The impact of viral gene expression and cellular signalling upon the control of HCMV latency and reactivation

Lead Research Organisation: University College London
Department Name: Virology

Abstract

Human Cytomegalovirus is a herpes virus that co-exists within the human host for life. Usually benign in healthy adults, in certain patients whose body defence mechanism (immune system) against pathogens is switched off (i.e. transplant, cancer and AIDS patients) the virus spreads uncontrollably throughout the body. This results in severe disease and, if not brought back under control, can lead to death.

The ultimate aim is to understand how the virus remains dormant in humans and what host signals tell the virus to awaken (‘reactivate‘) from its‘ dormant state. By understanding what the signals are which reactivate the virus we can then design therapies to intercept that signal and prevent it from telling the virus to reactivate until the patients‘ immune system has recovered sufficiently to regain control over the virus. Furthermore, if we can understand how the virus hijacks the cells it resides in during its‘ dormant phase we can hopefully, by determining what the virus needs to survive, design novel therapies that could allow us to understand what the virus needs for long term survival in the host. Such an approach may kill the virus and result in the eradication of this pathogen from humans.

Technical Summary

Although primary infection with human Cytomegalovirus (HCMV) is usually asymptomatic in healthy individuals, infection and reactivation of immuno-suppressed AIDS, cancer and transplant patients, causes severe morbidity and mortality. Additionally, infection in utero occurs in approximately 1-2% of all live births and is associated with severe long-term consequences including blindness, deafness and mental retardation.

Previous studies on HCMV latency and reactivation suggest that the myeloid lineage is important. HCMV establishes latency in bone-marrow progenitor cells and reactivation is linked with the differentiation state of myeloid cells whereby myeloid cell maturation induces chromatin remodelling of the major lytic (‘reactivation‘) promoter into an active form concomitant with reactivation. However, the apparent contradiction between efficiency of HCMV reactivation in vivo compared with studies in vitro suggests that further, yet undefined, factors augment reactivation in vivo.

Firstly, I propose to study the impact of chromatin remodelling of the MIEP during differentiation in the context of inflammatory signalling induced by myeloid cell maturation. Specifically, I intend to identify and characterise signalling pathways involved in reactivation. Broad spectrum chemical inhibitors will be used to identify potential pathways involved and then more detailed analyses using adenoviral vectors to introduce mutant forms of signalling proteins, siRNA technology and more specific inhibitors of points in the pathway will be carried out. These analyses will be performed in an in vitro model that has been used in previously to analyse the molecular mechanisms of HCMV reactivation.

Secondly, I propose the further study of a recently identified transcript, UL81-82ast (LUNA), expressed during both lytic and latent infection. Preliminary data suggests that LUNA can disrupt ND10 bodies which maybe due to a potential de-sumoylase activity of the protein. To as the role of the de-sumoylase function a mutant virus within the putative active site will be characterised for its ability to establish latency and reactivate using the in vitro system. Furthermore, expression of a SUMO protease would allow HCMV to target a number of diverse processes, thus I will perform a yeast 2 hybrid assay and then characterise positive interactions to give further insight into the precise role of this protein during latency, reactivation and lytic infection.

It is hoped that these studies will increase our understanding of how HCMV manipulates the host cell environment to so successfully establish both lytic and latent infections, and ultimately lead to the identification of key signalling pathways that promote reactivation providing a target for therapeutic intervention.

Publications

10 25 50
publication icon
Alsaady I (2019) Downregulation of the Central Noradrenergic System by Infection. in Infection and immunity

publication icon
Baraniak I (2018) Protection from cytomegalovirus viremia following glycoprotein B vaccination is not dependent on neutralizing antibodies. in Proceedings of the National Academy of Sciences of the United States of America

publication icon
Griffiths P (2015) The pathogenesis of human cytomegalovirus. in The Journal of pathology

 
Description BRC rapid response
Amount £195,000 (GBP)
Organisation National Institute for Health Research 
Department NIHR Biomedical Research Centre
Sector Public
Country United Kingdom
Start 10/2016 
End 09/2019
 
Description Bench to Bedside Studentship
Amount £99,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 09/2018
 
Description CTRF
Amount £185,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2016
 
Description FEMS Travel Award
Amount £600 (GBP)
Organisation Federation of European Microbiological Societies (FEMS) 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2011 
End 04/2011
 
Description Leonardo Da Vinci Scholarship
Amount £2,000 (GBP)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 04/2012 
End 06/2012
 
Description MRC Confidence in Concepts
Amount £840,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2017 
End 09/2018
 
Description MRC Project Grant
Amount £393,350 (GBP)
Funding ID MR/R021384/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 05/2018 
End 04/2021
 
Description Peter Samuel Fund
Amount £9,375 (GBP)
Organisation Royal Free Hospital 
Sector Hospitals
Country United Kingdom
Start 01/2014 
End 12/2014
 
Description Royal Free Bursary
Amount £4,750 (GBP)
Organisation Royal Free Hospital 
Sector Hospitals
Country United Kingdom
Start 06/2014 
End 09/2014
 
Description Wellcome Collaborator Award
Amount £2,567,000 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Bloomsbury Centre
Sector Charity/Non Profit
Country United Kingdom
Start 07/2017 
End 06/2022
 
Description Wellcome Trust Studentship
Amount £2,000 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Bloomsbury Centre
Sector Charity/Non Profit
Country United Kingdom
Start 06/2014 
End 09/2014
 
Description Anti-viral activity of an MSK inhibitor 
Organisation St George's University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided research expertise in chromatin biology and transcription to help define the MoA of this compound
Collaborator Contribution They approached us to assist them with their study as they did not have the technical expertise in the area - they performed the initial characterisation of anti-viral activity
Impact A paper was published in 2017 PMID:28100301
Start Year 2016
 
Description Cyclophilins and HCMV 
Organisation University College London
Department School of Life and Medical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We are pursuing the role of cyclophilins in HCMV biology and how we can modulate their function to expose the virus to the activity of anti-viral therapeutics. This is part of a broader aim to develop novel strategies for targeting viruses by enhancing the immune response against them.
Collaborator Contribution This work was led by Prof Towers and his studies on HIV. By studying HCMV in parallel we aim to identify commonalities between different virus families to direct future strategies for pan anti-viral treatments
Impact This collaboration is part of a larger collaborative project that simultaneously seeks to define the molecular biology of cyclophilins in multiple viruses whilst generating new compounds as therapeutics and is collaboration between virology and chemistry. These studies have been submitted for Wellcome Trust SDDI grant/ MRC DPFS awards We have secured BRC funding to explore this work for 3 years (£199,500)
Start Year 2014
 
Description Immune Protection against BK virus 
Organisation University College London
Department Division of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We are hosting and training a clinical lecturer to perform some pilot studies to address the long term aim of studying BK pathogenesis in kidney transplantation
Collaborator Contribution Our partners are clinicians who are nephrologists and thus provide samples and the clinical interface
Impact The clinical Lecturer has submitted Clinical PhD training grants. If successful she will be hosted in my lab Said clinician has secured a PhD Studentship
Start Year 2017
 
Description Novel HCMV anti-virals 
Organisation University College London
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution We will characterise a series of anti-virals for their activity against HCMV
Collaborator Contribution Our colleagues have provided the chemistry
Impact A paper is in preparation and the collaboration is supported with an MRC Confidence in Concepts Award to develop them further
Start Year 2016
 
Description Role of Creb response Elements in HCMV reactivation 
Organisation University of Iowa
Department Department of Internal Medicine
Country United States 
Sector Academic/University 
PI Contribution Testing the recombinant viruses in our experimental latency and reactivation systems. Using the viruses to help our ongoing work delineating the pathway of HCMV reactivation
Collaborator Contribution They provided the recombinant viruses
Impact Oral presentation of work at scientific meetings Work published in PLoS Pathogens (2014) - see publications list Follow up work supported by MRC Grant
Start Year 2010
 
Description Role of LUNA in HCMV latency and reactivation 
Organisation University of Cambridge
Department School of Clinical Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We are providing the biochemical characterisation of the LUNA protein and studies in HCMV reactivation
Collaborator Contribution They are generating the recombinant viruses and characterisation of the protein function during latency
Impact Oral presentations at scientific meetings Manuscript being prepared Led to an MRC PhD studentship to pursue this work further
Start Year 2009
 
Description Role of ion channels in herpes virus reactivation 
Organisation University of Leeds
Department School of Molecular & Cellular Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution We will study the role of ion channels in herpes virus reactivation with our expertise in HCMV being our contributions
Collaborator Contribution Our collaborator has the electrophysiology experience to study the role of channels in reactivation. They are focussed on KSHV.
Impact Potential ion channel targets in HCMV reactivation have been identified for further study We have contributed to studies of KSHV reactivation that is being prepared for submission
Start Year 2013
 
Description Role of ion channels in lytic viral replication 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution We are studying the role of chloride ion channels in HCMV infection - we have identified a novel target that could be a potential therapeutic target to control CMV infection in vivo
Collaborator Contribution Academic input on the initial triage of ion channel inhibitors - Electrophysiology expertise
Impact We have initiated a second collaboration with the Translational Research Office at UCL to synthesise next generation analogues of the tool compound to determine if we can identify active component and b) improve activity of the compound This work is now supported by an MRC PhD studentship
Start Year 2013
 
Description Role of monocyte subsets in HCMV biology 
Organisation University College London
Department School of Life and Medical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We are studying the carriage of HCMV in monocytic cell sub-populations using our expertise in the study of HCMV latency in naturally latent individuals
Collaborator Contribution The collaborator is an expert in monocyte biology and defines and isolates the cell populations for our studies
Impact We have established preliminary data that is being prepared for a joint Wellcome Investigator grant application This project is on hold
Start Year 2014
 
Description Structural insight into the biology of HCMV encoded protein 
Organisation University College London
Department Division of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide the biological knowledge on the protein. We identified the protein function and the key motifs responsible for it's activity
Collaborator Contribution The partners are cloning and purifying the protein for structural studies to determine a) the structure of the protein b) the structure of the functional mutant we have identified and c) the structural relationship of the protein with functional homologues encoded by the cell
Impact No direct outputs yet
Start Year 2015
 
Description T Gondii and CMV 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution We performed analyses with CMV to address whether changes seen with T. Gondii were specific or triggered by any pathogen
Collaborator Contribution They led the study and published the research
Impact Paper published: PMID: 30510101
Start Year 2017
 
Description The natural history of HCMV and impact of latency 
Organisation International Agency for Research on Cancer (IARC)
Department Virology Department
Country France 
Sector Academic/University 
PI Contribution We are applying our knowledge of HCMV latency and reactivation to ongoing studies of the immune response to HCMV in transplant patients
Collaborator Contribution The partners provide the clinical lead on this wih emphasis on the characterising the immune response to HCMV in transplant patients.
Impact This work is just beginning but a manuscript detailing the natural history of HCMV and the correlation with latency is being prepared we have been awarded a 5 year Wellcome Collaborator Award in which HCMV latency is an integral part (£2.67m)
Start Year 2013
 
Description Understanding the function of UL138 gene expression during latency 
Organisation Charité - University of Medicine Berlin
Department Paediatric and Adolescent Medicine
Country Germany 
Sector Hospitals 
PI Contribution Using recombinant viruses to assess the regulation of TNFRI expression during latency in our experimental systems
Collaborator Contribution Provision of recombinant viruses and intellectual input
Impact No published outputs
Start Year 2011
 
Description Viral modulation of neutrophil viability 
Organisation University of Cambridge
Department Department of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided our expertise in the understanding of HCMV biology to use the virus as a tool to probe the cellular functions that are intrinsic for the viability and function of neutrophils.
Collaborator Contribution Expertise with neutrophil isolation and assaying of function
Impact Abstract submitted for Oral presentation at International respiratory meeting Manuscript submitted to Blood The research has led to support for two PhD positions supported by the Gates Foundation and a CTRF from the MRC to pursue aspects of this study further
Start Year 2011
 
Description Viral regulaiton of 41BBL expression for immune evasion 
Organisation University of Cambridge
Department School of Clinical Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual input in to PhD project and providing supervision and molecular biology training
Collaborator Contribution Primary supervisor and immunology expertise
Impact Oral presentations at scientific meetings Manuscript in preparation PhD thesis being prepared
Start Year 2012
 
Title Nanobiosensor 
Description A nanobiosensor to detect hcmv in baby saliva to detect viraemia and report congenital infection. This will allow universal screening which will could have a big impact on NHS healthcare. Supported by an NIHR grant to develop. 
Type Diagnostic Tool - Non-Imaging
Current Stage Of Development Refinement. Non-clinical
Year Development Stage Completed 2018
Development Status Under active development/distribution
Impact It has become clear we may be able to multiplex the tech for multiple viruses. Implications for routine screening in transplants for example. 
 
Description Hosting School visiting UCL 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Local schools are hosted at UCL (organised by Richard Milne & Greg Towers) where they come to meet Infection & Immunity Researchers. They learn about infective agents - have hands on practicals demonstrating the spread of disease by sneezing and failure to hand wash (talc and hair gel, respectively) and then after a talk about what makes a pathogen a pathogen they work with members of the research community to design their own pathogens and describe them to the rest of the group. This gives them the chance to interact with different members of research teams (my lab was represented by undergraduates, post graduates and myself).

This activity occurs frequently in the UCL cycle and is part of a greater effort for UCL researchers to inspire the next generation of researchers
Year(s) Of Engagement Activity 2015
 
Description Institute Immunity & Transplantation open day 
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 The open days are multiple and are designed to give local people the opportunity to find out what research is being performed. A similar workshop is held for A-Level students to again inspire the next generation of researchers and tell them about the current scientific research being performed at the Institute.
Year(s) Of Engagement Activity 2014,2015
 
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 PhD student went to a school to talk to GCSE students about science and his research. Involved Q&A. Teacher hosting reported increased interest in pursuing STEM subjects at A levels amongst students.
Year(s) Of Engagement Activity 2017