The role of ubiquitin and ubiquitin-like proteins during viral infection and host-cell immunity

Lead Research Organisation: MRC Virology Unit

Abstract

During infection viruses must evade multiple host defence mechanisms in order to establish a productive infection. These include antibody and cell mediated acquired immunity, interferon regulated innate immunity, and intrinsic cellular immunity. In order to counteract these host defences many viruses have evolved strategies to engage with cellular ubiquitin and ubiquitin-like modification pathways to modulate their intracellular environment in favour of viral replication. This programme of research aims to define how specific viruses engage with components of these pathways in order to design and develop alternative therapeutic intervention strategies.
Our research has identified unique aspects relating to cellular immune defence and associated viral counter measures. Our current work aims to determine whether these pathways affect other clinically important human viruses, and to establish a platform of biochemical techniques that will facilitate the screening of compounds to inhibit viral counter measures of these pathways.

Technical Summary

The post-translational modification of proteins by ubiquitin and ubiquitin-related polypeptides regulates many essential cellular processes including protein degradation, transcription, DNA repair and host-cell anti-viral immunity. Consequently, many human viruses have evolved strategies to utilise or suppress these modification pathways in order to enhance their replication, thereby maintaining their presence within the human population. Utilising a variety of techniques; library screening, lentiviral expression, RNAi technologies, in vitro biochemistry and mass spectrometry, the aim of this programme is to identify and biochemically define how human viruses engage with components of these pathways during infection with respect to disarming host-cell anti-viral immunity. This work will provide a platform of biochemistry in order to develop novel strategies for future therapeutic intervention. The lab focuses on three clinically important human viruses: Herpes Simplex virus type-1 (HSV-1), Influenza A virus, and Hepatitis C Virus (HCV).

HSV-1 causes a variety of clinically important diseases, including herpes keratitis and encephalitis, and provides an ideal model system for studying multiple aspects of intracellular immunity in response to DNA virus infection. An important regulator of HSV-1 infection is ICP0, a viral ubiquitin ligase that targets a subset of cellular proteins implicated in anti-viral immune defecne for ubiquitination and proteasome-dependent degradation. Our research aims to identify and characterise the cellular regulators associated with nuclear immunity in response to DNA virus infection, both at a molecular and biochemical level, by virtue of identifying ICP0’s cellular substrates and biochemical specificity.

Recent progress in our understanding of the molecular mechanisms associated with the regulation of intrinsic immunity in response to HSV-1 infection are also being investigated in during the nuclear replication of RNA viruses, specifically Influenza A virus, in order to determine the specificity of these host defence mechanisms. Influenza virus infection is the leading causative agent associated with acute viral respiratory illnesses and causes significant annual morbidity and mortality within humans.

HCV infection is a leading cause of non-alcohol related liver disease within the UK, including decompensated cirrhosis and hepatocellular carcinoma. HCV induces the re-distribution of intracellular ubiquitin conjugates during infection, a phenotype that is dependent upon viral RNA replication. However, little is known about how HCV engages with components of the ubiquitin-pathway or the functional importance of these interactions with respect to viral replication. This research aims to identify and characterise the role of ubiquitin and ubiquitin-like modifications during HCV infection and their relationship to anti-viral defence.

Publications

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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

 
Title in vitro SUMO biochemistry update 
Description The cloning, expression, and purification of various proteins implicated in the regulation of host SUMOylation. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2016 
Provided To Others? Yes  
Impact Conn KL, Wasson P, McFarlane S, Tong L, Brown JR, Grant KG, et al. Novel Role for Protein Inhibitor of Activated STAT 4 (PIAS4) in the Restriction of Herpes Simplex Virus 1 by the Cellular Intrinsic Antiviral Immune Response. Journal of virology. 2016;90(9):4807-26. doi: Brown JR, Conn KL, Wasson P, Charman M, Tong L, Grant K, et al. SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1. Journal of virology. 2016;90(13):5939-52. doi: 10.1128/JVI.00426-16. PubMed PMID: 27099310; PubMed Central PMCID: PMCPMC4907222. 
 
Title in vitro ubiquitin biochemsitry updated 
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 2016 
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 updated 2016 
Description Primary cell lines that have been transduced with lentiviral vectors expressing short hairpin RNAs (shRNAs) to deplete components of the ubiquitin and SUMO conjugation pathways. 
Type Of Material Cell line 
Year Produced 2016 
Provided To Others? Yes  
Impact Research ongoing. Conn KL, Wasson P, McFarlane S, Tong L, Brown JR, Grant KG, et al. Novel Role for Protein Inhibitor of Activated STAT 4 (PIAS4) in the Restriction of Herpes Simplex Virus 1 by the Cellular Intrinsic Antiviral Immune Response. Journal of virology. 2016;90(9):4807-26. doi: Brown JR, Conn KL, Wasson P, Charman M, Tong L, Grant K, et al. SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1. Journal of virology. 2016;90(13):5939-52. doi: 10.1128/JVI.00426-16. PubMed PMID: 27099310; PubMed Central PMCID: PMCPMC4907222. 
 
Description Cellular restriction of Influenza virus 
Organisation University of Glasgow
Country United Kingdom 
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 
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 Intrinsic immunity to Influenza A Virus 
Organisation University of Glasgow
Department MRC - University of Glasgow Centre for Virus Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision or reagents and research support to establish a role for intrinsic immunity against IAV.
Collaborator Contribution Provision or reagents and research support to establish a role for intrinsic immunity against IAV.
Impact We have identfied the TRIM protein 22 to play a role in the cellular restriction of IAV prior to the activation of innate immune signalling, thereby confirming a role for intrinsic immunity in the intracellular restriction of IAV. A manuscript is currently in preparation.
Start Year 2015
 
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 
Sector Academic/University 
PI Contribution biochemical analysis
Collaborator Contribution Cell biology analysis
Impact Cell biology Ubiquitin SUMOylation Biochemistry
Start Year 2012
 
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 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