Understanding host factors that regulate the hepatitis B viral epigenome

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.

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.

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.