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Combined Small Molecule and Loss-Of-Function Screen Uncovers Estrogen Receptor Alpha and CAD As Host Factors for HDV Infection A

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Hepatology ORIGINAL ARTICLE Combined small molecule and loss-of-function screen Gut: first published as 10.1136/gutjnl-2018-317065 on 4 March 2019. Downloaded from uncovers estrogen receptor alpha and CAD as host factors for HDV infection and antiviral targets Eloi R Verrier,1 Amélie Weiss,2 Charlotte Bach,1 Laura Heydmann,1 Vincent Turon-Lagot,1 Arnaud Kopp,2 Houssein El Saghire,1 Emilie Crouchet,1 Patrick Pessaux ,1,3 Thomas Garcia,4 Patrick Pale,4 Mirjam B Zeisel,1 Camille Sureau,5 Catherine Schuster,1 Laurent Brino,2 Thomas F Baumert 1,3,6 1Université de Strasbourg, ABSTRact Inserm, Institut de Recherche Objective Hepatitis D virus (HDV) is a circular RNA Significance of this study sur les Maladies Virales et virus coinfecting hepatocytes with hepatitis B virus. Hépatiques UMR_S1110, F- What is already known on this subject? 67000 Strasbourg, France Chronic hepatitis D results in severe liver disease and 2IG BMC, Plateforme de Criblage an increased risk of liver cancer. Efficient therapeutic ► Chronic hepatitis D is the most severe form of Haut-débit, UMR7104 CNRS approaches against HDV are absent. viral hepatitis. U1258 Inserm, Illkirch, France Design Here, we combined an RNAi loss-of-function ► Efficient therapeutic strategies are absent. 3Institut Hospitalo-universitaire, ► Hepatitis D virus is a small hepatitis B virus Pôle Hépato-digestif, Nouvel and small molecule screen to uncover host-dependency Hôpital Civil, Strasbourg, France factors for HDV infection. satellite virus. 4Laboratoire de Synthèse, Results Functional screening unravelled the hypoxia- ► Knowledge about HDV–hepatocyte interactions Réactivité Organiques et inducible factor (HIF)-signalling and insulin-resistance is limited. Catalyse, Institut de Chimie, pathways, RNA polymerase II, glycosaminoglycan ► HDV host-dependency factors are largely UMR 7177 CNRS, Université de unknown. Strasbourg, Strasbourg, France biosynthesis and the pyrimidine metabolism as virus- 5 INTS, Laboratoire de Virologie hepatocyte dependency networks. Validation studies in What are the new findings? Moléculaire, Paris, France primary human hepatocytes identified the carbamoyl- 6 ► A RNAi screen identified oestrogen receptor 1 Institut Universitaire de France, phosphatesynthetase 2, aspartate transcarbamylase Paris, France (ESR1) and CAD as novel host factors for HDV and dihydroorotase (CAD) enzyme and estrogen infection. http://gut.bmj.com/ Correspondence to receptor alpha (encoded by ESR1) as key host factors ► The inhibition of CAD restricts HDV infection Dr Eloi R Verrier and Pr Thomas for HDV life cycle. Mechanistic studies revealed that through uridine depletion. F Baumert, Inserm UMR_S1110, the two host factors are required for viral replication. ► ESR1 and CAD are functionally linked by Institut de Recherche sur les Inhibition studies using N-(phosphonoacetyl)-L-aspartic Maladies Virales et Hépatiques, transcriptional activation of gene expression. 3 rue Koeberlé, 67000 acid and fulvestrant, specificCA D and ESR1 inhibitors, ► ESR1 and CAD inhibitors fulvestrant and PALA, Strasbourg, France; respectively, uncovered their impact as antiviral targets. respectively, specifically inhibit HDV replication e. verrier@ unistra. fr, Conclusion The discovery of HDV host-dependency in a dose-dependent manner in human on September 24, 2021 by guest. Protected copyright. thomas. baumert@ unistra. fr factors elucidates the pathogenesis of viral disease hepatocytes. biology and opens therapeutic strategies for HDV cure. Received 26 June 2018 ► CAD and ESR1 are previously undiscovered Revised 24 January 2019 targets for antiviral therapies. Accepted 10 February 2019 How might it impact on clinical practice in the INTRODUCTION foreseeable future? Hepatitis delta virus (HDV) infects human hepato- ► The discovery of HDV host-dependency factors cytes and causes acute acerbation of liver disease in opens the door for novel therapeutic strategies patients chronically infected with hepatitis B virus against chronic hepatitis D - a major unmet (HBV).1 Among HBV-infected patients, 5%–10% medical need. 2 are coinfected with HDV. HDV coinfection or ► Fulvestrant and PALA-like molecules are surinfection in HBV carriers induces more severe candidate compounds for HDV antivirals to © Author(s) (or their disease compared with infection with HBV alone. enter preclinical development. employer(s)) 2020. Re-use This includes a greater likelihood of experiencing permitted under CC BY-NC. No liver failure and a more rapid progression to liver commercial re-use. See rights and permissions. Published cirrhosis, with a markedly increased risk of devel- allows efficient cure of hepatitis D with eradica- 3 4–6 by BMJ. oping hepatocellular carcinoma. Consequently, tion of the virus and its associated disease. Thus, chronic hepatitis D is considered as one of the most there is a major unmet medical need for curative To cite: Verrier ER, Weiss A, severe forms of viral hepatitis. Treatment with HBV therapies. Bach C, et al. Gut Epub ahead of print: [please nucleos(t)ide analogues is not effective at reducing HDV is a small, circular RNA HBV satellite virus include Day Month Year]. HDV replication or disease. Moreover, the virus related to plant viroids using HBV envelope proteins doi:10.1136/ is only poorly controlled by the current inter- to assemble its infectious particles.1 5 6 Conse- gutjnl-2018-317065 feron (IFN)-based therapies.3 To date, no treatment quently, it can only be propagated by HBV-infected Verrier ER, et al. Gut 2019;0:1–10. doi:10.1136/gutjnl-2018-317065 1 Hepatology hepatocytes producing HBV envelope proteins (Hepatitis B transfected with siRNAs pools 48 hours before inoculation with surface antigen or HBsAg). The HDV genome is a circular 1.7 kb HDV (figure 1A) with an optical immunostaining-based viral single-stranded negative-sense RNA molecule (for a review protein (HDAg) readout 7 days after infection. The silencing of Gut: first published as 10.1136/gutjnl-2018-317065 on 4 March 2019. Downloaded from on HDV virology and pathogenesis, see ref 5). The HDV life expression of HDV host factor NTCP was used as functional cycle starts with virus entry into hepatocyte after attachment to positive control (figure 1B-C). The results of the primary screen heparan sulfate proteoglycans (HSPG) at the cell surface.5 7 As are presented in online supplementary table S1 . HBV and HDV share the same envelope proteins, they appear Using the hit selection algorithm described in online supple- to share the same entry pathway and receptor(s), including the mentary material and methods and figure 1E, we identified sodium taurocholate cotransporting polypeptide (NTCP) and 191 host factor genes showing a robust correlation between glypican 5 (reviewed in refs 5 6 8). The HDV genome is then gene silencing and level of HDV infection, moderate toxicity of imported in the nucleus, and three types of RNAs are tran- gene-specific siRNA and their expression in the liver (figure 1D,E scribed by the host RNA polymerase II: replication is initiated and online supplementary table S2). The threshold of hit selec- by synthesis of an antigenomic RNA that serves as a template for tion (described in figure 1E) was based on the functional effect the production of new HDV genomic RNA and HDV mRNA of SLC10A1 silencing (approximately 45% decrease in HDV from genomic RNA.5 mRNA transcripts contain one unique infection, online supplementary table S1). The hit rate of 191 open reading frame encoding the hepatitis delta antigen (HDAg) genes corresponds to 2.5% of total genes, which is similar to that exists in two forms (small version [S-HDAg] and large screens for other virus host factor-dependency screens.15 16 version [L-HDAg]) which play differential roles in the HDV life Functional pathway analysis using KEGG pathways identi- cycle.5 6 Indeed, S-HDAg is essential to HDV replication, while fied a network of host factors belonging to HSPG biosynthesis, prenylation of L-HDAg leads to the inhibition of HDV replica- including EXT1 and EXT2 (figure 2A,B). These genes encode tion and is crucial for binding to HBV envelope proteins and two major exostosins, key HSPG-related enzymes exhibiting virion assembly.6 In the nucleus, L-HDAgs bind to the de novo glycosyltransferase activities and involved in the elongation of HDV genomic RNA to form a new ribonucleoprotein, which heparan sulfate chains.17 Moreover, several subunits of the RNA is exported to the cytoplasm and interacts with HBsAgs at the polymerase II were identified in our screen (figure 2B), including endoplasmic reticulum to form new infectious virions that are specific subunits such as POLR2G or POLR2I, encoding Rpb7 then secreted to propagate further rounds of HDV infection.6 and Rpb9, which play key roles in the initiation of transcription While the molecular virology of HDV has been elucidated, the and binding to DNA template for an accurate selection of the role of host-dependency factors for HDV infection remains transcription start sites, respectively.18 19 The identification of largely unknown.5 HSPG enzymes and RNA polymerase II as known HDV-related A unique feature of HDV is the apparent lack of non-struc- pathways5 7 validates our approach for host factor discovery tural protein encoded by the viral genome (including viral poly- (figure 2). Importantly, the screen identified several previously merase), making it highly dependent of the host machinery for its unknown HDV-related networks, such as the hypoxia inducible replication. Targeting host factors is an emerging
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