To investigate the role of autophagy in the replication cycles of avian viruses
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
Autophagy is a conserved cellular pathway whereby upon amino acid starvation, cytoplasmic content is engulfed into an autophagosome and targeted to the lysosome for degradation. This allows the recycling of nutrients and continuation of cellular processes, promoting cell survival during stress conditions. The pathway is also implicated in innate immunity to intracellular pathogens, including viruses. Several viruses encode proteins that inhibit autophagy to prevent targeting of viral particles for degradation. However, other viruses induce autophagy but divert the pathway or prevent its completion to allow more efficient replication. A commonly used marker protein to study autophagy is microtubule associated protein 1B- light chain 3 (LC3). During previous work, the avian homologue of LC3 was cloned, GFP tagged and inserted into a replication deficient, recombinant adenovirus vector, allowing autophagy to be studied in avian cells. Infectious Bronchitis Virus (IBV) had previously been shown to induce autophagy upon infection of mammalian cells. Furthermore, expression of viral protein, nsp6, alone resulted in induction of autophagy. However, infection of avian cells did not result in induction of the pathway. This may suggest that either IBV is capable of inhibiting autophagy in avian cells or that IBV does not induce autophagy in avian cells. This project will further develop an understanding of autophagy in avian cells and to investigate the role of autophagy in the replication cycles of avian viruses.
Planned Impact
unavailable
People |
ORCID iD |
| Paul Britton (Principal Investigator) |
Publications
Maier HJ
(2012)
Involvement of autophagy in coronavirus replication.
in Viruses
Maier HJ
(2013)
Infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes.
in mBio
Maier H
(2014)
Visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus
in Autophagy
Maier HJ
(2014)
Spherules and IBV.
in Bioengineered
Kint J
(2015)
Activation of the chicken type I interferon response by infectious bronchitis coronavirus.
in Journal of virology
Dent SD
(2015)
The proteome of the infectious bronchitis virus Beau-R virion.
in The Journal of general virology
Kint J
(2015)
Quantification of infectious bronchitis coronavirus by titration in vitro and in ovo.
in Methods in molecular biology (Clifton, N.J.)
Maier HJ
(2015)
Preface. Coronaviruses.
in Methods in molecular biology (Clifton, N.J.)
Kint J
(2015)
Infectious Bronchitis Coronavirus Inhibits STAT1 Signaling and Requires Accessory Proteins for Resistance to Type I Interferon Activity.
in Journal of virology
Dent SD
(2015)
The proteome of the infectious bronchitis virus Beau-R virion.
in The Journal of general virology
Staines K
(2016)
A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR.
in PloS one
Maier HJ
(2016)
Extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity.
in Scientific reports
Kint J
(2016)
Infectious Bronchitis Coronavirus Limits Interferon Production by Inducing a Host Shutoff That Requires Accessory Protein 5b.
in Journal of virology
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
Piekarski A
(2018)
AMP-Activated Protein Kinase Mediates the Effect of Leptin on Avian Autophagy in a Tissue-Specific Manner.
in Frontiers in physiology
| Title | 3D spherules |
| Description | Electron microscopy images of virus induced cellular membrane rearrangements and virus particles were false coloured to provide artwork images. In addition, 3D electron tomography data was used to generated 3D printed images of virus induced structures in plastic. |
| Type Of Art | Artwork |
| Year Produced | 2015 |
| Impact | The false coloured electron microscopy images and 3D printed images of virus induced membrane structures have been used as props to explain scientific research during public engagement activities. |
| Title | False-coloured EM images |
| Description | Electron microscopy images of virus particles and of IBV induced cellular membrane rearrangements (spherules, zippered ER and double membrane vesicles) false coloured to create artworks. |
| Type Of Art | Image |
| Year Produced | 2015 |
| Impact | Images are used on presentations as well as the group webpage. |
| Description | The aim of this project was to characterise infectious bronchitis virus (IBV) replication organelles and identify interactions between IBV and the host cell important for successful virus replication. During this project we performed a thorough analysis of replication organelles induced by different strains of IBV, including pathogenic and apathogenic lab adapted strains, vaccine strains and field isolates. This detailed comparison of replication organelles induced by different virus strains has not been possible for other coronaviruses due to the more limited range of strains available and therefore provides valuable information about possible variation in replication organelle structure. We demonstrated that all strains of the virus induced double membrane vesicles, highlighting their importance during virus replication. In addition, most IBV strains induced both zippered ER and spherules, again indicating that these structures are also performing an important function. However, the pathogenic lab strain, M41, was found to have a low spherule phenotype. Although spherules were found in cells infected with this virus, they were present at a significantly lower frequency. This phenotype was observed in both primary chick kidney cells and ex vivo tracheal organ cultures demonstrating it is not a cell type specific observation. In addition, the low spherule phenotype was not associated with any defect in viral RNA synthesis when compared with apathogenic lab strain BeauR. Based on previous data, we can conclude that the genetic determinant for the low spherule phenotype is present between the 5'end of the genome and nsp13 coding region of ORF1ab. Future work will aim to identify which regions of ORF1ab are responsible for this phenotype and M41 now provides a useful tool to understand the function of spherules during virus replication. A new area of work initiated during this project is the characterisation of the cellular machinery required for IBV assembly and budding. We focused on determining whether the cellular ESCRT machinery is required for IBV budding and exit and tried several different approaches to address this question. Unfortunately, due to a range of technical challenges, there was limited success from this project. However, using this experience, the project will be continued in the future using a more unbiased genetic screen approach. |
| Exploitation Route | Is being used for new grants and has been used to form collaborations |
| Sectors | Agriculture Food and Drink Pharmaceuticals and Medical Biotechnology |
| URL | http://www.iah.ac.uk/research/coronavirus/Default.aspx |
| Description | The results are being used to obtain further funding |
| Sector | Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Societal |
| Description | Fellowship |
| Amount | £175,115 (GBP) |
| Organisation | The Pirbright Institute |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 01/2016 |
| End | 06/2019 |
| Description | Industrial placement studentship |
| Amount | £5,000 (GBP) |
| Organisation | The Pirbright Institute |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 07/2015 |
| End | 07/2016 |
| Description | PhD studentship |
| Amount | £96,072 (GBP) |
| Funding ID | JXG10710 Dept2 RH |
| Organisation | The Pirbright Institute |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 09/2013 |
| End | 09/2017 |
| Description | Responsive mode grant - new investigator scheme |
| Amount | £451,084 (GBP) |
| Funding ID | BB/N002350/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 06/2016 |
| End | 06/2019 |
| Description | Small project research grant |
| Amount | £5,000 (GBP) |
| Organisation | The Houghton Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 09/2014 |
| End | 10/2016 |
| 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 | 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 Bioimaging day event |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Other audiences |
| Results and Impact | I was asked to present data at a bioimaging day event held at Pirbright to showcase recent studies using bioimaging facilities. I presented and discussed my data with other researchers. |
| Year(s) Of Engagement Activity | 2016 |
| 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 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 | Presentation of data at the 13th International Nidovirus symposium. Data was discussed with other scientists. As a result of this presentation, a new collaboration with M Barcena was established. |
| Year(s) Of Engagement Activity | 2014 |
| Description | Presentation of data at Roslin Institute |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Other audiences |
| Results and Impact | I was invited to present a research seminar at the Roslin Institute. I presented data and discussed results with researchers. |
| Year(s) Of Engagement Activity | 2015 |
| Description | Presentation of data at University of Wageningen |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Other audiences |
| Results and Impact | I was invited to present a research seminar at University of Wageningen, the Netherlands. Data was presented and discussed with researchers. |
| Year(s) Of Engagement Activity | 2013 |
| 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 | School visit (Compton, West Berkshire) |
| 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 school visits included working with small groups of a-level biology students demonstrating a technique in the curriculum. They were encouraged to ask us questions about our work and how we use the technique as well as trying it out for themselves. |
| Year(s) Of Engagement Activity | 2012,2013,2014,2015 |
| 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 |