TP17: Contribution of innate immune responses to persistence versus clearance of hepatitis C and hepatitis A virus infections

We study the role of ESCRT in dsRNA secretion and unravel the cellular determinants of HCV recognition by TLR3. For HAV, we aim to identify protease-independent mechanism(s) blunting the cell intrinsic innate immunity in vivo and in vitro, with a focus on the viral protein 2B. Finally, we will study the contribution of viral replication fitness on the outcome of HCV and HAV infections. Our results will ultimately disentangle the complex relationship between viral replication, immune induction and counteraction goverining persistence versus clearance of hepatotropic viruses.

APL Prof. Dr. Volker Lohmann

. Grunvogel O, Colasanti O, Lee JY, Kloss 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, 2237-2251 e2216. doi:10.1053/j.gastro.2018.03.020.

Colasanti O, Burm R, Huang HE, Riedl T, Traut J, Gillich N, Li TF, Corneillie L, Faure-Dupuy S, Grunvogel O, Heide D, Lee JY, Tran CS, Merle U, Chironna M, Vondran FWR, Esser-Nobis K, Binder M, Bartenschlager R, Heikenwalder 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. doi:10.1016/j.jhep.2023.04.023.

Heuss C*, Rothhaar P*, Burm R, Lee JY, Ralfs P, Haselmann U, Stroh LJ, Colasanti O, Tran CS, Schafer N, Schnitzler P, Merle U, Bartenschlager R, Patel AH, 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 Pathog. 18, e1010472. doi:10.1371/journal.ppat.1010472.

Jo J, Aichele U, Kersting N, Klein R, Aichele P, Bisse E, Sewell AK, Blum HE, Bartenschlager R, Lohmann V*, Thimme R.* (2009) Analysis of CD8+ T-cell-mediated inhibition of hepatitis C virus replication using a novel immunological model. Gastroenterology. 136, 1391-1401. doi: 10.1053/j.gastro.2008.12.034.

Pollmann J, Gotz JJ, Rupp D, Strauss O, Granzin M, Grunvogel O, Mutz P, Kramer C, Lasitschka F, Lohmann V, Bjorkstrom 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, 421-430. doi:10.1016/j.jhep.2017.10.021.

Kloss V, Grunvogel O, Wabnitz G, Eigenbrod T, Ehrhardt S, Lasitschka F, Lohmann V, Dalpke AH. (2017) Interaction and Mutual Activation of Different Innate Immune Cells Is Necessary to Kill and Clear Hepatitis C Virus-Infected Cells. Front Immunol. 8, 1238. doi:10.3389/fimmu.2017.01238.

14. Chen P*, Wojdyla JA*, Colasanti O*, Li Z, Qin B, Wang M, Lohmann V*, Cui S.* (2022) Biochemical and structural characterization of hepatitis A virus 2C reveals an unusual ribonuclease activity on single-stranded RNA. Nucleic Acids Res. 50, 9470-9489. doi:10.1093/nar/gkac671.

Harak C, Meyrath M, Romero-Brey I, Schenk C, Gondeau C, Schult P, Esser-Nobis K, Saeed M, Neddermann P, Schnitzler P, Gotthardt D, Perez-Del-Pulgar S, Neumann-Haefelin C, Thimme R, Meuleman P, Vondran FW, De Francesco R, Rice CM, Bartenschlager R, Lohmann V. (2016) Tuning a cellular lipid kinase activity adapts hepatitis C virus to replication in cell culture. Nat Microbiol. 2, 16247. doi:10.1038/nmicrobiol.2016.247.

20. Costa R , Todt D, Zapatero-Belinchon F, Schenk C, Anastasiou OE, Walker A, Hertel B, Timmer L, Bojkova D, Ruckert M, Sarrazin C, Timm J, Lohmann V, Manns MP, Steinmann E, von Hahn T, Ciesek S. (2019) SEC14L2, a lipid-binding protein, regulates HCV replication in culture with inter- and intra-genotype variations. J Hepatol. 70, 603-614. doi:10.1016/j.jhep.2018.11.012.

Chen Q, Coto-Llerena M, Suslov A, Teixeira RD, Fofana I, Nuciforo S, Hofmann M, Thimme R, Hensel N, Lohmann V, Ng CKY, Rosenberger G, Wieland S, Heim MH. (2021) Interferon lambda 4 impairs hepatitis C viral antigen presentation and attenuates T cell responses. Nat Commun. 12, 4882. doi:10.1038/s41467-021-25218-x.

Project in detail +

The central aim of this project is to gain a mechanistic understanding of how induction and counteraction of innate immunity in infected cells contributes to the infection outcome of hepatitis C virus (HCV; primarily persistent infection) and hepatitis A virus (HAV; exclusively acute infection). With respect to HCV, in the first funding period, we have identified TLR3 as the main source of cell intrinsic innate immune responses induced by HCV and secretion of double stranded RNA (dsRNA) as a novel escape mechanism. In the current funding period, we confirmed the induction of ISGs in an in vivo model and substantiated the role of TLR3 as a main sensor. At the molecular level, we identified several components of ESCRT involved in dsRNA secretion, but not in TLR3 recognition, suggesting that both pathways are at least partially independent. Therefore, we performed an unbiased Crispr/Cas9 screen using authentic induction of TLR3 by HCV dsRNA and identified several interesting candidate host factors contributing to TLR3 induction by HCV. Regarding HAV, during the ongoing funding period we unexpectedly found a moderate but robust induction of cell intrinsic innate immune response via the PRRs MDA5-LGP2, which was not impacted by previously described proteolytic cleavage of adaptor proteins. Finally, we recently identified a region in HCV NS5A governing viral RNA replication efficiency in vivo, allowing to dramatically change viral replication fitness with a few mutations. Importantly, highly replicating variants are primarily found in immunocompromised hosts, e.g. liver transplant patients, suggesting that immune pressure selects for low HCV replication, thereby favouring persistence. Since HAV generally replicates at higher levels than HCV in the liver, e.g. of human liver chimeric mice (2), we hypothesize that viral replication fitness might have a decisive role in persistence versus clearance.
We have 3 aims, based on our recent novel findings. In aim 1, we will continue to study the role of ESCRT in dsRNA secretion and unravel the cellular determinants of HCV recognition by TLR3. We will focus on validation of host factors identified in the Crispr/Cas9 screen and characterize their mechanistic contribution to the TLR3 response against HCV (with Binder/Beisel, Bartenschlager). Aim 2 is dedicated to the identification of protease-independent mechanism(s) developed by HAV to blunt the cell intrinsic innate immunity, with viral protein 2B as the main candidate. We will further include unbiased screening approaches selecting for HAV variants escaping innate immunity (with Pichlmair, Binder/Beisel). This will be complemented with analyses of the spatiotemporal induction of interferon responses by HAV and viral counteraction by using physiological cell culture models and in vivo systems (with Wohlleber, Dao Thi, Z02, Z03). Aim 3 focuses on the contribution of viral replication lev-els on infection outcomes for HCV and HAV. Based on permissive cell culture models recently optimized for authentic interferon responses, we will build on ex vivo co-culture systems with virus specific T-cells and PMBCs, allowing a comprehensive analysis of the impact of replication fitness on innate and adaptive immune responses of HCV and HAV infections (with Binder/Beisel, Protzer/Kosinska, Cerwenka, Thimme, Hofmann, Böttler). This will ultimately disentangle the complex relationship between viral replication, immune induction and viral counteraction and the role of these factors for persistence and clearance of hepatotropic viruses.