Analysis of IRF9-dependent IFNbeta induction in response to viral dsRNAs

• Applicant: Dr. Katharina Schulz
• Subject Area: Virology ; Immunology
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 263073072

Double stranded (ds) RNAs, which are generated during the replication cycle of most viruses, are sensed by the innate immune system. The cellular receptors recognizing dsRNA activate the transcription factor Interferon (IFN) regulatory factor 3 (IRF3), which induces type I IFN production and the antiviral immune response. However, most viruses render their environment IRF3 deficient, raising the question, how the antiviral response is induced instead. Interestingly, in the absence of IRF3, long dsRNAs (greater or equal 1000 bp) but not short ones (less or equal 500 bp), are able to induce type I IFN production. This effect was not observed in the absence of IRF9 (DeWitte-Orr et al.; J. Immunol. 183, 2009), indicating that IRF9 might substitute for IRF3. The aim of my project is to analyze the signaling cascade induced by long dsRNAs in the absence of IRF3. The major topics hereby are investigating (i) how IRF9 is activated in response to long dsRNAs, (ii) how the transcription of IFNbeta is induced by IRF9 and (iii) why this pathway is only triggered by long dsRNAs. To elucidate activation of IRF9, first post-translational modification of IRF9 will be analyzed by 2D-gel electrophoresis, respectively co-immunoprecipitation and mass spectrometry, and the kinases modifying IRF9 will be studied using siRNA-assays. IRF9 most likely forms dimers upon activation. Confocal microscopy and native gel electrophoresis will give first indications whether homo- or heterodimers are formed. Subsequently, I will use co-immunoprecipitation combined with mass spectrometry for detailed analysis.To investigate how IFNbeta transcription is induced, a DNA-pull down assay and chromatin immunoprecipitation (ChiP) analyses will be applied. To assure functionality of the cascade, IFNbeta production will be measured by quantitative real-time PCR (qRT-PCR) after certain steps of the cascade are blocked. In addition, the supernatants of stimulated cells will be transferred to wild-type cells, and functional IFNbeta will be analyzed with standard antiviral assays. The experiments will be performed after treatment with long, as well as short dsRNAs to elucidate why short dsRNAs cannot activate this signaling pathway. Results of these analyses will help to better understand how the innate immune system responds to and controls viral infections and potentially reveal targets for new and better antivirals.

• DFG Programme: Research Fellowships
• International Connection: Canada
• Host: Professorin Dr. Karen Mossman