TP11: Effects of persistent antiviral signalling on tissue homeostasis and antiviral immunity

The cell-intrinsic antiviral system is highly potent, but imposes a massive perturbation onto cellular homeostasis, which is why it evolved to be strictly acute and transient. We investigate the consequences of prolonged stimulation of this defence program, as it occurs in the course of persistent virus infection, such as HCV. We examine both, the virus infected cell itself (counter regulation, refractoriness), as well as the effects of the cytokines, which it continuously secretes, onto non-infected neighbouring cells and onto cells of the immune system. We will address how this inflammatory milieu might contribute to the establishment of persistence of the viral infection.

Dr. Marco Binder

Prof. Dr. Dr. Andreas Pichlmair

Schulien I, Kemming J, Oberhardt V, Wild K, Seidel LM, Killmer S, Sagar, Daul F, Salvat Lago M, Decker A, Luxenburger H, Binder B, Bettinger D, Sogukpinar O, Rieg S, Panning M, Huzly D, Schwemmle M, Kochs G, Waller CF, Nieters A, Duerschmied D, Emmerich F, Mei HE, Schulz AR, Llewellyn-Lacey S, Price DA, Boettler T, Bengsch B, Thimme R, Hofmann M, Neumann-Haefelin C. Characterization of pre-existing and induced SARS-CoV-2-specific CD8+ T cells. Nat Med. 2020 Nov 12. doi: 10.1038/s41591-020-01143-2. (TP01, TP02, TP11, TP20, TP21)

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)

Urban C, Welsch H, Heine K, Wüst S, Haas DA, Dächert C, Pandey A, Pichlmair A, Binder M. Persistent Innate Immune Stimulation Results in IRF3-Mediated but Caspase-Independent Cytostasis. Viruses. 2020 Jun 11;12(6):635. doi: 10.3390/v12060635. (TP11)

Lampl S, Janas MK, Donakonda S, Brugger M, Lohr K, Schneider A, Manske K, Sperl LE, Kläger S, Küster B, Wettmarshausen J, Müller C, Laschinger M, Hartmann D, Hüser N, Perocchi F, Schmitt-Kopplin P, Hagn F, Zender L, Hornung V, Borner C, Pichlmair A, Kashkar H, Klingenspor M, Prinz M, Schreiner S, Conrad M, Jost PJ, Zischka H, Steiger K, Krönke M, Zehn D, Protzer U, Heikenwälder M, Knolle PA, Wohlleber D. Reduced mitochondrial resilience enables non-canonical induction of apoptosis after TNF receptor signaling in virus-infected hepatocytes. J Hepatol. 2020 Jun 26:S0168-8278(20)30398-6. doi: 10.1016/j.jhep.2020.06.026. (TP05, TP06, TP10, TP11, TP13, TP16, TP18)

Namineni S, O'Connor T, Faure-Dupuy S, Johansen P, Riedl T, Liu K, Xu H, Singh I, Shinde P, Li F, Pandyra A, Sharma P, Ringelhan M, Muschaweckh A, Borst K, Blank P, Lampl S, Durantel D, Farhat R, Weber A, Lenggenhager D, Kündig TM, Staeheli P, Protzer U, Wohlleber D, Holzmann B, Binder M, Breuhahn K, Assmus LM, Nattermann J, Abdullah Z, Rolland M, Dejardin E, Lang PA, Lang KS, Karin M, Lucifora J, Kalinke U, Knolle PA, Heikenwalder M. 2020. A dual role for hepatocyte-intrinsic canonical NF-κB signaling in virus control. J Hepatol. 72(5):960-975. (TP05, TP06, TP11, TP13, TP14)

Binder B, Thimme R. CD4+ T cell responses in human viral infection: lessons from hepatitis C. J Clin Invest. 2020 Feb 3;130(2):595-597. doi: 10.1172/JCI133222. (TP01, TP11)

A. Cadena C, Ahmad S, Xavier A, Willemsen J, Park S, Park JW, Oh SW, Fujita T, Hou F, Binder M, Hur S. 2019. Ubiquitin-Dependent and -Independent Roles of E3 Ligase RIPLET in Innate Immunity. Cell. 177(5):1187–1200

B. Hubel P, Urban C, Bergant V, Schneider WM, Knauer B, Stukalov A, Scaturro P, Mann A, Brunotte L, Hoffmann HH, Scho-ggins JW, Schwemmle M, Mann M, Rice CM, Pichlmair, A. 2019. A protein interaction network of interferon stimulated genes extends the innate immune system landscape. Nat Immunol. 20(4):493–502

C. Scaturro P, Stukalov A, Haas DA, Cortese M, Draganova K, Płaszczyca A, Bartenschlager R, Götz M, Pichlmair A. 2018. An orthogonal proteomic survey uncovers novel Zika virus host factors. Nature 561(7722):253-257.

D. Grünvogel O, Colasanti O, Lee J-Y, 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 Re-duces Activation of Toll-Like Receptor 3 in Hepatocytes. Gastroenterology 154(8):2237–2251.e16.

E. 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 Protect HCV From Antiviral Activity of Interferon. Gastroenterology 154(6):1791–1804.e22.

F. Haas DA, Meiler A, Geiger K, Vogt C, Preuss E, Kochs G, Pichlmair A. 2018. Viral targeting of TFIIB impairs de novo poly-merase II recruitment and affects antiviral immunity. PLoS Pathogens 14(4):e1006980.

G. Holze C, Michaudel C, Mackowiak C, Haas DA, Benda C, Hubel P, Pennemann FL, Schnepf D, Wettmarshausen J, Braun M, Leung DW, Amarasinghe GK, Perocchi F, Staeheli P, Ryffel B, Pichlmair A. 2018. Oxeiptosis, a ROS-induced caspase-independent apoptosis-like cell-death pathway. Nat Immunol. 19(2):130-140.

H. Goonawardane N, Gebhardt A, Bartlett C, Pichlmair A, Harris M. 2017. Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions. J Virol 91(17):e00805-17

I. Willemsen J, Wicht O, Wolanski JC, Baur N, Bastian S, Haas DA, Matula P, Knapp B, Meyniel-Schicklin L, Wang C, Barten-schlager 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 Kinome-wide siRNA Screening. Mol Cell. 65(3):403-415.e8.

J. Grünvogel O, Esser-Nobis K, Windisch MP, Frese M, Trippler M, Bartenschlager R, Lohmann V, Binder M. 2016. Type I and type II interferon responses in two human liver cell lines (Huh-7 and HuH6). Genomics Data 7:166-70

Project in detail +

Viral infections trigger cell-intrinsic innate antiviral immune responses that are key to controlling the infec-tion. Beside direct antiviral effects, they contribute to the induction of adaptive immunity, and are involved in both, tissue damage and repair, as well as modulation of cellular functions such as metabolic activity. Although most of these effects are well studied for acute viruses, significantly less is known about infec-tions with persistent viruses such as Hepatitis C virus (HCV), a small RNA virus. All along its persistence HCV has to continuously replicate its genetic material, leading to ongoing production of RNA structures that are recognized by host cells as non-self and thereby trigger an antiviral immune response. In the first fund-ing period, we investigated how such continuous antiviral signalling affects host cell function and homeo-stasis. We set up an experimental system closely mimicking the physiological low but long-term stimulation of the cellular RNA-sensor RIG-I, however without uncontrolled interference by virus encoded antagonists. Unexpectedly, we realized that even this gentle stimulation of the RIG-I system led to significantly reduced cell growth, while stronger stimulation even triggered cell death. We propose that this represents a defence strategy of an infected tissue to limit the overall presence of viral RNA (and other viral structures). Moreo-ver, it corroborates the notion that HCV requires constant re-infection of uninfected liver cells in order to successfully persist in a patient. Interestingly, we further found that long term exposure of cells to type I interferon (IFN)– the major cytokine produced by infected cells– does not significantly affect cell growth and induces an expression pattern of IFN stimulated genes (ISGs) that is different from the canonical re-sponse pattern seen after acute, transient cytokine exposure. In the course of this project we also identified a negative feedback loop that limits the activation of RIG-I and, hence, the secretion of IFN. These two un-expected facets of the innate immune response indicate more complex regulations on inter- and intracellular level than we previously anticipated.
In the next funding period, we will further exploit the established methods to analyse the effects of pro-longed stimulation of antiviral responses in the context of a mixed infected-uninfected population of cells. An important aspect will be to understand the regulatory basis responsible for the observed changes in the pattern of induced ISGs over time. We will study whether regulatory events occur on a transcriptional, trans-lational or post-translational level. This will comprise a multi-omics approach covering transcriptomics as well as proteomics, but also epigenetics together with Z03N. For proteomics, we have successfully estab-lished a SILAC-based pulse/chase labelling approach in mass-spectrometry (MS) allowing us to determine half-lifes and synthesis rates of individual proteins. Identification of candidate proteins that are temporally regulated over the course of an (experimental) persistent infection will guide us towards underlying princi-ples of protein regulation as well as potential functional consequences of this dynamically regulated re-sponse. We are going to separately assess the effects, both, directly in “infected” cells as well as in unin-fected bystander cells, also, amongst other methods, by single cell RNA sequencing together with Z03N. In this context, we will further investigate the importance of cell-cell-communication using the recently devel-oped method of AHA-click-labelling and MS of secreted proteins (secretome). The functional impact of prolonged exposure to these secreted factors on bystander cells as well as infiltrating and tissue resident immune cells (together with Thimme, Böttler and Cerwenka) will be investigated, with a particular focus on its contributions to creating a milieu permissive for HCV persistence.
In order to validate and corroborate these processes in relevant hepatic models, we will extend our toolbox of cell culture systems to physiologically more authentic models: stem-cell derived hepatocyte-like cells (HLC) will be explored together with Dao Thi/Boulant; spheroid culture of liver cell lines or primary cells will be developed further with Heikenwälder; primary human hepatocytes (PHH) will be used in collaboration with Bartenschlager, Klingmüller/Timmer and Protzer. We are furthermore collaborating with Wohlleber and Knolle to assess the proteomic profile induced in the livers of mice infected with an Adeno-associated virus ex-pressing IFN. Preliminary experiments indicate that proteins are regulated in an unappreciated way in this model, likely leading to an expansion of our knowledge of the innate immune response in the liver.

Milestones of the first fnding period:
- Cell culture models of persistent virus infection and continuous innate immune stimulation
- Functional characterization of innate immune responses in cells with continuous stimulation of innate immune pathways
- Assessing the global effects of continuous innate immune stimulation on the cellular signalling landscape
- Bioinformatic analysis of proteomics data, and validation and functional characterization of identified processes