Mechanisms of immune evasion by hepatitis C virus and their role in establishment of viral persistence
Final Report Abstract
An estimated 170 million people worldwide are chronically infected with hepatitis C virus (HCV), leading in many cases to severe liver damage. The reasons for the high rate of persistence are not fully understood yet, but it is clear that the failure of innate and adaptive immune responses to eliminate the virus is of particular importance. Although not originally planned, we decided early after the onset of funding, that Hepatitis A virus (HAV) would perfectly complement our studies due to the similar replication cycle, but lack of persistent outcomes. We therefore aimed at generating a homogenous cell culture model for both viruses and found that replication indeed was equally efficient in Huh-7 cells. Using this model, we found that HCV and HAV replicases are similarly sensitive to type I, II and III IFNs. We further identified the Dead-box helicase DDX60L as an important contributor to the IFN response against HCV, but not HAV. Our results pointed to DDX60L as a direct effector protein mediating innate immune responses. Therefore, the different impact on HAV and HCV might be linked to differences in the configuration of the replication organelles, e.g. due to accessibility of viral RNA. While HCV and HAV have not developed countermeasures to interfere with the inhibitory action of IFNs, both viruses have established the same means to counteract induction of the IFN responses by cleaving the adaptor proteins MAVS and TRIF. Indeed, most of the HCV response mediated via MAVS is blunted by proteolytic cleavage. In contrast, expression of TLR3 mounted a strong ISG response against HCV, partially inhibiting viral replication. Interestingly, cell lines harboring persistent replicons of HAV and HCV and expressing TLR3 were still fully responsive to exogenous polyI/C, suggesting that TRIF cleavage was not efficient in blocking TLR3 response for both viruses. However, HAV replication did not stimulate TLR3 at all in contrast to HCV, despite similar amounts of intracellular viral RNA, suggesting that HCV replication intermediates reach the endosomal compartment to stimulate TLR3, whereas this is not the case for HAV. Taken together, our data suggested that a main difference between HCV and HAV in induction of innate immune responses is the efficient stimulation of TLR3 responses by HCV and not by HAV. Our data indicate that this difference is not due to variations in efficient TRIF cleavage, but rather due to the lack of induction by HAV, which is most likely based on differences in the architecture of the viral replication sites and the turnover and intracellular fate/ transport of viral replication intermediates. To assess differences in immune induction in more complex environments we furthermore established co-culture models to study the recognition of HCV by pDCs and Monocytes, allowing side-by-side comparisons of HAV and HCV. In addition, we developed an antigen presentation model for the quantitative assessment of the impact of T-cell recognition on viral replication This model has been further extended to other cells lines (Huh6, HepG2), now also allowing the quantitative assessment of antigen presentation due to authentic HLA-A2 expression. The results of these studies contributed to a better understanding of the strategies used by HCV and HAV to overcome innate immune responses. In particularly, widening the scope towards HAV and the observed differences in TLR3 response provided the essential basis for the successful application of this project within the TRR "Determinants and dynamics of elimination versus persistence of hepatitis virus infection".
Publications
- 2010. Protective effect of human leukocyte antigen B27 in hepatitis C virus infection requires the presence of a genotype-specific immunodominant CD8+ T-cell epitope. Hepatology 51:54-62
Neumann-Haefelin, C., J. Timm, J. Schmidt, N. Kersting, K. Fitzmaurice, C. Oniangue-Ndza, M. N. Kemper, I. Humphreys, S. McKiernan, D. Kelleher, V. Lohmann, P. Bowness, D. Huzly, H. R. Rosen, A. Y. Kim, G. M. Lauer, T. M. Allen, E. Barnes, M. Roggendorf, H. E. Blum, and R. Thimme
(See online at https://doi.org/10.1002/hep.23275) - 2011. Human leukocyte antigen B27 selects for rare escape mutations that significantly impair hepatitis C virus replication and require compensatory mutations. Hepatology 54:1157-1166
Neumann-Haefelin, C., C. Oniangue-Ndza, T. Kuntzen, J. Schmidt, K. Nitschke, J. Sidney, C. Caillet- Saguy, M. Binder, N. Kersting, M. W. Kemper, K. A. Power, S. Ingber, L. L. Reyor, K. Hills-Evans, A.Y. Kim, G. M. Lauer, V. Lohmann, A. Sette, M. R. Henn, S. Bressanelli, R. Thimme, and T. M. Allen
(See online at https://doi.org/10.1002/hep.24541) - 2011. Immunodominance of HLA-A2-restricted hepatitis C virus-specific CD8+ T cell responses is linked to naive-precursor frequency. J. Virol. 85:5232-5236
Schmidt, J., C. Neumann-Haefelin, T. Altay, E. Gostick, D. A. Price, V. Lohmann, H. E. Blum, and R. Thimme
(See online at https://doi.org/10.1128/JVI.00093-11) - 2011. Molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-I (RIG-I). J. Biol. Chem. 286:27278-27287
Binder, M., F. Eberle, S. Seitz, N. Mucke, C. M. Huber, N. Kiani, L. Kaderali, V. Lohmann, A. Dalpke, and R. Bartenschlager
(See online at https://doi.org/10.1074/jbc.M111.256974) - 2012. Low perforin expression of early differentiated HCV-specific CD8+ T cells limits their hepatotoxic potential J. Hepatology 57(1):9-16
Jo, J, B. Bengsch; B. Seigel, S. J. Rau, J. Schmidt, E. Bisse, P. Aichele, U. Aichele, L. Joeckel, C. Royer, K. Sa Ferreira, C. Borner, T. F. Baumert, H. E. Blum, V. Lohmann, R. Fischer, R. Thimme
(See online at https://doi.org/10.1016/j.jhep.2012.02.030) - 2013. Analysis of hepatitis C virus resistance to silibinin in vitro and in vivo points to a novel mechanism involving nonstructural protein 4B. Hepatology. 57:953-63
Esser-Nobis K, Romero-Brey I, Ganten TM, Gouttenoire J, Harak C, Klein R, Schemmer P, Binder M, Schnitzler P, Moradpour D, Bartenschlager R, Polyak SJ, Stremmel W, Penin F, Eisenbach C, Lohmann V
(See online at https://doi.org/10.1002/hep.26260) - 2014. Monocytes activate natural killer cells via inflammasome-induced interleukin 18 in response to hepatitis C virus replication. Gastroenterology, Vol. 147. 2014, Issue 1, pp. 209-220.e3.
Serti ., Werner J.M., Chattergoon M., Cox A.L., Lohmann V., Rehermann B.
(See online at https://doi.org/10.1053/j.gastro.2014.03.046) - 2015. Control of temporal activation of hepati-tis C virus-induced interferon response by domain 2 of nonstructural protein 5A. 1. J Hepatol. 63(4):829-37
Hiet MS, Bauhofer O, Zayas M, Roth H, Tanaka Y, Schirmacher P, Willemsen J, Grünvogel O, Bender S, Binder M, Lohmann V, Lotteau V, Ruggieri A, Bartenschlager R
(See online at https://doi.org/10.1016/j.jhep.2015.04.015) - 2015. Dead box helicase 60-like (DDX60L) is an interferon stimulated gene restricting hepatitis C virus replication in cell culture. J Virol 89:10548-68
Grünvogel O, Esser-Nobis K, Reustle A, Schult P, Müller B, Metz P, Trippler M, Windisch MP, Frese M, Binder M, Fackler O, Bartenschlager R, Ruggieri A, Lohmann V
(See online at https://doi.org/10.1128/JVI.01297-15) - 2015. Novel perspectives for hepatitis A virus therapy revealed by comparative analysis of hepatitis C virus and hepatitis A virus RNA replication. Hepatology. 62(2):397-408
Esser-Nobis K, Harak C, Schult P, Kusov Y, Lohmann V
(See online at https://doi.org/10.1002/hep.27847) - 2015. Type I and type II interferon responses in two human liver cell lines (Huh-7 and HuH6). Genom Data. 2015 Dec 28;7:166-70
Grünvogel O, Esser-Nobis K, Windisch MP, Frese M, Trippler M, Bartenschlager R, Lohmann V, Binder M
(See online at https://doi.org/10.1016/j.gdata.2015.12.017) - 2016. The cyclophilin-inhibitor alisporivir stimulates antigen presentation thereby promoting antigen-specific CD8(+) T cell activation. J Hepatol. 64(6):1305-14
Esser-Nobis K, Schmidt J, Nitschke K, Neumann-Haefelin C, Thimme R, Lohmann V
(See online at https://doi.org/10.1016/j.jhep.2016.02.027) - 2018. Secretion of Hepatitis C Virus Replication Intermediates Reduces Activation of Toll Like Receptor 3 in Hepatocytes. Gastroenterology. 2018 Mar 10. pii: S0016-5085(18)30309-3
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
(See online at https://doi.org/10.1053/j.gastro.2018.03.020)
