TP09: Control of hepatitis C virus infection by the interferon system and virus – host co-evolution

A hallmark of hepatitis C virus (HCV) infections is the high propensity to establish persistence. This is achieved by active suppression and perturbation of both innate and adaptive immune responses. However the mechanistic details are still poorly understood and the relative contributions of the various immune escape strategies for persistence are unknown. Here we want to address three complementary aspects: (i) how LGP2 modulates RIG-I and MDA5 triggered IFN responses, (ii) how HCV persists in IFN-activated cells and (iii) the long-term co-evolution of hepatitis viruses with their hosts.

Prof. Dr. Dr. hc. Ralf Bartenschlager

Pfister S, Rabl J, Wiegand T, Mattei S, Malär AA, Lecoq L, Seitz S, Bartenschlager R, Böckmann A, Nassal M, Boehringer D, Meier BH. (2023) Structural conservation of HBV-like capsid proteins over hundreds of millions of years despite the shift from non-enveloped to enveloped life-style. Nat Commun. 14:1574. doi.org/10.1038/s41467-023-37068-w.

Colasanti O, Burm R, Huang HE, Riedl T, Traut J, Gillich N, Li TF, Corneillie C, Faure-Dupuy S, Grünvogel O, Heide D, Lee JY, Tran CS, Merle U, Chironna M, Vondran FWF, Esser-Nobis K, Binder M, Bartenschlager R, Heikenwälder M, Meuleman P, Lohmann V. (2023) Comparison of HAV and HCV infections in vivo and in vitro reveals distinct patterns of innate immune evasion and activation. J Hepatol. 28;S0168-8278(23)00309-4. doi: 10.1016/j.jhep.2023.04.023.

Esser K, Cheng X, Wettengel JM, Lucifora J, Hansen-Palmus L, Austen K, Roca Suarez AA, Heintz S, Testoni B, Nebioglu F, Pham MT, Yang S, Zernecke A, Wohlleber D, Ringelhan M, Broxtermann M, Hartmann D, Hüser N, Mergner J, Pichlmair A, Thasler WE, Heikenwalder M, Gasteiger G, Blutke A, Walch A, Knolle PA, Bartenschlager R, Protzer U. (2023) Hepatitis B virus targets lipid transport pathways to infect hepatocytes,Cellular and Molecular Gastroenterology and Hepatology, 2023,ISSN 2352-345X,https://doi.org/10.1016/j.jcmgh.2023.03.011.

Isken O, Pham MT, Schwanke H, Schlotthauer F, Bartenschlager R, et al. (2022) Characterization of a multipurpose NS3 surface patch coordinating HCV replicase assembly and virion morphogenesis. PLOS Pathogens 18(10): e1010895. https://doi.org/10.1371/journal.ppat.1010895.

Richards CM, Jabs S, Qiao W, Varanese LD, Schweizer M, Mosen PR, Riley NM, Klüssendorf M, Zengel JR, Flynn RA, Rustagi A, Widen JC, Peters CE, Ooi YS, Xie X, Shi PY, Bartenschlager R, Puschnik AS, Bogyo M, Bertozzi CR, Blish CA, Winter D, Nagamine CM, Braulke T, Carette JE. (2022) The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection. Science. 7;378(6615):eabn5648. doi: 10.1126/science.abn5648.

Gillich N, Zhang Z, Binder M, Urban S, Bartenschlager R. (2022) Effect of variants in LGP2 on MDA5-mediated activation of interferon response and suppression of hepatitis D virus replication. J Hepatol. 21:S0168-8278(22)03113-0. doi: 10.1016/j.jhep.2022.08.041.

Zhang Z, Ni Y, Lempp FA, Walter L, Mutz P, Bartenschlager R, Urban S. (2022) Hepatitis D virus-induced interferon response and administered interferons control cell division-mediated virus spread. J Hepatol. 77(4):957-966. doi: 10.1016/j.jhep.2022.05.023.

Qu B, Nebioglu F, Leuthold MM, Ni Y, Mutz P, Beneke J, Erfle H, Vondran FWR, Bartenschlager R, Urban S. (2022) Dual role of neddylation in transcription of hepatitis B virus RNAs from cccDNA and production of viral surface antigen. JHEP Rep. 100551, ISSN 2589-5559, https://doi.org/10.1016/j.jhepr.2022.100551.

Heuss C, Rothhaar P, Burm R, Lee JY, Ralfs P, Haselmann U , Ströh LJ, Colasanti O, Tran CS, Schäfer N, Schnitzler P, Merle U, Bartenschlager R, Patel A, Graw F, Krey T, Laketa V, Meuleman P, Lohmann V. (2022) A Hepatitis C virus genotype 1b post-transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo. PLOS Pathogens 18(6): e1010472. https://doi.org/10.1371/journal.ppat.1010472.

Neufeldt CJ, Cerikan B, Cortese M, Frankish J, Lee JY, Plociennikowska A, Heigwer F, Prasad V, Joecks S, Burkart SS, Zander DY, Subramanian B, Gimi R, Padmanabhan S, Iyer R, Gendarme M, El Debs B, Halama N, Merle U, Boutros M, Binder M, Bartenschlager R. (2022) SARS-CoV-2 infection induces a pro-inflammatory cytokine response through cGAS-STING and NF-κB. Commun Biol. 12;5(1):45. doi: 10.1038/s42003-021-02983-5.

Tabata K, Prasad V, Paul D, Lee JY, Pham MT, Twu WI, Neufeldt CJ, Cortese M, Cerikan B, Stahl Y, Joecks S, Tran CS, Lüchtenborg C, V'kovski P, Hörmann K, Müller AC, Zitzmann C, Haselmann U, Beneke J, Kaderali L, Erfle H, Thiel V, Lohmann V, Superti-Furga G, Brügger B, Bartenschlager R. (2021) Convergent use of phosphatidic acid for hepatitis C virus and SARS-CoV-2 replication organelle formation. Nat Commun. 14;12(1):7276. doi: 10.1038/s41467-021-27511-1.

Twu WI, Lee JY, Kim H, Prasad V, Cerikan B, Haselmann U, Tabata K, Bartenschlager R. (2021) Contribution of autophagy machinery factors to HCV and SARS-CoV-2 replication organelle formation. Cell Rep. 23;37(8):110049. doi: 10.1016/j.celrep.2021.110049.

Stadler D, Kächele M, Jones AN, Hess J, Urban C, Schneider J, Xia Y, Oswald A, Nebioglu F, Bester R, Lasitschka F, Ringelhan M, Ko C, Chou WM, Geerlof A, van de Klundert MA, Wettengel JM, Schirmacher P, Heikenwälder M, Schreiner S, Bartenschlager R, Pichlmair A, Sattler M, Unger K, Protzer U. (2021) Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20. EMBO Rep. 4;22(6):e49568. doi: 10.15252/embr.201949568.

Kamel W, Noerenberg M, Cerikan B, Chen H, Järvelin AI, Kammoun M, Lee JY, Shuai N, Garcia-Moreno M, Andrejeva A, Deery MJ, Johnson N, Neufeldt CJ, Cortese M, Knight ML, Lilley KS, Martinez J, Davis I, Bartenschlager R, Mohammed S, Castello A. (2021) Global analysis of protein-RNA interactions in SARS-CoV-2-infected cells reveals key regulators of infection. Mol Cell. 1;81(13):2851-2867.e7. doi: 10.1016/j.molcel.2021.05.023.

Hensel N, Gu Z, Sagar, Wieland D, Jechow K, Kemming J, Llewellyn-Lacey S, Gostick E, Sogukpinar O, Emmerich F, Price DA, Bengsch B, Boettler T, Neumann-Haefelin C, Eils R, Conrad C, Bartenschlager R, Grün D, Ishaque N, Thimme R, Hofmann M. (2021) Memory-like HCV-specific CD8+ T cells retain a molecular scar after cure of chronic HCV infection. Nat Immunol. 22(2):229-239. doi: 10.1038/s41590-020-00817-w.

Cortese M, Lee JY, Cerikan B, Neufeldt CJ, Oorschot VMJ, Köhrer S, Hennies J, Schieber NL, Ronchi P, Mizzon G, Romero-Brey I, Santarella-Mellwig R, Schorb M, Boermel M, Mocaer K, Beckwith MS, Templin RM, Gross V, Pape C, Tischer C, Frankish J, Horvat NK, Laketa V, Stanifer M, Boulant S, Ruggieri A, Chatel-Chaix L, Schwab Y, Bartenschlager R. (2020) Integrative imaging reveals SARS-CoV-2-induced reshaping of subcellular morphologies. Cell Host Microbe. 17:S1931-3128(20)30620-X. doi: 10.1016/j.chom.2020.11.003.

Vassilaki N, Frakolaki E, Kalliampakou KI, Sakellariou P, Kotta-Loizou I, Bartenschlager R, Mavromara P. (2020) A novel Cis-acting RNA structural element embedded in the core coding region of the Hepatitis C virus genome directs internal translation initiation of the overlapping Core+1 ORF. Int J Mol Sci. 22;21(18):E6974. doi: 10.3390/ijms21186974.

Robichon K, Maiwald T, Schilling M, Schneider A, Willemsen J, Salopiata F, Teusel M, Kreutz C, Ehlting C, Huang J, Chakraborty S, Huang X, Damm G, Seehofer D, Lang PA, Bode JG, Binder M, Bartenschlager R, Timmer J, Klingmüller U. Identification of Interleukin1β as an Amplifier of Interferon alpha-induced Antiviral Responses. PLoS Pathog. 2020 Oct 1;16(10):e1008461. doi: 10.1371/journal.ppat.1008461. (TP09, TP10, TP11)

Seitz S, Habjanič J, Schütz AK, Bartenschlager R. 2020. The Hepatitis B Virus Envelope Proteins: Molecular Gymnastics Throughout the Viral Life Cycle. 2020. Annu Rev Virol. 2020 Jun 29. doi: 10.1146/annurev-virology-092818-015508 (TP09,TP23)

Che C Colpitts, Ridewood S., Schneiderman B., Warne J., Tabata K., Caitlin F Ng, Bartenschlager R, Selwood DL, Towers. GJ 2020. Hepatitis C virus exploits cyclophilin A to evade PKR. eLife. 2020; 9: e52237. doi: 10.7554/eLife.52237 (TP09)

Stanifer ML, Kee C, Cortese M, Zumaran CM, Triana S, Mukenhirn M, Kraeusslich HG, Alexandrov T, Bartenschlager R, Boulant S. 2020. Critical Role of Type III Interferon in Controlling SARS-CoV-2 Infection in Human Intestinal Epithelial Cells. Cell Rep. 2020 Jul 7;32(1):107863. doi: 10.1016/j.celrep.2020.107863 (TP09, TP22)

Kinast V, Plociennikowska A, Anggakusuma, Bracht T, Todt D, Brown RJ, Boldanova T, Zhang Y, Brueggemann Y, Friesland M, Engelmann M, Vieyres G, Broering R, Vondran FWR, Heim MH, Sitek B, Bartenschlager R, Pietschmann T, Steinmann E. 2020. C19orf66 is an interferon-induced inhibitor of HCV replication that restricts formation of the viral replication organelle. J Hepatol. 2020 Apr 12. pii: S0168-8278(20)30209-9. doi: 10.1016/j.jhep.2020.03.047. (TP09)

Lee JY, Cortese M, Haselmann U, Tabata K, Romero-Brey I, Funaya C, Schieber NL, Qiang Y, Barten-schlager M, Kallis S, Ritter C, Rohr K, Schwab Y, Ruggieri A, Bartenschlager R. 2019. Spatiotemporal Coupling of the Hepatitis C Virus Replication Cycle by Creating a Lipid Droplet- Proximal Membranous Replication Compartment. Cell Rep. 18;27(12):3602-3617.e5..

Zhang Z, Filzmayer C, Ni Y, Sültmann H, Mutz P, Hiet MS, Vondran FWR, Bartenschlager R, Urban S. 2018. Hepatitis D Virus replication is sensed by MDA5 and induces IFN-β/λ responses in hepatocytes. J Hepatol. 69(1):25-35.

Grünvogel O, Colasanti O, Lee JY, Klöss V, Belouzard S, Reustle A, Esser-Nobis K, Hesebeck-Brinckmann J, Mutz P, Hoffmann K, Mehrabi A, Koschny R, Vondran FWR, Gotthardt D, Schnitzler P, Neumann-Haefelin C, Thimme R, Binder M, Bartenschlager R, Dubuisson J, Dalpke AH, Lohmann V. 2018. Secretion of Hepatitis C Virus Replication Intermediates Reduces Activation of Toll Like Receptor 3 in Hepatocytes. Gastroenterology. 154(8):2237-2251.

Mutz P, Metz P, Lempp FA, Bender S, Qu B, Schöneweis K, Seitz S, Tu T, Restuccia A, Frankish J, Dächert C, Schusser B, Koschny R, Polychronidis G, Schemmer P, Hoffmann K, Baumert TF, Binder M, Urban S, Bartenschlager R. 2018. HBV Bypasses the Innate Immune Response and Does not Pro-tect HCV From Antiviral Activity of Interferon. Gastroenterology. 154(6):1791-1804.

Pollmann J, Götz JJ, Rupp D, Strauss O, Granzin M, Grünvogel O, Mutz P, Kramer C, Lasitschka F, Lohmann V, Björkström NK, Thimme R, Bartenschlager R, Cerwenka A. 2018. Hepatitis C virus-induced natural killer cell proliferation involves monocyte-derived cells and the OX40/OX40L axis. J Hepatol. 68(3):421-430.

Lauber C, Seitz S, Mattei S, Suh A, Beck J, Herstein J, Börold J, Salzburger W, Kaderali L, Briggs J, Bartenschlager R. 2017. Deciphering the origin and evolution of hepatitis B viruses by means of a family of nonenveloped fish viruses. Cell Host & Microbe, 22(3):387-399.e6.

Harak C, Meyrath M, Romero-Brey I, Schenk C, Gondeau C, Schult P, Esser-Nobis K, Saeed M, Ned-dermann P, Schnitzler P, Gotthardt D, Perez-Del-Pulgar S, Neumann-Haefelin C, Thimme R, Meuleman P, Vondran FW, Francesco R, Rice CM, Bartenschlager R, Lohmann V. 2017. Tuning a cellular lipid ki-nase activity adapts hepatitis C virus to replication in cell culture. Nature Microbiology 23;2:17012.

Pfaender S, Walter S, Grabski E, Todt D, Bruening J, Romero-Brey I, Gather T, Brown RJ, Hahn K, Puff C, Pfankuche VM, Hansmann F, Postel A, Becher P, Thiel V, Kalinke U, Wagner B, Bartenschlager R, Baumgärtner W, Feige K, Pietschmann T, Cavalleri JM, Steinmann E. 2017. Immune protection against reinfection with nonprimate hepacivirus. Proc Natl Acad Sci U S A. 114(12):E2430-E2439.

Willemsen J, Wicht O, Wolanski JC, Baur N, Bastian S, Haas DA, Matula P, Knapp B, Meyniel-Schicklin L, Wang C, Bartenschlager R, Lohmann V, Rohr K, Erfle H, Kaderali L, Marcotrigiano J, Pichlmair A, Binder M. 2017. Phosphorylation-Dependent Feedback Inhibition of RIG-I by DAPK1 Identified by Ki-nome-wide siRNA Screening. Molecular Cell. 65(3):403-415.

Senís E, Mockenhaupt S, Rupp D, Bauer T, Paramasivam N, Knapp B, Gronych J, Grosse S, Windisch MP, Schmidt F, Theis FJ, Eils R, Lichter P, Schlesner M, Bartenschlager R, Grimm D. 2016. TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus. Nucleic Acids Research 45(1):e3.

Project in detail +

Infections with the hepatitis C virus (HCV) are major risk factors for serious liver diseases and account for a total of ~71 million chronic infections worldwide. HCV has a high propensity to establish persistence by overcoming innate and adaptive immunity, but the underlying mechanisms are still incompletely understood. Importantly, HCV can be eliminated in most infected individuals by antiviral therapy and therefore, HCV has become a unique system to study antiviral immune responses and viral countermeasures not only in vitro, but also in infected patients1. During the last funding period, we have characterized the induction of the interferon (IFN) response by HCV, involved pattern recognition receptors and how the virus evades the IFN induced antiviral state. In addition, we compared HCV and HBV regarding escape from the IFN response and possible viral cross-protection in co-infected cells. Moreover, as part of a new research line and in close collaboration with Seitz, we employed a new bioinformatics approach to search for ancestral viruses related to HBV. By the discovery of a new hepadnavirus-related virus group we were able to trace the evolu-tionary origin of HBV, which provided new insights into hepadnavirus – host coevolution and a possible explanation why HBV escapes innate antiviral defense so efficiently. Building on obtained results, and with the long-term goal to decipher the molecular mechanisms by which HCV blocks innate and adaptive immune responses and how this compares to other hepatitis viruses, during the next funding period we will continue and extend these studies along three major directions. First, with respect to the induction of the IFN re-sponse, we identified MDA5 as the main pattern recognition receptor sensing HCV (and HDV) RNA (Zhang et al., 2018). This response is enhanced profoundly by LGP2 (also known as DHX58 or RIG-I-like receptor 3) in a dose-dependent manner, but suppressed when induced by RIG-I, arguing for a regulatory role of LGP2. In the next funding period we will continue to study the role of LGP2 in modulating RIG-I and MDA5 trig-gered IFN responses and determine the time- and concentration-dependency of this process (with Klingmül-ler/Timmer) as well as the possible regulation of LGP2 by cofactors and post-translational modifications (with Binder/Pichlmair). In addition, we will employ biochemical assays to probe for possible sequence or structure specificity of LGP2 binding to RNA. Studies on LGP2 will be conducted with HCV and, in collabo-ration with Urban with HDV. The second aspect of this project deals with the question how HCV, which is highly susceptible to IFN, can persist in the face of an IFN-induced antiviral state. We had earlier proposed that HCV might exploit the stochasticity of the IFN response2. Alternatively, HCV might induce a low-level IFN resistance or be less susceptible to IFN effectors once the virus has established its membranous repli-cation organelle. To address these possibilities, together with Z03N and Lohmann we will employ single cell (sc) RNAseq to differentiated and persistently HCV-infected cells in order to identify determinants account-ing for the heterogeneity of the IFN response and/or HCV permissiveness of a cell. These studies include the possible effect of the cellular context of a given cell by using spatial transcriptomics and its epigenetic status as determined by ATAC-seq. In addition, we will search for IFN-stimulated genes (ISGs) counteracting the formation of the HCV replication organelle, designated membranous web. The reason is that the web provides a shielding compartment against hostile cellular attacks and therefore, viral RNA prior to web for-mation might be very vulnerable. Consistently, we have earlier identified the ISG C19orf66 as one ISG inter-fering with web formation, thus providing a proof-of-concept. As third aspect of this project, we will expand our earlier studies on the evolutionary origin of HBV (Lauber, Seitz et al., 2017) and propose two subpro-jects related to virus – host co-evolution. The first one relates to hepaciviruses. Together with Schütz/Seitz we have already identified numerous novel members of this group and thus far established the entire poly-protein-encoding regions for more than a dozen novel hepaciviral genomes. These were identified in various species, including fishes. Together with Schütz/Seitz and Lohmann, we aim to determine the evolutionary conservation of key features of the viral replication cycle of selected newly identified viruses and how they counteract innate immune responses. We will study basic features of their viral replication cycle in homolo-gous and human cells, including the formation of a membranous replication organelle, the inhibition of key signalling molecules of the IFN pathway (e.g. MAVS cleavage), IFN sensitivity and utilization of microRNAs, especially miRNA122 that protects HCV RNA from rapid degradation by host cell nucleases. The second subproject builds on the recent report that HCV can act as helper virus of HDV and the discovery of HDV-like viruses in the absence of HBV-like helper viruses. Based on these results we will closely collaborate with Urban to dissect the cross-talk between HDV and HCV co-replication in cells and compare single vs. HCV/HDV co-infection regarding activation of the IFN system and viral countermeasures. In addition, to-gether with Schütz/Seitz we will conduct searches in unprocessed NGS data to identify novel HDV-like vi-ruses that can be found in association with HCV-like viruses. In conclusion, our studies will provide new insights into fundamental aspects of the IFN system that reach far beyond HCV and shed light on HCV co-evolution with its host and possibly also other viruses.

Milestones of the first funding period:
- deciphering how and where within the cell Mda5 is activated by HCV RNA, what the ligand of Mda5 is, how activation is mediated despite MAVS cleavage and how NS5A counteracts this IFN induction pathway;
- IFN response activation dynamics and the role of IFN-λ SNPs by using single cell approaches and, on the long run, mathematical modelling at the level of the activation of the effector phase (i.e. ISG induction);
- NOS1 and the mechanism how it contributes to a vigorous IFN response that suppresses the replication of HCV and possibly other hepatitis viruses at the level of the IFN-induced antiviral programme
- Characterization of the impact of long-term IFN-λ exposure on CD8+ T cells and activity of other immune cells at the level of innate/adaptive cross-talk