Analysing the Role of Ubiquitin and Ubiquitin-like Modification During Herpesvirus Infection and Host Immunity

Lead Research Organisation: University of Glasgow

Abstract

Dr Chris Boutell, Lead Investigator, MRC-University of Glasgow Centre for Virus Research (CVR), University of Glasgow
The modification of proteins regulates many important aspects of cell biology, including host immunity to infection. Viruses have therefore evolved strategies to utilise or suppress pathways that regulate protein modification to overcome host defences to infection, thereby enhancing their replication, spread, and disease burden. However, these key interactions remain largely undefined at a biochemical level. Work within this programme focuses on two pathways, namely ubiquitin and SUMO (Small Ubiquitin-like MOdifier), which we and others have shown to play an important role in the regulation of host immunity. Our aim is to define the activity of core enzymes that influence the outcome of infection, using herpes simplex virus 1 (HSV-1) as a model DNA virus. These studies will reveal unique insight into the mechanisms that regulate host immunity during DNA virus infection, as well as uncovering fundamental principles that regulate other important areas of cell biology relevant to human disease, for example carcinogenesis. By understanding these principles we aim to develop new targeted approaches for future therapeutic intervention to treat viral infections pertinent to public health.

Technical Summary

The post-translational modification (PTM) of proteins by ubiquitin and ubiquitin-like (UBL) polypeptides regulates many fundamental aspects of cell biology, including multiple facets relating to host immunity. Viruses have therefore evolved mechanisms to utilise or suppress these PTM pathways to overcome host immune defence mechanisms during infection, thereby promoting their replication and spread. Understanding the biochemical interplay between viral and host factors which utilise these pathways will therefore lead to new insights into the regulation of host immunity, processes which restrict the replication of many clinically important viruses. This programme aims to define the biochemistry of key viral host-cell interactions that utilise ubiquitin and SUMO PTM pathways to influence the outcome of host immunity during herpesvirus infection, using HSV-1 as a model system. By defining the biochemistry of these interactions we aim to: significantly advance our understanding of the signalling mechanisms that mediate the cellular restriction of herpesviruses and the establishment of viral latency; uncover fundamental principles relating to the regulation of ubiquitin and SUMO ligases that mediate key aspects of cellular immunity during HSV-1 infection; develop methodologies suitable for the identification of viral ubiquitin ligase inhibitors using ICP0 as a model ligase.
This programme will add significant value to the MRC, CVR, and its associated research partners through the delivery of an interdisciplinary and internationally competitive programme of research. This programme will strengthen the study of ubiquitin and ubiquitin-related pathways within the CVR in other virus systems, including influenza A virus (IAV), Hepatitis C Virus (HCV), and selected arboviruses, through the dissemination of research reagents and expert knowledge. Our goal is to define the functional significance and host-cell regulation of these important PTM pathways during infection, thereby revealing fundamental insight into the biochemical mechanisms that influence the outcome of viral infection and pathogenesis

Publications

10 25 50

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Boutell C (2013) Regulation of alphaherpesvirus infections by the ICP0 family of proteins. in The Journal of general virology

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Busnadiego I (2014) Host and viral determinants of Mx2 antiretroviral activity. in Journal of virology

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Everett RD (2013) Interplay between viruses and host sumoylation pathways. in Nature reviews. Microbiology

 
Description ASM Conference on Viral Manipulation of Nuclear Processes
Amount $1,000 (USD)
Organisation American Society for Microbiology (ASM) 
Sector Charity/Non Profit
Country United States of America
Start 11/2014 
End 11/2014
 
Description BBSRC DTP PhD studentship
Amount £10,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2015 
End 10/2019
 
Description ISSF Univeristy of Glasgow polyomics funding scheme
Amount £5,000 (GBP)
Organisation University of Glasgow 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 11/2016 
 
Description King Abdulaziz University Scholarship scheme
Amount £100,000 (GBP)
Organisation King Abdulaziz University 
Sector Academic/University
Country Saudi Arabia, Kingdom of
Start 10/2014 
End 10/2017
 
Description NIH Chromatin Control of Viral Infection
Amount $500 (USD)
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States of America
Start 09/2014 
End 09/2014
 
Title in vitro SUMO biochemistry 
Description The cloning, expression, and purification of various proteins implicated in the regulation of SUMOylation. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2012 
Provided To Others? Yes  
Impact multiple ongoing collaborations 
 
Title in vitro ubiquitin biochemsitry 
Description The cloning, expression, and purification of various proteins implicated in ubiquitin biochemistry. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2010 
Provided To Others? Yes  
Impact 1. Lilley, C. E., M. S. Chaurushiya, C. Boutell, S. Landry, J. Suh, S. Panier, R. D. Everett, G. S. Stewart, D. Durocher, and M. D. Weitzman. 2010. A viral E3 ligase targets RNF8 and RNF168 to control histone ubiquitination and DNA damage responses. Embo J 29:943-55. 2. Morris, J. R., C. Boutell, M. Keppler, R. Densham, D. Weekes, A. Alamshah, L. Butler, Y. Galanty, L. Pangon, T. Kiuchi, T. Ng, and E. Solomon. 2009. The SUMO modification pathway is involved in the BRCA1 response to genotoxic stress. Nature 462:886-90. 3. Morris, J. R., L. Pangon, C. Boutell, T. Katagiri, N. H. Keep, and E. Solomon. 2006. Genetic analysis of BRCA1 ubiquitin ligase activity and its relationship to breast cancer susceptibility. Hum Mol Genet 15:599-606. 
 
Title shRNA lentiviral cell lines 
Description Primary cell lines that have been transduced with lentiviral vectors expressing short hairpin RNAs (shRNAs) to deplete components of the SUMO conjugation pathway. 
Type Of Material Cell line 
Year Produced 2010 
Provided To Others? Yes  
Impact none currently. Research ongoing. 
 
Title single genome labelling technologies to study virus host-cell interactions 
Description viral nucleic acid represents a major immune stimulus. However, visualization of viral nucleic acid upon entry to host cells in relation to cellular proteins which mediate immune signalling has been problematic. We have optimized an efficient methodology for labeling viral DNA that has enabled us to detect viral nucleic acid within cells 15 minutes post-infection. This technique will prove extremely valuable in the identification of cellular processes that directly contribute to regulation of host immunity to virus infection. 
Type Of Material Biological samples 
Year Produced 2017 
Provided To Others? Yes  
Impact We are currently writing two manuscripts which describe the use of this technique 
 
Description Cellular restriction of Influenza virus 
Organisation University of Glasgow
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Provision of reagents to study SUMOylation and ubiquitination during Influenza virus infection
Collaborator Contribution Provision of reagents and expert knowledge within this area of research
Impact multi-disciplinary virology research
Start Year 2012
 
Description Defining the role of SUMOylation during arbovirus infection 
Organisation The Pirbright Institute
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Defining the biochemical activity of host SUMOylation enzymes expressed in Aedes aegepti.
Collaborator Contribution Cloning of cDNAs that express Aedes aegepti SUMOylation enzymes
Impact Research ongoing. Student selected for national presentation (microbiology society)
Start Year 2014
 
Description Defining the role of the SUMO ligase ZNF451 in the regulation of PML NBs and intrinsic antiviral immunity 
Organisation Max Planck Society
Department Max Planck Institute of Immunobiology and Epigenetics
Country Germany, Federal Republic of 
Sector Public 
PI Contribution Examining the role of ZNF451 during HSV-1 infection and host-cell intrinsic antiviral immunity
Collaborator Contribution expression plasmids and polyclonal antibodies
Impact none
Start Year 2015
 
Description Reactivation of Latent HSV Requires a Specific Function encoded in RL2 
Organisation Beckman Researh Institute, City of Hope
Country United States of America 
Sector Academic/University 
PI Contribution Provision of research reagents
Collaborator Contribution Provision of reagents
Impact Ongoing research. No outputs recorded yet
Start Year 2014
 
Description Regulation of the DNA damage response during viral infection 
Organisation Children's Hospital of Philadelphia
Department Center for Cellular and Molecular Therapeutics
Country United States of America 
Sector Academic/University 
PI Contribution Provision of biochemical expertise in the study of viral ubiquitin ligases
Collaborator Contribution Providing a detailed understanding of DNA repair mechanisms initiated in response to infection
Impact See assoc. publications list
Start Year 2008
 
Description SUMO modification and centromeres 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Department Center for Molecular Genetics (CGM)
Country France, French Republic 
Sector Academic/University 
PI Contribution biochemical analysis
Collaborator Contribution Cell biology analysis
Impact Cell biology Ubiquitin SUMOylation Biochemistry
Start Year 2012
 
Description The role of ICP0 in HSV-1 viral pathogenesis 
Organisation University of Kansas
Department Department of Molecular Biosciences
Country United States of America 
Sector Academic/University 
PI Contribution Provision of reagents, experimentation and publication
Collaborator Contribution Provision of reagents and model system for viral pathogenesis
Impact 1. Boutell, C., R. Everett, J. Hilliard, P. Schaffer, A. Orr, and D. Davido. 2008. Herpes simplex virus type 1 ICP0 phosphorylation mutants impair the E3 ubiquitin ligase activity of ICP0 in a cell type-dependent manner. J Virol 82:10647-56. 2. Smith, M. C., C. Boutell, and D. J. Davido. 2011. HSV-1 ICP0: paving the way for viral replication. Future Virol 6:421-429.
Start Year 2006
 
Description X-ray crystallography studies of the purified ICP0 RING-finger domains from HSV-1 and VZV 
Organisation York University Toronto
Country Canada 
Sector Academic/University 
PI Contribution Provision of reagents for the purification of RING-domain containing viral E3 ubiquitin ligases expressed during HSV-1 and VZV infection
Collaborator Contribution Purification of recombinant proteins and X-ray crystallography trials
Impact none
Start Year 2015
 
Description Glasgow Virology Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact This annual one day meeting aims to encourage interactions between virologists within Scotland and across the UK. It is usually very well attended (over 200 delegates last year), with groups from Glasgow, Edinburgh, St. Andrews, Dundee, Leeds, as well as other universities from around the UK. We attract a diverse audience, including local biotechnology companies, professional peers, undergraduates, and post-graduate students.
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017
URL http://www.gla.ac.uk/researchinstitutes/iii/cvr/events/headline_430158_en.html
 
Description Kelvingrove Museum Science Weekend 2014 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact We ran our 'Build a virus' and 'One Infection' activities - offering participants the opportunity to meet CVR scientists, learn about the structure of viruses and also key messages related to the transmission of viruses, and control measures including hygiene and vaccination.

Participants were asked to provide feedback on the activities to help us determine how to improve the activity in future. We have been asked to submit a proposal to run an activity during the Glasgow Science Festival in June 2015.
Year(s) Of Engagement Activity 2014
 
Description PCR workshop 
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 Since 2006, this activity has been delivered to more than 700 Scottish high school students. The teaching resources developed have been adopted by Learning Teaching Scotland to teach higher biology. Workshop has been praised in an HMIE report. Evaluation student feedback is very positive each year.

Our PCR workshop run in partnership with Glasgow Science Centre is a flagship/exemplar activity in high-school education. The PCR workshop has been cited by HMiE as an example of excellence. School pupils from all over Scotland travel to Glasgow to participate.
Year(s) Of Engagement Activity 2013,2014
 
Description Scientific consultant for BBC 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Scientific consultant for a BBC public engagement program explaining the cause of cold sores
Year(s) Of Engagement Activity 2017
 
Description Scout's Science Day - 2014 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact 200 local scouts and guides attended a day of science engagement at the University of Glasgow where they had the opportunity to take part in a range of activities. All attendees took part in our 'Build a Virus' activity and meet with CVR scientists. These interactions gave the attendees the opportunity to ask questions and also learn a little more about viruses, their structure, transmission and control through group discussion with individual researchers. The activity was supported by 8 CVR researchers, providing an excellent opportunity for individual public engagement training and development.

The local Scout and Guide leaders are keen to facilitate another event in 2015.
Year(s) Of Engagement Activity 2014