Dissecting the molecular pathways of MDV oncoprotein Meq for understanding pathogenesis and aid vaccine development

Lead Research Organisation: The Pirbright Institute
Department Name: Avian Oncogenic Viruses

Abstract

Infectious diseases result in direct and indirect losses at various steps of poultry farming and amongst them neoplastic disease caused by viruses is a major economic problem faced by the poultry industry worldwide. The oncogenic viruses causing neoplastic infection in chickens are herpesviruses comprising of Marek's disease virus (MDV) and retroviruses comprising of reticuloendotheliosis virus (REV) and avian leucosis virus (ALV). Marek's disease (MD) is a common disease of chickens involving paralysis and commonly death from the growth of highly malignant T lymphomas (cancers of white blood cells). MD is caused by a transmissible agent MDV. MDV is very contagious and is a major threat to the poultry industry worldwide. The estimated total loss from this disease worldwide is up to $2 billion. Presently, it is controlled by vaccination, and nearly 22 billion vaccine doses a year are used in an attempt to control the disease. Despite widespread vaccination, the threat from this disease is on increase due to continued evolution of MDV towards greater virulence, and more fundamental studies to understand the mechanisms by which this virus causes cancer is needed to develop more effective control programmes. Meq is the major oncoprotein in MDV induced tumorigenesis. Previously, we have identified Meq targetome in cancer cells. In this new grant proposal, to be carried out jointly between the Pirbright Institute and Roslin Institute, we want to extend these studies to obtain detailed information on the role of Meq and its targets in MDV induced oncogenesis using a number of advanced approaches including CRISPR/Cas9 genome editing we have recently established in the lab and rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for Meq interactome identification. The study is very important to understand the mechanisms by which this virus induces cancer, some of which are valuable in understanding cancer in other species including humans. Finally, the findings from the project will be very valuable in developing new approaches for the control of cancers caused by oncogenic viruses.

Technical Summary

The objectives of this project are to dissect the molecular pathways of MDV oncoprotein Meq for understanding pathogenesis and aid vaccine development. Meq is the major oncoprotein in MDV induced tumorigenesis. Yet the underlying molecular mechanisms are not fully understood. Our overall aim is to dissect the molecular events during Meq-induced neoplastic transformation exploiting the recent technological advances such as the CRISPR/Cas9 genome editing and rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME). Specifically, we will investigate the role of Meq and its cancer related targets in maintaining the transformed phenotype of MDV1 cell line by deleting/mutating Meq/Meq-targets using CRISPR/Cas9 genome editing tool in MDV transformed cell line and perform proliferation assay for cell growth and RNA-seq for gene expression and pathways involved. RIME will be carried out to identify Meq interactome. Finally, we will delete Meq from very virulent plus (vv+) MDV1 virus and also to replace vv+ Meq with vaccine strain CVI988 Meq using CRISPR/Cas9 system and ask the question "Are the mutant viruses could be used as vaccine?" The data obtained will allow us to understand the role of Meq in maintaining the transformed phenotype and molecular interactions and pathways involved during MDV-induced oncogenesis.

Planned Impact

Poultry industry is a rapidly growing sector crucial for the global food security, acting as a major source of protein for the growing world population. Marek's disease (MD) is one of the major disease of poultry which causes serious economic losses and the global estimate of losses from MD is approximately $2 billion annually. Detailed understanding of the molecular basis of MDV induced oncogenesis, as the current proposal aims to achieve, will benefit development of new strategies for control.The beneficiaries of this research will include academic scientists, the poultry breeding companies and vaccine production companies, the Pirbright Institute, the BBSRC and its stakeholders such as Defra and the UK farming industry.

The research will have general impact with the wider scientific community, veterinary and medical practitioners, students and general public. Engagement with these diverse groups will be achieved via meetings, articles in the trade press, tailored web pages, press releases to the media and outreach events in schools.

In the longer term the research may lead to medical benefits by improving control of human virus pathogens which will benefit the UK MRC and UK department of Health, the pharmaceutical industry and international organization such as the World Health Organization.

If the proposed studies lead to new approaches for controlling MDV or other viral diseases, additional funding will be sought from relevant funding agencies and other sources for further development. There is extensive experience within the Pirbright Institute of patent applications and commercialisations, new opportunities will feed into an established system for technology development and knowledge transfer by the Pirbright Business Development group.
 
Description In situ CRISPR/Cas9 gene editing tools for functional genomics in ALV-transformed cell lines: ALV is a typical retrovirus which induces the pathogenicity by integration into the host genome. After integration, LTR acts as promoter to activate the expression of the oncogene and induce tumors. The development of lymphoid leukosis is a result of cooperation of c-Myc and c-Bic (encoding the oncogenic miRNA mir-155) integration and activation by ALV. Using the CRISPR/Cas9 editing system, we have shown that both LTR and c-Myc but not miR-155 are essential for the continued proliferation of ALV transformed B cell line HP45.
The role of c-Myc oncogene in regulating the transcriptional machinery of ALV transformed cells: c-Myc is a transcription factor that binds to specific signature sequences in the different promoters in regulating expression. To examine the role of Myc in ALV-induced transformation, we determined the global Myc-binding sites in the genome of ALV cell line HP45 using Myc-ChIP (chromatin immunoprecipitation) analysis after tagging c-Myc with AM tag which has got a good Chippable antibody.
Speci?c high-sensitivity enzymatic reporter unlocking (SHERLOCK) platform for diagnosis of ALV: As vaccines are not available for ALV, the current practice of eradication involves early detection and removal of infected birds to reduce contact and the incidence of horizontal spread. To aid the eradication program, rapid, sensitive, specific, easy-to-use and cost effective on-site diagnostic method is needed. We have developed a lateral flow detection method using Cas13a-based SHERLOCK platform for rapid detection of the most common ALV subgroups A, B, and J.
Exploitation Route Establishment of the pipeline for in situ CRISPR editing of lymphoma-derived cell lines, gives the opportunity for the identification of critical viral and host genes involved in features such as virus-host interactions, neoplastic transformation, and virus latency.
Determination of the global myc-binding profiles in transformed macrophages could provide insights into its role in transformation.
Achieving rapid detection of ALV infection is imperative in effective control of the spread of ALVs.
Sectors Agriculture, Food and Drink

 
Description BBSRC IAA The Pirbright Institute
Amount £300,000 (GBP)
Funding ID BB/S506680/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 03/2021
 
Description CRISPR/Cas system-based molecular diagnostics for avian viral pathogens
Amount £21,263 (GBP)
Organisation The Pirbright Institute 
Sector Academic/University
Country United Kingdom
Start 01/2020 
End 03/2020
 
Description Collaborative partnership for establish of PhysioMimix™ OOC system, part of Pirbright Institute Flexible Talent Mobility Account
Amount £180,000 (GBP)
Funding ID BB/S507945/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2020 
End 12/2020
 
Description Development of CVI-988 based recombinant vaccine
Amount £240,000 (GBP)
Organisation Eco Animal Health Ltd 
Sector Private
Country United Kingdom
Start 08/2019 
End 03/2021
 
Description Development of avian herpesvirus vector vaccines for poultry
Amount £432,000 (GBP)
Organisation MSD Animal Health 
Sector Private
Country United Kingdom
Start 01/2021 
End 12/2023
 
Description Development of improved HVT based vaccines using CRISPR/Cas9 system
Amount £258,820 (GBP)
Organisation Bill and Melinda Gates Foundation 
Sector Charity/Non Profit
Country United States
Start 01/2021 
End 12/2022
 
Description PhD studentship
Amount £91,830 (GBP)
Organisation The Pirbright Institute 
Sector Academic/University
Country United Kingdom
Start 10/2019 
End 03/2023
 
Description Production of HVT vectored VhH construct
Amount £155,000 (GBP)
Organisation Eco Animal Health Ltd 
Sector Private
Country United Kingdom
Start 01/2020 
End 06/2020
 
Description Seeding Catalyst Award
Amount £29,683 (GBP)
Funding ID ISCF-TFPSA-Pirbright 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2018 
End 02/2019
 
Title The establishment of an efficient pipeline for in situ CRISPR editing of the Marek's disease virus (MDV) genome in lymphoma-derived cell lines 
Description The lymphoblastoid cell lines (LCLs) derived from MD lymphomas have served as valuable resources to study virus-host molecular interactions in transformed cells. However, detailed investigations into the functional role of different viral and host determinants in these cells have been difficult due to the lack of tools for in situ manipulation of viral/host genomes in MDV-transformed cell lines. Our recent success in efficient CRISPR/Cas9 editing of the MDV genome in LCLs has demonstrated the potential for targeted editing to dissect the regulatory pathways involved in latency, transformation, reactivation and lytic switch. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact Using this approach, we have demonstrated viral genes such as pp38 and MDV-miR-M4 are not essential for maintaining the transformed phenotype. On the other hand, deletion of MDV-encoded oncogene Meq or mutations preventing its interactions resulted in cell death demonstrating the essential role of Meq in transformation. 
URL https://www.mdpi.com/1999-4915/10/6/279
 
Description Finn Frey 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Worked together to identify the host factors involved in MDV latency
Collaborator Contribution Provided chicken gRNA library for screening of MDV cell line
Impact Not yet
Start Year 2018
 
Description Mick Watson 
Organisation University of Edinburgh
Department The Roslin Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Provide samples for host-virus interaction study of MDV
Collaborator Contribution Data analysis of RNA-seq on CRISPR/Cas9 edited MDV cell lines.
Impact One publication from this collaboration: Zhang Y, Tang N, Luo J, Teng M, Moffat K, Shen Z, Watson M, Nair V#, Yao Y#. Marek's disease virus-encoded miR-155 ortholog critical for the induction of lymphomas is not essential for the proliferation of transformed cell lines. J Virol. 2019 Jun 12. pii: JVI.00713-19. doi: 10.1128/JVI.00713-19.
Start Year 2018
 
Description Professor Guozhong Zhuang 
Organisation Henan Agricultural University
Country China 
Sector Academic/University 
PI Contribution Provided knowledge and expertise on MDV research
Collaborator Contribution Provided samples for MDV research
Impact One publication has been generated: Sun A, Yang S, Luo J,Teng M, Xu Y, Wang R, Zhu X, Zheng L, Wu Y, Yao Y, Nair V, Zhang G, Zhuang G. UL28 and UL33 homologs of Marek's disease virus terminase complex involved in the regulation of cleavage and packaging of viral DNA are indispensable for replication in cultured cells. Veterinary Research. 2021, 52:20. 10.1186/s13567-021-00901-5
Start Year 2019
 
Description Professor Luo 
Organisation Henan Academy of Agricultural Sciences
Country China 
Sector Academic/University 
PI Contribution Provided knowledge, reagent and information on MDV research
Collaborator Contribution Provided samples and reagent for MDV research
Impact 6 publications have been generated: 1. Sun A, Yang S, Luo J,Teng M, Xu Y, Wang R, Zhu X, Zheng L, Wu Y, Yao Y, Nair V, Zhang G, Zhuang G. UL28 and UL33 homologs of Marek's disease virus terminase complex involved in the regulation of cleavage and packaging of viral DNA are indispensable for replication in cultured cells. Veterinary Research. 2021, 52:20. 10.1186/s13567-021-00901-5 2. Zhu Z, Teng M, Li H, Zheng L, Liu J, Yao Y, Nair V, Zhang G, Luo J. Virus-encoded miR-155 ortholog in Marek's disease virus promotes cell proliferation via suppressing apoptosis by targeting tumor suppressor WWOX. Veterinary Microbiology. 7 November 2020, https://doi.org/10.1016/j.vetmic.2020.108919 3. Zhu Z, Teng M, Li H, Zheng L, Liu J, Chai S, Yao Y, Nair V, Zhang G, Luo J. Marek's disease virus (Gallid alphaherpesvirus 2, GaHV-2)-encoded miR-M2-5p simultaneously promotes cell proliferation and suppresses apoptosis through RBM24 and MYOD1-mediated signaling pathways. Frontiers in Microbiology. 03 November 2020 | https://doi.org/10.3389/fmicb.2020.596422 4. Luo J, Teng M, Zai X, Tang N, Zhang Y, Mandviwala A, Reddy VRAP, Baigent S, Yao Y, Nair V. Efficient Mutagenesis of Marek's Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System. Viruses. 2020 Apr 20;12(4): E466. doi: 10.3390/v12040466. 5. Zhang Y, Tang N, Luo J, Teng M, Moffat K, Shen Z, Watson M, Nair V#, Yao Y#. Marek's disease virus-encoded miR-155 ortholog critical for the induction of lymphomas is not essential for the proliferation of transformed cell lines. J Virol. 2019 Jun 12. pii: JVI.00713-19. doi: 10.1128/JVI.00713-19. 6. Zhang Y, Luo J, Tang N, Teng M, Reddy VRAP, Moffat K, Shen Z, Nair V#, Yao Y#. Targeted Editing of the pp38 Gene in Marek's Disease Virus-Transformed Cell Lines Using CRISPR/Cas9 System. Viruses. 2019 Apr 26;11(5). pii: E391. doi: 10.3390/v11050391.
Start Year 2018
 
Description AN INTERVIEW WITH PROFESSOR VENUGOPAL NAIR 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Professor Venugopal Nair is a Research Group Leader at The Pirbright Institute, a visiting Professor of Avian Virology at the Department of Zoology, and a Jenner Investigator at the Jenner Institute, University of Oxford. He is also a member of the Microbiology Society, and in this interview, he tells us more about his research into viral diseases of livestock.
Year(s) Of Engagement Activity 2020
URL https://microbiologysociety.org/membership/meet-our-members/focus-area-viruses/an-interview-with-pro...
 
Description Interview by CGTN 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Increased profile of both Institute and The UK-China Centre of Excellence for Research on Avian Diseases (CERAD)
Year(s) Of Engagement Activity 2019
URL https://newseu.cgtn.com/news/2020-01-28/British-and-Chinese-scientists-join-forces-to-fight-avian-di...
 
Description School visit (Tilingborne) 
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 Stimulating increased interest in science and research
Year(s) Of Engagement Activity 2019
 
Description School visit (Woking) 
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 Stimulating increased interest in science and research
Year(s) Of Engagement Activity 2019
 
Description Science Festival (Cheltenham) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Stimulating increased interest in science and research
Year(s) Of Engagement Activity 2019