Identification of essential host proteins for the control of nidovirus replication
Lead Research Organisation:
THE PIRBRIGHT INSTITUTE
Department Name: UNLISTED
Abstract
Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.
Technical Summary
The aim of my research is to “identify essential host proteins for the control of nidovirus replication” using avian coronavirus, infectious bronchitis virus (IBV), as a model, and making comparisons initially to porcine and human coronaviruses and more distantly related nidovirus, porcine reproductive and respiratory syndrome virus. More diverse positive sense RNA (+RNA) viruses will be incorporated in the future through collaboration with other groups at TPI. By adopting a comparative virology and “One Health” approach to understand key conserved steps in virus replication, development of pan-virus control measures may be possible. The virus lifecycle can be simply divided into three stages – entry, replication and exit. Although entry varies between nido- and coronaviruses, virus induction of cellular membrane rearrangements during replication and virus budding to gain a lipid envelope during exit are conserved steps. Using cell biology, ‘omics and new bioimaging technologies, fundamental understanding of virus replication and interactions between the virus and host with a particular emphasis on host factors will be achieved. Subsequently, potential to control virus replication through altering virus-host interactions will be investigated. The programme consists of three work areas.
Planned Impact
unavailable
Organisations
- THE PIRBRIGHT INSTITUTE (Lead Research Organisation)
- University of Edinburgh (Collaboration)
- Leiden University Medical Center (Collaboration)
- UNIVERSITY OF BRISTOL (Collaboration)
- University of Surrey (Collaboration)
- Diamond Light Source (Collaboration)
- UNIVERSITY OF READING (Collaboration)
- Texas A&M University–Texarkana (Collaboration)
Publications
Batra A
(2017)
Selection of reference genes for gene expression analysis by real-time qPCR in avian cells infected with infectious bronchitis virus.
in Avian pathology : journal of the W.V.P.A
Bickerton E
(2018)
The S2 Subunit of Infectious Bronchitis Virus Beaudette Is a Determinant of Cellular Tropism.
in Journal of virology
Brownsword M
(2019)
Infectious bronchitis virus regulates cellular stress granule signaling
Brownsword MJ
(2020)
Infectious Bronchitis Virus Regulates Cellular Stress Granule Signaling.
in Viruses
Doyle N
(2018)
Infectious Bronchitis Virus Nonstructural Protein 4 Alone Induces Membrane Pairing.
in Viruses
Doyle N
(2021)
Coronavirus RNA Synthesis Takes Place within Membrane-Bound Sites.
in Viruses
Kint J
(2016)
Infectious Bronchitis Coronavirus Limits Interferon Production by Inducing a Host Shutoff That Requires Accessory Protein 5b.
in Journal of virology
Maier HJ
(2016)
Extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity.
in Scientific reports
Staines K
(2016)
A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR.
in PloS one
| Title | Canvas print of IBV infected TOC |
| Description | A printed canvas of a confocal microscopy image showing an ex vivo tracheal organ culture infected with IBV and labelled with anti-dsRNA, anti-tubulin and DAPI. |
| Type Of Art | Artwork |
| Year Produced | 2020 |
| Impact | The canvas was awarded as a prize following entry into a image competition. |
| Description | The aim of this project was to identify interactions between viral and cellular proteins that coronaviruses require to allow successful replication. During this project we initiated a new area of research bringing together data generated during several other pieces of work to study the regulation of cellular stress granule signalling by infectious bronchitis virus (IBV). We found that IBV replication induces the formation of stress granules in around 25% of infected cells. The induction of stress granules requires virus replication as infection with inactivated virus particles did not result in stress granule formation. In addition, these stress granules contained multiple stress granule markers, indicating that they are likely to be typical cellular stress granules. Interestingly, however, IBV replication was able to effectively inhibit stress granule signalling when induced with potent chemical stressor sodium arsenite, indicating that IBV must employ mechanisms to regulate this important cellular pathway. This work provided preliminary data for a grant application and has also formed the basis for a new PhD project. Finally, through this work, a new collaboration has been established with Nicolas Locker, University of Surrey. In addition, we began to investigate the role of lipids in IBV replication organelle formation. Like all positive strand RNA viruses, IBV induces the rearrangement of cellular membranes during replication to provide a platform for the assembly of the viral RNA synthesis machinery. Using a variety of stains and other detection methods, we visualised the cellular location and abundance of several lipid species in IBV infected cells. This preliminary data indicated that IBV does not appear to utilise cholesterol transport pathways used by other positive strand RNA viruses and instead other pathways and lipid species may be important for coronavirus replication. This work is forming the basis for future planned experiments to continue to develop this research area. |
| Exploitation Route | Output from this work has already formed the basis for a further funding application. In addition, we are establishing new areas of research allowing expansion of the research programme and new questions to be addressed. Ultimately we hope our findings will be of interest to agriculture and pharmaceutical companies for development of novel vaccines for protection against IBV or other coronaviruses and development of cross-protective anti-virals. |
| Sectors | Agriculture Food and Drink Pharmaceuticals and Medical Biotechnology |
| Description | Data was used to form the basis of an application for further funding. |
| Sector | Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Societal |
| Description | Characterising the role of cellular proteins in coronavirus replication |
| Amount | £5,000 (GBP) |
| Organisation | The Pirbright Institute |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 07/2022 |
| End | 08/2023 |
| Description | Common host proteins required for replication organelle function across coronaviruses |
| Amount | £371,708 (GBP) |
| Funding ID | BB/T014571/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 03/2021 |
| End | 02/2024 |
| Description | Coronavirus regulation of cellular antiviral responses and cross species transmission |
| Amount | £52,000 (GBP) |
| Organisation | The Pirbright Institute |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 09/2022 |
| End | 09/2026 |
| Description | Identifying pro-viral cellular proteins in chicken cells that can be exploited to better control avian endemic viruses |
| Amount | £104,000 (GBP) |
| Organisation | The Pirbright Institute |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 01/2021 |
| End | 09/2024 |
| Description | Understanding coronavirus replication using soft x-ray and electron tomography |
| Amount | £62,600 (GBP) |
| Organisation | The Pirbright Institute |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 09/2020 |
| End | 09/2024 |
| Description | Andrew Davidson - Proteome of viral replication organelles |
| Organisation | University of Bristol |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Projects are being initiated to study the cellular proteins associated with coronavirus replication organelle membranes and a funding application has been submitted to BBSRC. |
| Collaborator Contribution | Andrew has provided assistance with experimental planning and drafting of a grant application. He has also provided protocols to allow preliminary data to be generated. Once protocols have been established, Andrew will facilitate mass spectrometry data generation and will analyse the resulting data. |
| Impact | No outcomes to date, projects being initiated and funding applications in progress. |
| Start Year | 2018 |
| Description | Ben Neuman - Study of IBV induced membrane rearrangements |
| Organisation | Texas A&M University–Texarkana |
| Country | United States |
| Sector | Academic/University |
| PI Contribution | My team provide samples for electron microscopy analysis. In addition, we provide expertise in molecular virology and cell biology and experience of membrane rearrangements induced by infectious bronchitis virus. I contribute intellectual input in data analysis and project development. |
| Collaborator Contribution | Ben Neuman and his team perform electron microscopy sample processing and imaging as well as experience in data processing. Ben Neuman also contributes intellectual input in data analysis and project development. |
| Impact | Maier et al. Extensive coronavirus induced membrane rearrangements are not a determinant of pathogenicity. Sci. Rep. 6: 27126. |
| Start Year | 2014 |
| Description | Ben Neuman - Study of IBV induced membrane rearrangements |
| Organisation | University of Reading |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | My team provide samples for electron microscopy analysis. In addition, we provide expertise in molecular virology and cell biology and experience of membrane rearrangements induced by infectious bronchitis virus. I contribute intellectual input in data analysis and project development. |
| Collaborator Contribution | Ben Neuman and his team perform electron microscopy sample processing and imaging as well as experience in data processing. Ben Neuman also contributes intellectual input in data analysis and project development. |
| Impact | Maier et al. Extensive coronavirus induced membrane rearrangements are not a determinant of pathogenicity. Sci. Rep. 6: 27126. |
| Start Year | 2014 |
| Description | Dan Clare - cryo-electron microscopy |
| Organisation | Diamond Light Source |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | The project aims to characterise proteins complexes associated with coronavirus replication organelles. My team provides significant expertise in replication organelles induced by coronaviruses. Laboratory work is performed by my team. The project is funded by a joint PhD studentship working, predominantly in my laboratory. |
| Collaborator Contribution | Dan provides expertise in cryo-electron microscopy and is advising on experimental design and data analysis as well as providing training. |
| Impact | The student has generated recombinant viruses expressing fluorescently-tagged proteins to allow direct visualisation of replication organelle membranes. These have been validated and cryo-electron microscopy experiments are planned shortly. |
| Start Year | 2020 |
| Description | David Matthews - Proteomics informed by transcriptomics |
| Organisation | University of Bristol |
| Department | Proteomics Facility |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Projects are being initiated to study the cellular proteins associated with coronavirus replication organelle membranes and a funding application has been submitted to BBSRC. |
| Collaborator Contribution | David has assisted in project design and drafting of a funding application to BBSRC. We are in the process of generating transcriptomics data, which David will process to provide an expressed protein database for peptide identification following mass spectrometry. |
| Impact | No outcomes to date, a funding application has been submitted to BBSRC. |
| Start Year | 2018 |
| Description | Finn Grey - CRISPR and ISG screens |
| Organisation | University of Edinburgh |
| Department | The Roslin Institute |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | My research team is providing expertise in coronavirus biology and regulation of the host cell environment. Laboratory experiments are being performed by my team. |
| Collaborator Contribution | Finn's team have provided reagents and are advising on experimental design and data analysis. |
| Impact | Cas-9 expressing avian cells have been generated and validated. Optimisation of the CRISPR screen is underway. |
| Start Year | 2021 |
| Description | Maria Harkiolaki - soft xray tomography |
| Organisation | Diamond Light Source |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | The project aims to characterise coronavirus replication organelles in whole infected cells. My team provides significant experience in coronavirus replication organelles. The project is funded by a joint studentship and the student is, predominantly based in my laboratory. |
| Collaborator Contribution | Maria's team provide extensive expertise in soft Xray tomography and are advising on experimental design and dat aanalysis. |
| Impact | Preliminary experiments have been performed to visualise replication organelle membranes. Optimisation of experimental set up is underway. |
| Start Year | 2020 |
| Description | Montse Barcena - Studying the role of coronavirus membrane rearrangements |
| Organisation | Leiden University Medical Center |
| Country | Netherlands |
| Sector | Academic/University |
| PI Contribution | My team has provided reagents and biological samples for the study of the role of IBV induced membrane rearrangements. In addition we have provided expertise and knowledge for the set up of protocols and assisted in data analysis. |
| Collaborator Contribution | M Barcena has provided extensive experience in electron microscopy including use of specialised protocols. She has provided expertise in data analysis and protocol development. |
| Impact | Several presentation of data have been made at international conferences. |
| Start Year | 2014 |
| Description | Nicolas Locker - Investigation of translation regulation by infectious bronchitis virus |
| Organisation | University of Surrey |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | My team provide experience in molecular virology and cellular interactions of coronaviruses including previous experience of accessory proteins regulating cellular translation. One PhD student has successfully completed his studies working jointly between our labs and a second is due to begin in October 2022. |
| Collaborator Contribution | Dr Locker's team provide expertise in viral regulation of cellular translation and stress granule signalling. Dr Locker is also providing protocols, advice and training for techniques including ribopuromycylation and ribosome profiling. |
| Impact | PhD student funding (studentship Oct 2017- Sept 2021) - Control of host cell translation by infectious bronchitis virus PhD student funding (studentship Oct 2022- Mar 2026) - Coronavirus regulation of cellular antiviral responses and cross species transmission |
| Start Year | 2016 |
| Description | Paul Verkade - Electron tomography and CLEM |
| Organisation | University of Bristol |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Nicole Doyle has performed electron tomography to analyse the three dimensional structure of membrane rearrangements induced by the expression of IBV non-structural proteins 4 or 3 and 4 in the absence of other viral components. Sample set up was performed at Pirbright and analysis was subsequently performed at University of Bristol followed by additional analysis at Pirbright. |
| Collaborator Contribution | Paul has assisted with electron tomography data generation providing access to electron microscopes in Bristol as well as training for Nicole Doyle in performing experiments and analysing and modelling data. He is also advising Nicole while establishing correlative light electron microscopy at Pirbright. |
| Impact | doi: 10.3390/v10090477 |
| Start Year | 2018 |
| Description | Coronavirus lecture MSc students (Surrey) |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Postgraduate students |
| Results and Impact | A lecture was presented on the replication, cellular interactions and pathogenesis of coronaviruses to 15-20 MSc students from University of Surrey. The students were engaged and interested, answering and asking questions during the session. |
| Year(s) Of Engagement Activity | 2016,2018 |
| Description | Lecture on entereic coronaviruses to MSc Students (Surrey) |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Postgraduate students |
| Results and Impact | A lecture was presented on the pathology, diagnosis and detection of enteric coronaviruses, including virus replication and interaction with the host. The students were engaged and asked/answered question throughout the session. We were invited to repeat the lecture for the subsequent student intakes. |
| Year(s) Of Engagement Activity | 2015,2016,2018 |
| Description | Presentation at Avian coronaviruses meeting |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | Data was presented at the Avian coronaviruses and other complicating pathogens meetings in 2014 and 2016. Data was then discussed with other scientists in the field. |
| Year(s) Of Engagement Activity | 2014,2016 |
| Description | Presentation at Avian viruses focussed meeting - Sept 2018 ND |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | Presentation of data as an offered paper at the molecular biology and pathogenesis of avian viruses meeting, Oxford Sept 2018. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Presentation at Inter-institute Bioimaging networking meeting |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Other audiences |
| Results and Impact | An event was held to enable improved collaboration opportunities between scientists working with bioimaging techniques at the different research institutes within the UK. I presented data from the last 10 years describing our work characterising replication organelles induced by IBV, describing the different techniques and approaches we have used and the outlook for the future. The talk aimed to showcase the bioimaging facilities at Pirbright to encourage new collaborations. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Presentation at RIVR meeting |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Other audiences |
| Results and Impact | Oral presentation of my work to other researchers at the Recently Independent Virology Researchers meeting. I discussed my data with other researchers and potential for future collaboration. |
| Year(s) Of Engagement Activity | 2016,2017,2019,2020 |
| Description | Presentation by ND at Nidovirus symposium |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | The post-doctoral researcher presented our data at the 14th international Nidovirus symposium. She discussed data and potential collaboration with other scientisits from the field. |
| Year(s) Of Engagement Activity | 2017 |
| Description | Presentation by RH at COST meeting |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | PhD student presented our data at the COST workshop for tackling infectious bronchitis virus. He discussed results and potential collaboration with other scientists. |
| Year(s) Of Engagement Activity | 2016 |
| Description | Presentation by RH at Nidovirus symposium |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | PhD student presented our data at the 14th international Nidovirus symposium. He discussed results and potential for future collaboration with other scientists in the field. |
| Year(s) Of Engagement Activity | 2017 |
| Description | Presentation by RH at the Microbiology Society Annual meeting |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Other audiences |
| Results and Impact | PhD student presented our data at the Annual Microbiology Society meeting, He discussed results and potential for collaboration with other scientists. As a result of this presentation, a new collaboration with N Locker at University of Surrey was established. |
| Year(s) Of Engagement Activity | 2016 |
| Description | Reverse genetics seminar to MSc students |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | A seminar was presented describing reverse genetics of viruses, including infectious bronchitis virus and the application of reverse genetics in research. The students were interested and engaged, participating in activities and asking questions. We were invited to present the seminar in subsequent years for new student intakes. |
| Year(s) Of Engagement Activity | 2013,2014,2015,2016,2018,2019 |
| Description | Surrey vet school open day - MJB |
| 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 | The event was an open day for prospective students for the University of Surrey vet school. The course includes a research placement, which can be conducted at Pirbright. Therefore there was a stand from Pirbright to describe the research we perform generally as well as a discussion of our specific research. The audience were generally interested and the information helped inform them when selecting a university course. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Talk at Avian viruses focussed meeting - Sept 2018 HM |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | Presentation of data as an offered paper at the Molecular biology and pathogenesis of avian viruses meeting in Oxford, September 2018. |
| Year(s) Of Engagement Activity | 2018 |
| Description | The Hurst careers event - HJM |
| 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 | The Hurst school held a careers event for GCSE students to attend throughout the day. The Pirbright stand contained a variety of engagement activities to demonstrate the research we perform and to stimulate discussion. The students seemed interested in the activities and those more interested in a science career asked a variety of questions about the research and entry into scientific research as a career. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Tillingbourne School bug hunt - ND |
| 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 | Primary school students were involved in an event to identify insects in the school grounds. |
| Year(s) Of Engagement Activity | 2018 |