I. Understanding the dynamics and regulation of cell-intrinsic antiviral signaling

Panel (A) shows examples of temporally highly resolved measurements of activation of two different stages of antiviral signaling. Similarly, we characterized many further steps of RIG-I and interferon signaling in order to obtain a full understanding of the kinetics of the antiviral response. (B) schematically shows the regulator role of DAPK1 in the termination of RIG-I antiviral signaling in a negative feedback loop.
© dkfz.de

In order to sense a viral infection and trigger an appropriate defensive response, the cell has developed a set of sensor molecules. One of the most prominent sensors is retinoic acid inducible gene I (RIG-I, aka DDX58). Upon recognition of viral RNA, RIG-I triggers a signaling cascade through the crucial innate adapter molecule MAVS, eventually leading to activation of the latent transcription factors IRF-3 and NFkB. These transcription factors in turn drive the production and secretion of type I and III interferons, which are highly potent mediators of the antiviral response, driving the infected as well as surrounding cells into an alert antiviral state, which, in most cases, is sufficient in restricting the replication and spread of the virus.

We are very much interested in understanding the dynamics of this vital signaling system and how it is modulated by cellular regulators as well as virus encoded antagonists. For the body, on the one hand, it is extremely important to tightly regulate these potent responses as aberrant activation or prolonged / overshooting activity of these systems are known to cause severe damage to the organism. On the other hand, upon recognition of only a tiny amount of non-self, virus-derived molecules, the defense system has fire up instantly in order to stop the fast exponential replication of the pathogen. We have established different technological approaches to study the dynamics of these processes (figure 1A) and to identify and characterize molecular regulators. 

In one branch of this research line, we have performed a high-throughput screen for cellular kinases regulating the RIG-I signaling pathway. We described one kinase, DAKP1, to be an induced inhibitor of the sensor molecular RIG-I, contributing to the proper termination of the antiviral defense program (Willemsen 2017, Figure 1B). Further >20 kinases were identified in this study and await in-depth characterization.

We have further studied the role of ubiquitin-ligases TRIM25 and RIPLET in the activation of the RIG-I / IRF3 signaling pathway and found a surprisingly impactful role for RIPLET in to collaborative studies (Cadena 2019; Hayman 2019). Together with the group of Ursula Klingmüller here at DKFZ, we also studied complex regulatory events in the downstream interferon signaling system (Robichon 2020; Kok 2020).

  • Willemsen, J., Wicht, O., Wolanski, J.C., Baur, N., Bastian, S., Haas, D.A., Matula, P., Knapp, B., Meyniel-Schicklin, L., Wang, C., Bartenschlager, R., Lohmann, V., Rohr, K., Erfle, H., Kaderali, L., Marcotrigiano, J., Pichlmair, A. and Binder, M. (2017) Phosphorylation-Dependent Feedback Inhibition of RIG-I by DAPK1 Identified by Kinome-wide siRNA Screening. Molecular Cell 65 (3):403–415
  • 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.e16
  • Hayman TJ, Hsu AC, Kolesnik TB, Dagley LF, Willemsen J, Tate MD, Baker PJ, Kershaw NJ, Kedzierski L, Webb AI, Wark PA, Kedzierska K, Masters SL, Belz GT, Binder M, Hansbro PM, Nicola NA, Nicholson SE (2019) RIPLET, and not TRIM25, is required for endogenous RIG-I-dependent antiviral responses. Immunol Cell Biol 97(9):840-852
  • 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 (2020) Identification of Interleukin1β as an Amplifier of Interferon alpha-induced Antiviral Responses. PLoS Pathog, 16(10):e1008461
  • Kok F, Rosenblatt M, Teusel M, Nizharadze T, Gonçalves Magalhães V, Dächert C, Maiwald T, Vlasov A, Wäsch M, Tyufekchieva S, Hoffmann K, Damm G, Seehofer D, Boettler T, Binder M, Timmer J, Schilling M, Klingmüller U (2020) Disentangling molecular mechanisms regulating sensitization of interferon alpha signal transduction. Mol Syst Biol, 16(7):e8955

II. Involvement of cell-intrinsic antiviral signaling in tumor development and therapy

Treatment of A549 lung adenocarcinoma cells with doxorubicin (a chemotherapeutic) induces cell death. Remarkably, this cell death is largely dependent on the presence of intact RIG-I signaling, whereas signaling downstream of the interferon receptor appeared dispensable.
© dkfz.de

Cell-intrinsic antiviral immunity is pivotal to combat virus infections. Sensing of conserved pathogen-associated molecular patterns (PAMPS) by cellular receptors such as RIG-I results in the production and release of interferons and massive expression of interferon-stimulated genes (ISGs), conferring an antiviral state. Notably, this response not only comprises antiviral effectors but also displays cytostatic and anti-tumorigenic properties, inducing apoptosis, and suppressing proliferation and migration. Upon continued triggering of a very slight RIG-I signal, we observed significantly inhibited cell growth, which could be reversed by knockout of the transcription factor IRF3. Initial results indicated that this cytostatic effect was not (only) mediated by secreted interferons but rather was an intrinsic effect of the "infected" cell (Urban 2020). We are currently investigating whether prolonged stimulation of this antiviral system, as it occurs during chronic viral infection, would lead to selection and preferential outgrowth of interferon resistant cells. This could have significant implications for both viral persistence as well as the development of tumors in the long run. This project is part of a large DFG-funded "transregional" Collaborative Research Center, TRR179

Recent literature has furthermore shown that antiviral innate immune signaling is directly involved in tumor development and/or immune control of emerging tumors, albeit the molecular mechanisms are not at all understood yet. Surprisingly, two studies (Sistigu et al., Nature Medicine 2014; Ranoa et al., Oncotarget 2016) have also demonstrated a direct involvement of the RIG-I/interferon axis in the induction of tumor-cell death in at least a subset of chemotherapies, e.g. doxorubicin. Based on these anecdotal reports, our research aims to identify and characterize the components and the mechanism of RIG-I mediated cell-death upon doxorubicin-induced DNA damage. We generated A549 CRISPR/Cas9 knockout cell lines targeting different components of innate immunity and determined rates of cell death upon treatment with cytostatics or also γ-irradiation by life cell imaging. We confirmed that doxorubicin treatment induces cell death dependent on a functioning RIG-I/MAVS axis, including the transcription factors IRF3 and NFkB, while the interferon system appeared dispensable (see figure). Moreover, we have identified proteins that contribute to cell death but are not related to the canonical RIG-I pathway. We are currently investigating the mechanistic role of these proteins in DNA damage-induced cell death.

 

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

III. Understanding the dynamics of viral replication and its interplay with the cellular antiviral system

(A) Schematic representation of the extended full-lifecycle model of HCV replication. The intracellular core part of the model is based on our previously published work (Binder 2013). (B) The model was trained on experimental data acquired from authentic HCV infection experiments in our BSL-3 lab. We measured positive and negative strand viral RNA, viral protein production, secretion of offspring viral particles and monitored the number of productively infected cells over time. Circles in the plots represent actual measurements and the lines are model predictions.
© dkfz.de

The interplay between virus infection, establishment of replication and the induction of the cell-intrinsic antiviral defense program is at the core of the decision of whether a virus will be efficiently cleared or leads to productive infection and disease. The dynamics, i.e. promptness and vigor, of the two processes is critical– if the cell-intrinsic response is quick enough, it can kill off the virus before it enters its exponential replication phase. In the contrary, as most (if not all) viruses encode factors that potently antagonize the cellular defense program, once the virus is one step ahead, it may effectively block the induction of the RIG-I/interferon response and manage to establish robust replication within the host cell. In order to understand this complex interplay, we teamed up with biomathematicians who together with us establish mathematical models (based on systems of ordinary differential equations) accurately simulating molecular pathways or virus replication cycles.

Several years ago, we have established the most detailed model of intracellular replication of the Hepatitis C virus (HCV) (Binder 2013). This model comprises every single major step of genome replication, beginning with translation of viral proteins, setting up replication organelles and replicating the viral RNA genome. Recently, we have been extending this original model by the extracellular steps of the HCV life cycle, i.e. the production and secretion of infectious viral particles from the host cell, and the infection of new "naive" host cells (figure 3A). The mathematical model is trained and validated against ample sets of experimental data. Generating this data required tedious measurements of viral parameters in our BSL-3 laboratory. The model can very precisely predict the course of amplification of a given amount of viral inoculum (figure 3B), and can be used to predict and understand the effects of various antiviral substances. In the future, we propose such modeling approaches to be used for establishing optimal drug regimens, meaning combinations of optimal doses of differently acting antiviral compounds.

We are going to set up models for further viruses; a model for Dengue virus has already been set up in collaboration with the team of Ralf Bartenschlager (Zitzmann 2020). In parallel, we are setting up mathematical models of cellular antiviral pathways, most importantly the RIG-I signaling pathway. Based on previous biochemical studies of the pathway in our lab, we have recently published a model of RIG-I activation (Schweinoch 2020). Currently, we are finalizing a very large combined model of the RIG-I and the interferon JAK/STAT signaling cascades, allowing for the simulation of the full antiviral response over the course of 24 hours post infection.

Our longer-term goal is to combine the models of viral replication with models of cell-intrinsic antiviral responses. This will eventually permit studying the mutual interactions as described above and potentially allow for a better understanding of the essential parameters determining the outcome of infection. A particular focus will be the difference between acute, self-limiting infections and infections that establish persistence, such as HCV, where a delicate balance of viral and antiviral processes must exist.

  • Binder, M., Sulaimanov, N., Clausznitzer, D., Schulze, M., Hüber, C. M., Lenz, S. M., Schlöder, J. P., Trippler, M., Bartenschlager, R., Lohman, V. and Kaderali L. (2013). Replication vesicles are load- and choke-points in the hepatitis C virus lifecycle. PLoS Pathog 9, e1003561
  • Zitzmann C, Schmid B, Ruggieri A, Perelson AS, Binder M, Bartenschlager R, Kaderali L. (2020) A Coupled Mathematical Model of the Intracellular Replication of Dengue Virus and the Host Cell Immune Response to Infection. Front Microbiol. 11:725

IV. Our 2020 Special– Investigations into the innate immune response against SARS-CoV2

Having worked on RNA viruses and their interaction with the host cell-intrinsic immune system for many years, in the light of the rampant pandemic of the newly emerged SARS-Coronavirus 2, we started a fourth research line in 2020. We have devoted one of our two BSL-3 labs to research on SARS-CoV2 and began to study the interplay of this large RNA virus with the host cellular defense machinery. In a close collaboration with the team of Ralf Bartenschlager at the CIID Heidelberg, we were able to confirm that the virus very efficiently blocks the activation of the transcription factor IRF3 (see above), while still permitting activation of NFkB. This leads to a strongly biased transcriptional response, largely comprised of pro-inflammatory (but not antiviral) cytokines. This strong activation of pro-inflammtory factors was shown to be mediated through STING, an adapter molecule of the defense system usually triggered by DNA viruses (Neufeldt 2020). 

In a second collaboration, we contributed to a large single cell RNA-sequencing study performed at the Charité hospital in Berlin. They examined the effects of hypertension (HT) and other cardiovascular disease (CVD) in the clinical course of COVID-19. It was known that HT/CVD significantly worsens the outcome of disease, but interstingly, we found that treatment with anti-hypertensive drugs ameliorated these detrimental effects. In particular inhibitors of the angiotensin converting enzyme I (ACEi) almost completely reverted the increased risk, leading to viral clearance comparable to non hypertensive patients. We found that secretory cells (the target cells for SARS-CoV2) in ACEi treated patients show a clear signature of cell-intrinsic antiviral signaling, indicating that this response might contribute to the positive outcome in this cohort (Trump 2020).

Currently, we are investigating the pattern recognition receptors that sense SARS-CoV2 infection in lung epithlial cells, as well as the ensuing cell-intrinsic antiviral response and how exact timing of infection vs induction of this response is critical in determining wether the virus can establish productive replication.

  • Neufeldt CJ, Cerikan B, Cortese M, Frankish J, Lee J, Plociennikowska A, Heigwer F, Joecks S, Burkart SS, Zander DY, Gendarme M, El Debs B, Halama N, Merle U, Boutros M, Binder M, Bartenschlager R
    (2020) SARS-CoV-2 infection induces a pro-inflammatory cytokine response through cGAS-STING and NF-κB. bioRxiv (PPR189879) - under revision
  • Trump S, Lukassen S, Anker MS, Chua RL, Liebig J, Thürmann L, Corman VM, Binder M, Loske J, Klasa C, Krieger TG, Hennig BP, Messingschlager M, Pott F, Kazmierski J, Twardziok S, Albrecht JP, Eils J, Hadzibegovic S, [...] Lehmann I (2020) Delayed viral clearance and exacerbated airway hyperinflammation in hypertensive COVID-19 patients. medRxiv (PPR217861) - under revision

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