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Regulatory Functions of the DAP Kinase Family in Antiviral RIG-I Signalling

Wolanski, Julia Caroline

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Cytosolic recognition of viral replication intermediates by RIG-I, the founding member of the RIG-I-like receptor (RLR) family, initiates a signalling cascade which culminates in the activation of latent transcription factors IRF3 and IRF7, inducing the expression of type I and type III interferons (IFNs) and interferon-stimulated genes (ISGs). These work in concert to combat viral infection. Thus, regulation of RIG-I activity is crucial in mounting a balanced antiviral response strong enough to ward off infection, but strictly transient in order to avoid tissue damage. Initially, death-associated protein kinases (DAPKs) were described as initiators of cell death. Since then, they have been found to mediate a variety of cellular processes, such as cell growth and survival, cytoskeletal remodelling and inflammatory responses, but had not been linked to the regulation of innate antiviral signalling. We identified death-associated protein kinase 1 (DAPK1) as a negative feedback-regulator of RIG-I activity. The present study confirms that a minimal DAPK1 construct, DAPK1KCA, comprising only the kinase, calmodulin (CaM)-binding and Ankyrin repeats domains, inhibits RIG-I in a kinase-dependent manner. DAPK1KCA harbours residues targeted by growth factor signalling-related kinases. We could demonstrate, however, that inhibition of RIG-I signalling mediated by DAPK1 happened independently of its role in growth factor signalling. Nevertheless, DAPK1 expression was strongly influenced by cell growth and division. Since the kinase domains of the different DAPKs are homologous and share some substrate specificity, we investigated if, in addition to DAPK1, other DAPK family members would interfere with RIG-I signalling. Upon over-expression, all DAPK family members inhibited RIG-I signalling, except DRAK2 which, in our hands, was kinase-inactive. Reciprocally, siRNA-mediated knockdown of DAPK1 and DAPK3 resulted in increased antiviral signalling activity. Moreover, silencing of DAPK1 and DAPK3 reduced the replication of two different RNA viruses, emphasizing the physiological relevance of these kinases in the regulation of RIG-I signalling. Similar to what was observed for DAPK1, we found DAPK2 to inhibit RIG-I signalling in a kinase activity-dependent fashion. Moreover, the kinase domain of DAPK3 alone inhibited RIG-I signalling. While all DAPK family members phosphorylated RIG-I in vitro, interaction with RIG-I could only be shown for DAPK1 and DAPK2. Although we found DAPKs to physically interact with each other, we observed no interdependence of the kinases regarding their inhibitory function in RIG-I signalling. In order to further study DAPK functions in RIG-I signalling, we created single and combined knockout cell lines for DAPK1, DAPK2, and DAPK3. However, there was no change in the antiviral response in these cells, possibly due to up-regulation of other proteins compensating for loss of the DAPKs. We observed that inhibition of RIG-I signalling by DAPKs happened independently of their well-studied role in apoptotic as well as autophagic cell death induction. In fact, we did not detect any induction of autophagy upon DAPK expression. Ultimately, we discovered that regulation of antiviral signalling by DAPKs is not limited to an inhibition of RIG-I, but that they also inhibited MDA5-mediated signalling. Moreover, we found evidence that DAPKs additionally inhibit antiviral signalling at the level or downstream of IRF3-mediated gene transcription. In summary, the present study identifies a conserved role for DAPKs in the regulation of antiviral RLR signalling independent of known DAPK functions in cell death and survival.

Item Type: Dissertation
Supervisor: Bartenschlager, Prof. Dr. Ralf
Date of thesis defense: 22 September 2017
Date Deposited: 06 Oct 2017 10:10
Date: 2017
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
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