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Molecular Studies on Dengue Virus-Host Interaction
Dengue is the most prevalent mosquito-borne viral disease world-wide. The causative agent Dengue Virus (DENV) is a positive strand RNA virus replicating predominantly in monocyte-derived macrophages and dendritic cells. The severi-ty of the disease is strongly linked to the level of viral replication, which is de-termined, amongst others by pre-existing DENV-specific antibodies and genetic determinants of the virus and the host. The aim of my thesis was to characterize the role of nonstructural protein 5 (NS5) for the DENV replication cycle. NS5 is a multifunctional protein involved in viral RNA replication, 5’ end capping and blocking of interferon (IFN)-mediated signaling by degrading STAT2. Although DENV replicates in the cytoplasm, NS5 mostly accumulates in the nucleus of infected cells. This study investigated the determinants of NS5 nuclear transport and the effects of nuclear NS5 on viral replication and innate immunity. Nuclear accumulation of NS5 occurred inde-pendent from other viral proteins and was found in infected mammalian and mosquito cells arguing for an evolutionarily conserved property. A mutation analysis was used to identify amino acid residues in NS5 essential for nuclear accumulation. Replication analyses of these nuclear localization signal (NLS) mu-tants showed that even though a high level of nuclear NS5 is not required for efficient DENV replication, complete abrogation of nuclear transport significantly reduced viral replication. Interestingly, the poorly replicating NLS mutants could not be rescued by providing wild type protein in trans indicating that factors other than nuclear NS5 are responsible for their poor replication. To check whether these NLS mutations affect enzymatic activities of NS5, recombinant full length proteins were bacterially expressed and purified. Enzymatic assays showed that save for one, none of the NLS mutations impaired RNA-dependent RNA polymerase or methyl transferase activity. Moreover, IFN sensitivity of the NLS mutants was similar to wild type indicating that nuclear NS5 is not required to counteract IFN induced genes. Contrary to earlier observations no significant difference was observed in induction of interleukin-8 by wild type and mutant NS5. In the second part of my PhD study, I performed a genome-wide RNAi-based kinase screen to identify cellular kinases promoting or restricting DENV replication. An imaging based RNAi screening platform for DENV was developed, which included optimization of siRNA-mediated silencing in human hepatoma cells, viral infection, immunostaining, image acquisition and statistical data analysis. The primary screen was carried out with a kinase library targeting all known and putative cellular kinases with three siRNAs per gene. Approximately 100 kinases selected from the primary screen were validated in an infection based screen with a new siRNA library containing a different set of siRNAs against each gene. The screen identified 18 kinases essential for DENV replication and 15 kinases suppressing viral replication. The kinase siRNAs were later tested in a DENV subgenomic reporter replicon based screen to differentiate their role in viral en-try or replication. The effect of selected kinases on DENV infection was further validated using chemical inhibitors. The kinases identified by this study can serve as targets for developing novel antiviral compounds against dengue infection.
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570 Biowissenschaften, Biologie
570 Life sciences