PROJET HIT-HIDDEN-HBV

Towards HBV Cure: Understanding HBVccc DNA persistence and expression

Ce projet est porté par le CNRS DR Occitanie-Est – INSERM – CIRI, Lyon – IGH, Université de Montpellier.

CONTEXT

Despite the existence of a very efficient and safe prophylactic vaccine against HBV (hepatitis B virus) infection, around 296 million people worldwide are yet chronic HBV carriers, of whom 25% could die of liver cirrhosis decompensation or hepatocellular carcinoma (HCC), if left untreated. Current treatments for chronic hepatitis B (CHB) patients are ineffective at completely clearing the virus. This is due to the inability of the treatments to eliminate the nuclear viral DNA episome, called cccDNA (i.e. covalently-closed-circular DNA). This long-lived cccDNA accounts for long-term persistence of infection and is responsible for the viral rebound after cessation of antiviral treatment with nucleoside analogues (i.e. standard-of-care in industrialized countries). Moreover, chronically infected patients suffer from a profound dysfunction of both intrahepatic innate immunity and adaptive HBV-specific T and B cells responses, which render them unable to efficiently fight viral infection by immunological means. To better achieve a functional cure in CHB patients, which is defined by a durable loss of viremia and antigenemia, as well as an immune response recovery, new therapeutic approaches targeting the viral persistence and maintenance of cccDNA are urgently needed. This will require a deeper understanding of the molecular mechanisms involved in cccDNA formation, maintenance, and transcriptional activity leading to the synthesis of HBV RNAs that are in turn crucial to maintain an active replication of the virus.

OBJECTIVES

Our objectives are to identify and in-depth study the role of viral and host proteins associated with HBV cccDNA and RNAs and reveal their roles in cccDNA formation, maintenance and transcriptional activity, as well as in HBV RNA biogenesis at large, using state-to-the-art and unbiased technologies, combined to AI-driven multi-bio-informatic analyses. Such advances will allow defining novel and most relevant therapeutic targets for future and improved antiviral strategies.

To this end, 3 axes of research (with technological and biological coherences) were defined and aim at:

1. Identifying, and validating (overall validation) the role of host proteins associated with cccDNA and DNA- or RNA-intermediates of HBV replication;
2. Understanding the biology of HBV nuclear DNAs, with 3 different aspects, related to the role of viral/host proteins:

• in cccDNA formation, maintenance, and transcriptional activity (with spatio-temporal level of analyses);
• in HBV DNA integration into host genome, leading to functional and mutational events causing hepatocellular carcinogenesis;
• in escape mechanisms from DNA-induced innate immunity and viral intrinsic restriction systems;

3. Understanding the biology of HBV RNAs & its interplay with innate immune sensing, including 2 sub-aspects, related to the role of viral/host proteins:

•  in the biogenesis, splicing escape, stability/degradation escape and trafficking of HBV RNAs out of the nucleus; 
•  in escape mechanisms from RNA-induced innate immunity and viral intrinsic restriction systems.

RESULTS

After few months into the program, two high-value and unbiased technologies, namely PICh-MS (i.e. Proteomics of Isolated Chromatin segments obtained by Mass-Spectrometry) and ChIRP-MS (i.e. Comprehensive Identification of RNA-binding Proteins by Mass Spectrometry), were respectively performed to identify host factors bound to HBV DNA and RNA replication intermediates.

PiCh-MS has been performed using HBV infected hepatoma cells. Mass spectrometry analysis identified 198 proteins that were enriched in the eluate from infected cells compared to noninfected cells (ongoing bioinformatic analyses of based on functions, numbers or intensities of the identified peptides), including the viral capsid (HBc) protein known to bind cccDNA as well as host factors known to bind cccDNA or viral proteins (such as HMGA1, SRSF10, SRPK1). We are currently investigating the impact of these factors on HBV replication using small interfering RNA screens of pooled siRNAs targeting the 198 selected candidates. To identified pathways relevant for HBV replication/ biology, we will also cross this analysis with proteome performed using human primary hepatocytes infected by HBV or inactivated HBV or with HBc and HBx interactomes.

In the case of the ChIRP-MS approach two sets of data were independently obtained from two cell culture models. The most statistically relevant hits were then crossed with previous data sets (HBV protein interactomes and proteome/phosphor-proteome in HBV-infected hepatocytes), and a list of 30 genes was selected for a first round of validation via a loss of function approach in several relevant cell culture models (PHH, dHepaRG, and dHuh7.5-hNTCP). Out of this, one leader candidate (druggable target) is already being investigated by using genetic and pharmacologic means. Interestingly this factor was also identified recently as playing an important role in hepatocellular carcinogenesis, thus linking virology and oncology.