Understanding host factors that regulate the hepatitis B viral epigenome
Lead Research Organisation:
University of Birmingham
Department Name: Institute of Cancer and Genomic Sciences
Abstract
Hepatitis B virus (HBV) is a global cause of liver disease. At least 300 million individuals are chronically infected with HBV, leading to over 800,000 cases of life threatening liver cirrhosis and cancers every year. Current therapies limit HBV infections, but do not eradicate the virus from the body, leaving patients at risk of viral reactivation and developing liver disease. Upon infection of the target cell, viruses need to induce expression of viral encoded genes that are essential for replication of the viral genetic material and production of progeny virus. Persistent viruses, such as HBV, also need to manipulate the host cell environment to support long-term infection. This project aims to understand the molecular events that regulate HBV gene expression following infection and support the persistence of HBV DNA. Following viral entry, HBV DNA enters the host cell nucleus and is bound by host cell proteins called histones that serve to organise genetic material into areas where genes are switched on or off. The precise arrangement of these genetic hubs is vital for controlling gene expression and requires further investigation. We have identified the host cell protein CTCF as a key regulator of HBV gene expression. Since CTCF plays an important role in the formation of physical boundaries between active and inactive gene expression hubs within the host genome, we predict that CTCF plays a critical role in regulating HBV gene expression and establishment of chronic infection. We will dissect the molecular organisation of the HBV chromosome and determine the function of CTCF recruitment on HBV gene expression regulation. An in-depth analysis of how these host factors organise viral gene expression immediately after infection and in chronic, persistent HBV infection will provide a step-change in our understanding of this important human pathogen. The novel insights into the HBV life cycle that we will contribute throughout this study are vital in the future design of novel anti-HBV strategies that will be useful in the treatment of chronic HBV infection.
Technical Summary
Hepatitis B virus infection is a significant and global health burden. Over 300 million chronically infected individuals are at risk of developing life-limiting liver disease. Current therapies are effective at reducing viral load but do not clear the persistent HBV DNA, leaving these patients at significant risk of viral reactivation. Following host cell entry, HBV DNA is established as a persistent episome of covalently closed circular DNA (cccDNA). This episome serves as a template for virus transcription. It is known that HBV cccDNA recruits cellular histones but the dynamic epigenetic regulation of HBV and viral promoter usage is not understood. This project aims to dissect the temporal control of HBV transcription during de novo infection of primary human hepatocytes. The HBV epigenome at early and late stages of infection will be characterised by ChIP- and DNaseI-Seq of cccDNA to map epigenetic boundaries and the formation of regulatory elements. These data will be aligned with rigorous and time-resolved analysis of viral transcripts. Our preliminary data provide compelling evidence that the host cell transcription regulator CTCF binds the HBV genome to positively regulate enhancer activity. Using a combination of mutagenesis and shRNA methodologies to abrogate CTCF binding we will dissect the function of CTCF in the establishment and maintenance of the HBV epigenome. We also provide evidence that CTCF co-operates with the transcription regulator YY1 to stimulate enhancer activity. We aim to determine the interplay between CTCF and YY1 in the epigenetic regulation of HBV enhancer activity and virus transcription. This in-depth analysis of HBV transcription in a physiologically relevant HBV infection model, and molecular dissection of key host factors will provide a step-change in our understanding of the HBV life cycle. The outcomes of this project will impact on future anti-viral strategies that will be effective in the clearance of chronic HBV infections.
Planned Impact
Who will be impacted?
a) Our studies will generate new data describing the interaction between a relevant human cell and a pathogenic virus. This will benefit academic virologists and cell biologists. Because HBV infection represents a serious health burden this research will be of interest to patients and clinicians.
b) Much of the health care costs associated with HBV infection are due to progressive liver disease, cirrhosis and hepatocellular carcinoma. Currently there are no curative treatments for this virus and the discovery of novel targets would represent a significant impact to Industry who will benefit from the discovery of new drug targets and patients would benefit from new efficacious drugs.
c) Because HBV is related to other important viruses discoveries made may be relevant for other pathogens with impact to human health.
d) Combining our expertise on viral hepatitis and epigenetics provides a powerful systematic approach to studying complex biological problems. If successful we believe combining such orthogonal methodological approaches will be widely adopted.
e) The postdoctoral research fellows employed on the grant will learn new skills and expertise, which will be utilised in this research programme and benefit their future, thus enabling the transfer of knowledge between individuals and institutes. They will be offered the opportunity to take part in personal and research specific training courses to promote their professional development, in keeping with the principles set out in the Concordat to Support the Career Development of Researchers. Additionally, they will share their data at lab meetings, present research at international conferences and be encouraged to take part in public engagement activities.
Defining the route to impact.
i) The targets we discover will be knocked out or over-expressed in human liver cells and assessed for their ability to halt viral replication and their impact on the host anti-viral response. Any previously known chemical modifiers of these targets, such as the compounds from CRT, will also be tested at this stage.
ii) Findings from the proposed studies can be directly translated to humans. McKeating is coordinator of a EU consortia HepCAR that studies the impact of comorbidities in HCC and involves many leading clinicians and basic scientists working in this area as partners providing opportunities to progress to clinical trials.
a) Our studies will generate new data describing the interaction between a relevant human cell and a pathogenic virus. This will benefit academic virologists and cell biologists. Because HBV infection represents a serious health burden this research will be of interest to patients and clinicians.
b) Much of the health care costs associated with HBV infection are due to progressive liver disease, cirrhosis and hepatocellular carcinoma. Currently there are no curative treatments for this virus and the discovery of novel targets would represent a significant impact to Industry who will benefit from the discovery of new drug targets and patients would benefit from new efficacious drugs.
c) Because HBV is related to other important viruses discoveries made may be relevant for other pathogens with impact to human health.
d) Combining our expertise on viral hepatitis and epigenetics provides a powerful systematic approach to studying complex biological problems. If successful we believe combining such orthogonal methodological approaches will be widely adopted.
e) The postdoctoral research fellows employed on the grant will learn new skills and expertise, which will be utilised in this research programme and benefit their future, thus enabling the transfer of knowledge between individuals and institutes. They will be offered the opportunity to take part in personal and research specific training courses to promote their professional development, in keeping with the principles set out in the Concordat to Support the Career Development of Researchers. Additionally, they will share their data at lab meetings, present research at international conferences and be encouraged to take part in public engagement activities.
Defining the route to impact.
i) The targets we discover will be knocked out or over-expressed in human liver cells and assessed for their ability to halt viral replication and their impact on the host anti-viral response. Any previously known chemical modifiers of these targets, such as the compounds from CRT, will also be tested at this stage.
ii) Findings from the proposed studies can be directly translated to humans. McKeating is coordinator of a EU consortia HepCAR that studies the impact of comorbidities in HCC and involves many leading clinicians and basic scientists working in this area as partners providing opportunities to progress to clinical trials.
Publications
Wing PA
(2019)
A dual role for SAMHD1 in regulating HBV cccDNA and RT-dependent particle genesis.
in Life science alliance
D'Arienzo V
(2021)
A PCR assay to quantify patterns of HBV transcription.
in The Journal of general virology
Desombere I
(2021)
A Role for B Cells to Transmit Hepatitis C Virus Infection.
in Frontiers in immunology
Liu Y
(2020)
Accurate targeted long-read DNA methylation and hydroxymethylation sequencing with TAPS.
in Genome biology
Wing PAC
(2023)
An ACAT inhibitor suppresses SARS-CoV-2 replication and boosts antiviral T cell activity.
in PLoS pathogens
Ng E
(2023)
An enrichment protocol and analysis pipeline for long read sequencing of the hepatitis B virus transcriptome.
in The Journal of general virology
Smirnov A
(2023)
ASPP2 binds to hepatitis C virus NS5A protein via an SH3 domain/PxxP motif-mediated interaction and potentiates infection
in Journal of General Virology
Benedikz E
(2019)
Bacterial flagellin promotes viral entry via an NF-kB and Toll Like Receptor 5 dependent pathway
in Scientific Reports
Schmidt NM
(2020)
Cholesterol-modifying drugs in COVID-19.
in Oxford open immunology
Zhuang X
(2021)
Circadian control of hepatitis B virus replication.
in Nature communications
Dobrica M
(2024)
CTCF regulates hepatitis B virus cccDNA chromatin topology
in Journal of General Virology
Burley M
(2020)
Epigenetic regulation of human papillomavirus transcription in the productive virus life cycle.
in Seminars in immunopathology
Lythgoe K
(2020)
Estimating hepatitis B virus cccDNA persistence in chronic infection
Magon KL
(2021)
From infection to cancer: how DNA tumour viruses alter host cell central carbon and lipid metabolism.
in Open biology
Ko C
(2018)
Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels.
in Journal of hepatology
Schilling M
(2023)
Hypoxia dampens innate immune signalling at early time points and increases Zika virus RNA levels in iPSC-derived macrophages
in Journal of General Virology
Zhuang X
(2024)
Hypoxia inducible factors inhibit respiratory syncytial virus infection by modulation of nucleolin expression.
in iScience
Description | Alteration of host chromatin accessibility by carcinogenic human papillomavirus types 16 & 18 |
Amount | £4,887 (GBP) |
Organisation | University of Birmingham |
Sector | Academic/University |
Country | United Kingdom |
Start | 12/2020 |
End | 04/2021 |
Description | Exploring the potential of ACAT inhibition as a novel immunomodulatory and antiviral agent in HBV infection |
Amount | £90,970 (GBP) |
Organisation | Gilead Sciences, Inc. |
Sector | Private |
Country | United States |
Start | 01/2022 |
End | 10/2022 |
Description | Molecular characterization of the infection pathway of SARS-CoV-2 in polarized respiratory epithelial cells and its susceptibility to pharmaceutical inhibition |
Amount | £116,987 (GBP) |
Organisation | John Black Charitable Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 01/2022 |
End | 06/2023 |
Description | Society Conference Grant |
Amount | £250 (GBP) |
Organisation | Microbiology Society |
Sector | Learned Society |
Country | United Kingdom |
Start | 03/2020 |
End | 04/2020 |
Description | Analysis of HBV and HPV chromatin structure - Csilla |
Organisation | University of Birmingham |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Experimental analysis of viral and host chromatin - ChIP-Seq, ATAC-Seq |
Collaborator Contribution | Computational analysis of RNA-Seq, ChIP-Seq and ATAC-Seq data |
Impact | Multidisciplinary - molecular biology, virology and computational biology Supervision of MSc Bioinformatics student - graduated 2021 |
Start Year | 2021 |
Description | Analysis of HBV and HPV gene expression and splicing - Arnold |
Organisation | University of Birmingham |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Nanopore analysis of HBV and HPV transcription |
Collaborator Contribution | Computational analysis of Nanopore sequencing |
Impact | Multi-disciplinary; molecular biology, virology and computational biology |
Start Year | 2021 |
Description | CTCF dependent regulation of HBV - Testoni |
Organisation | National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS) |
Department | IN2P3-Lyon |
Country | France |
Sector | Academic/University |
PI Contribution | Collaboration with Barbara Testoni to study the functional role of CTCF in HBV transcription control using minicircle technology established in the Testoni Lab. |
Collaborator Contribution | Data generated through this collaboration contributed to our recent publication in Cellular microbiology. We have had frequent meetings between partners to discuss results and future experiments. |
Impact | D'Arienzo V*, Ferguson J*, Giraud G, Chapus F, Harris JM, Wing PAC, Claydon A, Begum S, Zhuang X, Balfe P, Testoni B, McKeating JA and Parish JL. The CCCTC-binding factor CTCF represses hepatitis B virus Enhancer I and regulates viral transcription. (2020) Cellular Microbiology. DOI:10.1111/cmi.13274 *Equal Contribution |
Start Year | 2018 |
Description | CTCF dependent regulation of the HBV epigenome |
Organisation | University of Oxford |
Department | Target Discovery Institute (TDI) |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | This MRC award was made to Parish and McKeating to support collaborative research into HBV transcription regulation. Both partners are actively engaged in this collaboration and contributing unique skills, knowledge and reagents. |
Collaborator Contribution | This MRC award was made to Parish and McKeating to support collaborative research into HBV transcription regulation. Both partners are actively engaged in this collaboration and contributing unique skills, knowledge and reagents. |
Impact | After securing MRC funding for this collaborative project we have recruited to the funded posts and have established a productive way of working. 1 publication has already arisen from this collaboration and we are in the process of writing a second publication. |
Start Year | 2018 |
Description | SARS-CoV-2 Diagnostic testing with Andrew Beggs |
Organisation | University of Birmingham |
Department | College of Medical and Dental Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Scientific input generation of research data |
Collaborator Contribution | project management manuscript writing analysis of research data |
Impact | https://doi.org/10.1016/j.jmoldx.2021.12.007 |
Start Year | 2020 |
Description | SARS-CoV-2 Testing with Alan McNally |
Organisation | University of Birmingham |
Department | College of Medical and Dental Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | management of COVID-19 testing sites overseeing the risk assessments of COVID-19 testing sites Analysis of data writing of manuscripts |
Collaborator Contribution | management of COVID-19 testing sites overseeing the risk assessments of COVID-19 testing sites Analysis of data writing of manuscripts project management Next-gen sequencing |
Impact | 1 - https://doi.org/10.1371/journal.pbio.3001216 2 - https://doi.org/10.1101/2021.11.17.21266297 |
Start Year | 2020 |
Description | Cancer prevention and healthy living workshop |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Other audiences |
Results and Impact | Workshop at local scout group to discuss health living and cancer prevention. |
Year(s) Of Engagement Activity | 2020 |
Description | External Seminar Invitation - University of Melbourne Veterinary School |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | I was invited to give a seminar on my research at the University of Melbourne, Australia. This covered my PhD subject and also research relating to Hepatitis B Virus. This was on the 10th October 2019. |
Year(s) Of Engagement Activity | 2019 |
Description | Family in Academia workshop |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Other audiences |
Results and Impact | Annaul presentation of my journey as an academic with a family. tips on how i manage my work/life balance and inspire others to realize it is possible to have a family and be a successful academic. |
Year(s) Of Engagement Activity | 2021,2022,2023 |
Description | Frontiers Science Presentation ay Association for Science Teachers Conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Schools |
Results and Impact | Delivered a presentation of my research area to a 'lay audience' of science teachers at an International conference in the Fronteirs Science lecture series |
Year(s) Of Engagement Activity | 2018 |
Description | Life Sciences in Six |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Presentation of Life Sciences in Six at the Birmingham Rep to an audience of approximately 250. Discussion afterwards was very lively and audience reported increased interest and changed perceptions. |
Year(s) Of Engagement Activity | 2018 |
URL | https://www.birmingham.ac.uk/university/colleges/mds/events/2017/11/life-sciences-six-2017/index.asp... |
Description | Presentation to lay audience |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Other audiences |
Results and Impact | 80 students and staff attended a day of talks called UBeEnlightened from across the University to showcase the breadth of our research. |
Year(s) Of Engagement Activity | 2020 |
Description | STEM Careers Talk - Y11 Coventry |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Gave a presentation on Careers in Academia to 97 Y11 pupils at a regional secondary high school as part of their STEM Careers Week. This was followed by a Q&A session where the pupils were able to ask questions on how to get into academic jobs through education and what the career entails. |
Year(s) Of Engagement Activity | 2021 |
Description | School Visit (Bromsgrove, Worcestershire) |
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 | Ran a science workshop focussed on genetics with school children aged 10-13 |
Year(s) Of Engagement Activity | 2018 |
Description | School Visit (Redditch, Worcestershire) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | School Science day including judging science competion, careers talk and hands on demonstrations. |
Year(s) Of Engagement Activity | 2018 |
Description | School Visit (Redditch, Worcestershire) |
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 | Science workshop based on DNA extraction and genetics for school children aged 10-13 |
Year(s) Of Engagement Activity | 2018 |
Description | Schools visit for Science workshop |
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 | Schools visit to Woodfield Academy, Redditch, Worcestershire to run science workshop focused on DNA with children aged 10-11. 120+ children attended. Very postitive feedback from students and teachers was received. This is part of a long term initiative to work with this school to develop a STEM club supported by academics at the University of Birmingham. |
Year(s) Of Engagement Activity | 2018 |
Description | Smoking and Cancer Workshop |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Other audiences |
Results and Impact | Workshop with Bromsgrove Scout group to discuss smoking and cancer/health. Scouts were very engaged with many questions. There was much discussion. Scouts reported increased awareness of health impact of smoking. |
Year(s) Of Engagement Activity | 2018 |