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

TP17 studies the determinants of clearance and persistence of hepatitis A virus (HAV) and hepatitis C virus (HCV) infections, focusing on toll-like receptor 3 (TLR3). We have unravelled a novel TLR3 escape mechanism employed by HCV, studied induction and counteraction of the TLR3 pathway by HCV and HAV and identified novel cellular factors involved in TLR3 response. In the next funding period we want to study the transport mechanisms of HCV and HAV double stranded RNA activating TLR3 or avoiding it. The project further aims to define TLR3 specific response patterns and interactomes to understand the contribution of this pathway to persistence of hepatotropic viruses.

APL Prof. Dr. Volker Lohmann

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.

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. 10:gkac671. doi: 10.1093/nar/gkac671.

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.

Silva DN, Chrobok M, Rovesti G, Healy K, Wagner AK, Maravelia P, Gatto F, Mazza M, Mazzotti L, Lohmann V, Sällberg Chen M, Sällberg M, Buggert M, Pasetto A. (2022) Process Development for Adoptive Cell Therapy in Academia: A Pipeline for Clinical-Scale Manufacturing of Multiple TCR-T Cell Products. Front Immunol. 13:896242. doi: 10.3389/fimmu.2022.896242.

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.

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;12(1):4882. doi: 10.1038/s41467-021-25218-x.

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.e16.

Lohmann V. 2019. Hepatitis C virus cell culture models: an encomium on basic research paving the road to therapy development. Med Microbiol Immunol. 208(1):3-24. Review

Costa R, Todt D, Zapatero-Belinchón 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(4):603-614.

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.

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, Francesco R, Rice CM, Bartenschlager R, Lohmann V. 2017. Tuning a cellu-lar lipid kinase activity adapts hepatitis C virus to replication in cell culture. Nat Microbiol. 2:17012.

Klöss V, Grünvogel 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.

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. Mol Cell. 65(3):403-415.e8.

Esser-Nobis K, Harak C, Schult P, Kusov Y, Lohmann V. 2015. Novel perspectives for hepatitis A virus therapy revealed by comparative analysis of hepatitis C virus and hepatitis A virus RNA rep-lication. Hepatology 62(2):397-408

Serti E, Werner JM, Chattergoon M, Cox AL, Lohmann V, Rehermann B. 2014. Monocytes activate natural killer cells via inflammasome-induced interleukin 18 in response to hepatitis C virus replication. Gastroenterology 147(1): 209-220.

Jo J, Aichele U, Kersting N, Klein R, Aichele P, Bisse E, Sewell AK, Blum HE, Bartenschlager R, Loh-mann V*, Thimme R*. 2009. Analysis of CD8+ T-cell-mediated inhibition of HCV replication using a novel immunological model. Gastroenterology 136(4):1391-401.*Co-corresponding authors.

Project in detail +

Hepatitis C virus (HCV, family Flaviviridae) and hepatitis A virus (HAV, family Picornaviridae) are both posi-tive strand RNA viruses with very similar mechanisms of RNA replication. However, HAV infections are al-ways cleared whereas HCV establishes persistence in ~80% of infected individuals. Clearance correlates with low levels of innate immune responses in the liver in case of HAV, whereas persistence in case of HCV is associated with a long-lasting induction of a variety of interferon-induced genes (ISGs), which mainly originate from infected cells. In the previous funding period we have focused on the contribution of Toll-like receptor 3 (TLR3) to the innate immune response against HAV and HCV. We found that both viruses are not capable of efficiently blocking the TLR3 pathway by proteolytic cleavage of TRIF, opposite to reports in literature. Instead, we identified a novel immune escape mechanism of HCV, which stimulates TLR3, but dampens recognition by exosomal secretion of double stranded replication intermediates (dsRNA) (cooper-ation with Thimme, Neumann-Haefelin, Bartenschlager, Binder). In contrast, HAV barely induces TLR3, likely because viral dsRNA does not reach the endosomal compartment. In addition, we have studied several general aspects of the TLR3 response in hepatocytes with Dao Thi/Boulant and Seitz/Schütz by (i) function-ally analysing single nucleotide polymorphisms reported in humans, (ii) performing a transcriptomic analysis of TLR3-induced genes compared to genes induced by cytosolic pattern recognition receptors, thereby validating hepatoma cells as an appropriate model and (iii) conducting a CRISPR/Cas9 knockout screen to identify novel genes involved in regulating the TLR3 pathway with Z2, revealing a set of validated candi-dates.
In the proposed next funding period we will focus on four major aims. First, we will clarify the cellular path-ways mediating the delivery of HCV replication intermediates to the endosomal compartment in cooperation with Bartenschlager. Second, we will address the fate of HAV dsRNA, which is synthesized at membranous vesicular structures similar to HCV, but seems to be deposited differently. Third, we aim to understand the specific function of the factors identified in our screen in regulation of the TLR3 response. Therefore, we will perform an interactome analysis with Binder/Pichlmair and validate the identified candidates. Finally, we aim to define a TLR3 specific gene signature in collaboration with Binder/Pichlmair, Seitz/Schütz and Z03N to understand the induction of innate immune responses by HCV in primary human hepatocytes, hepatocyte like cells (with Dao Thi/Boulant) and in vivo using well characterized patient specimen obtained from Neu-mann-Haefelin, Hofmann, Thimme and Z2.

Milestones of the first funding period:
- Clarification of the composition of viral replication complexes and the fate of viral replication intermediates, mainly dsRNA using proteomics, biochemical methods and microscopy
- Quantification of the efficiency of HAV and HCV proteases to cleave TRIF, MAVS and NEMO building on our recently established model allowing comparative analysis of both viruses