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

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.

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.

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.