Periodic Reporting for period 1 - HIPShot (HBV GenetIc and Proteomic Screen)
Reporting period: 2019-03-04 to 2021-03-03
Viral infections pose a constant threat to human societies by causing substantial morbidity and mortality worldwide. Over 500 million people worldwide, are still persistently infected with hepatitis B virus (HBV), hepatitis delta virus (HDV), and/or HCV. In fact, these viruses constitute many challenges to the medical community worldwide for several reasons. First, they specifically infect the liver, a vital detoxifying organ whose damage can have important consequences on patients’ lives. Second, no satisfactory treatment options are always available against these viruses. Third, the outcome of HBV, HCV or HBV/HDV persistent infections often manifests in a sequential progression from chronic liver disease, to fibrosis, then cirrhosis, culminating in hepatocellular carcinoma (HCC). Finally, the challenges these viruses pose are largely due to our inability to target them due to our lack of knowledge on fundamental aspects of their lifecycles. HIPSHOT aimed in gaining insights into the mechanisms by which HBV and HDV viral genomes exploit their host environment to promote their replication and persistence. By doing so, HIPSHOT not only revealed unforeseen biological knowledge, but also yielded new targets for host-directed antiviral therapies to combat infection and chronic inflammation.
The overall objective of this project was to discover and understand how HBV and HDV are able to infect and replicate in human hepatocytes through interactions with cellular host factors.The objective was reached through the following specific aims:
1. Comprehensive identification of proteins associated with the HBV and HDV genomes.
2. Uncovering pro- or anti-viral factors through genetics screens and dissecting their roles in viral replication.
In conclusion, through proteomics and genetic approaches, I was able to identify a list of genes associated with HBV and HDV infections. These genes encode hepatic host factors important during the course of viral replication, playing either pro-viral or antiviral roles. The identified host factors would help answering unresolved questions concerning HBV and HDV lifecycles (e.g. cccDNA formation, HDV sensing). Most importantly the identified factors constitute ideal drug targets, suitable for the development of novel antiviral therapies against these viruses.
The overall objective of this project was to discover and understand how HBV and HDV are able to infect and replicate in human hepatocytes through interactions with cellular host factors.The objective was reached through the following specific aims:
1. Comprehensive identification of proteins associated with the HBV and HDV genomes.
2. Uncovering pro- or anti-viral factors through genetics screens and dissecting their roles in viral replication.
In conclusion, through proteomics and genetic approaches, I was able to identify a list of genes associated with HBV and HDV infections. These genes encode hepatic host factors important during the course of viral replication, playing either pro-viral or antiviral roles. The identified host factors would help answering unresolved questions concerning HBV and HDV lifecycles (e.g. cccDNA formation, HDV sensing). Most importantly the identified factors constitute ideal drug targets, suitable for the development of novel antiviral therapies against these viruses.
Overview of the results
We first conducted Comprehensive Identification of RNA binding Proteins by Mass Spectrometry (ChIRP-MS) experiments in Huh7 cells to identify host proteins associated with HBV, HDV and HCV nucleic acids . This resulted in a total of 594 unique proteins co-purifying with the viral genomes . Peptides from 274, 295 and 293 proteins were found to be associated to HBV, HDV and HCV nucleic acids respectively . HBV ChIRP-MS results were obtained very recently (February 2021) and are being analyzed. HDV ChIRP-MS results were obtained before the french lockdown (March 2020) and were pursued further in our analysis. A majority of the top 50 enriched hits is known to have a nuclear localization. We found many new protein complexes that have never been associated with HDV infection before (e.g. MCM complex). Importantly, in parallel to these mechanistic studies we conducted a chemical screening to inhibit HDV replication using small drug compounds targeting factors identified by HDV ChIRP-MS. A library of 20 compounds from different families (e.g. anti-cancer drugs, antivirals) known to target factors identified by our ChIRP-MS (Fig. 3) is being tested to identify molecules with anti-viral properties. Many drugs are showing strong effects on HDV replication in hepatocytes and we are currently testing them as potential HDV antivirals. I was also able to contribute to a genome-scale gain-of-function HBV screen that was ongoing in the lab. Indeed, through a genome-wide gain-of-function screen uncovered HBV-related host-dependency factors (Eller et al 2020). I contributed to validation experiments, where I was able to generate CDKN2C KO cells that were crucial for the validation of CDKN2C as an important host factor for HBV replication. CDKN2C is overexpressed in permissive cells and HBV-infected patients. Mechanistic studies show a role for CDKN2C in inducing cell cycle G1 arrest through inhibition of CDK4/6 associated with the upregulation of HBV transcription enhancers. A correlation between CDKN2C expression and disease progression in HBV-infected patients suggests a role in HBV-induced liver disease. Taken together, our study identified a previously undiscovered clinically relevant HBV host factor, allowing the development of improved infectious model systems for drug discovery and the study of the HBV life cycle (Eller et al 2020).
Exploitation and dissemination of the results
I attended 3 scientific conferences where results of the project were disseminated either by oral presentation or by a poster (see report).
The fellowship also permitted the publication of 3 peer-reviewed scientific articles (see report for details).
During HIPSHOT I was heavily involved in Science outreach activities and communication to large Public. I participated to Nuit Européenes des Musées in Strasbourg where I explained to curious large public visiting the University of Strasbourg Zoologic Museum the work of research scientists and our projects to tackle viruses such as HBV in the lab. I also wrote an article in French about antiviral immunity in animals for the website Planet-Vie, that is a reference for biology teachers and educators in French High Schools (see report for link). I also participated to a Webinar organized by Mutualia Grand Ouest in French explaining to large public how vaccines work, vaccine technology and the benefit of vaccination to public health (see Youtube link in the report).
We first conducted Comprehensive Identification of RNA binding Proteins by Mass Spectrometry (ChIRP-MS) experiments in Huh7 cells to identify host proteins associated with HBV, HDV and HCV nucleic acids . This resulted in a total of 594 unique proteins co-purifying with the viral genomes . Peptides from 274, 295 and 293 proteins were found to be associated to HBV, HDV and HCV nucleic acids respectively . HBV ChIRP-MS results were obtained very recently (February 2021) and are being analyzed. HDV ChIRP-MS results were obtained before the french lockdown (March 2020) and were pursued further in our analysis. A majority of the top 50 enriched hits is known to have a nuclear localization. We found many new protein complexes that have never been associated with HDV infection before (e.g. MCM complex). Importantly, in parallel to these mechanistic studies we conducted a chemical screening to inhibit HDV replication using small drug compounds targeting factors identified by HDV ChIRP-MS. A library of 20 compounds from different families (e.g. anti-cancer drugs, antivirals) known to target factors identified by our ChIRP-MS (Fig. 3) is being tested to identify molecules with anti-viral properties. Many drugs are showing strong effects on HDV replication in hepatocytes and we are currently testing them as potential HDV antivirals. I was also able to contribute to a genome-scale gain-of-function HBV screen that was ongoing in the lab. Indeed, through a genome-wide gain-of-function screen uncovered HBV-related host-dependency factors (Eller et al 2020). I contributed to validation experiments, where I was able to generate CDKN2C KO cells that were crucial for the validation of CDKN2C as an important host factor for HBV replication. CDKN2C is overexpressed in permissive cells and HBV-infected patients. Mechanistic studies show a role for CDKN2C in inducing cell cycle G1 arrest through inhibition of CDK4/6 associated with the upregulation of HBV transcription enhancers. A correlation between CDKN2C expression and disease progression in HBV-infected patients suggests a role in HBV-induced liver disease. Taken together, our study identified a previously undiscovered clinically relevant HBV host factor, allowing the development of improved infectious model systems for drug discovery and the study of the HBV life cycle (Eller et al 2020).
Exploitation and dissemination of the results
I attended 3 scientific conferences where results of the project were disseminated either by oral presentation or by a poster (see report).
The fellowship also permitted the publication of 3 peer-reviewed scientific articles (see report for details).
During HIPSHOT I was heavily involved in Science outreach activities and communication to large Public. I participated to Nuit Européenes des Musées in Strasbourg where I explained to curious large public visiting the University of Strasbourg Zoologic Museum the work of research scientists and our projects to tackle viruses such as HBV in the lab. I also wrote an article in French about antiviral immunity in animals for the website Planet-Vie, that is a reference for biology teachers and educators in French High Schools (see report for link). I also participated to a Webinar organized by Mutualia Grand Ouest in French explaining to large public how vaccines work, vaccine technology and the benefit of vaccination to public health (see Youtube link in the report).
HIPSHOT permitted the identification of hepatic host factors essential for infection by HBV and HDV. The identified factors have never been associated to these viruses before. These can be leveraged to better dissect HBV and HDV viral lifecycles which is crucial in the fight of these uncurable viruses. Most importantly, the list of the identified cellular host factors provides valuable drug targets to develop new antiviral approaches to treat these viruses and address unmet medical needs. Indeed, the progress made by the project have several economic and societal implications. On the economic level, molecules currently screened for antiviral activity, targeting factors discovered through HIPSHOT will be protected by intellectual property (IP) and subjected to patent deposition. On the societal level, the prospect of developing therapies to cure HBV and HDV infections have important public health implications. Indeed, HBV and HDV infections are major causes of chronic liver disease, including liver cirrhosis, liver failure and HCC . An estimated 2 billion people have evidence of exposure to HBV and approximately 350 million people are chronically infected with HBV worldwide.Infected patients have an approximately 100-fold increased risk for HCC compared to uninfected patients. Efficient treatment options for advanced HCC are limited or absent. Thus, the burden of established, incurable HBV-induced liver disease represents a major challenge to public health. Efficient treatment strategies to cure chronic hepatitis B are thus greatly needed and HIPSHOT results will contribute to the developments of these much needed strategies which would save many human lives.