronless viral mRNAs. Moreover, we have shown that the ORF57-mediated RNP trafficks through the nucleolus and this is essential for efficient nuclear export. How ORF57 co-ordinates it's many is unknown, but we have preliminary data to suggest that the methylation status of ORF57 is important for its function. Specifically, we have demonstrated that ORF57 can be methylated but also interacts with a nucleolar demethylase enzyme, suggesting that it can also undergo demethylation. Moreover, nucleocytoplasmic shuttling of ORF57 is regulated by its methylation status, as inhibition of ORF57 methylation redistributes ORF57 into the cytoplasm. We now aim to further investigate these observations and determine the role of arginine methylation in the functioning of this key herpesvirus nucleocytoplasmic shuttle protein. We will initially aim to identify the residues within ORF57 which are methylated using mass spectroscopy. Moreover, we will identify the enzymes which are responsible for both the methylation and demethylation of the ORF57 protein. An ORF57 mutant protein will then be produced which cannot undergo methylation. This mutant will then be compared to the wild type ORF57 protein to assess what effect arginine methylation / demethylation has upon the multifunctional roles of ORF57 such as RNA binding, hTREX recruitment, nucleolar trafficking, nucleocytoplasmic shuttling and intronless mRNA export.
 
Description We have shown that the functioning of an essential herpesviral protein required for efficient replication of the virus interacts with a cellular protein arginine methyltransferase. This leads to the viral protein becoming post-translationally methylated. We have identified where upon the viral protein methylation occurs and produced mutant viral proteins which cannot become methylated. Functional analysis of these mutants hve shown that they have inferior RNA binding capacity and therefore methylation is required for specific functioning of the viral protein. In addition, we have shown that ORF57 interacts with another cellular protein that regulates this post-translational methylation and this interacts occurs in the host cell nucleolus. This provides the first evidence that the cellular nucleolus maybe a site for viral protein post-tranlsational modifications.
Exploitation Route Potential for new antiviral targets for oncogenic viruses Provides ew insights regarding the role of methylation in the regulation of nucleocytoplasmic shuttle proteins, formation of export competent ribonucleoprotein particles and mRNA nuclear export. In addition,provides information of an undescribed role of the nucleolus as a compartment for demethylating proteins. Moreover, insights gained would provide a better understanding of the host cellular interactions required for the functioning of a key herpesvirus nucleocytoplasmic shuttle protein - which may lead to novel antiviral agents in the future.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
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 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