Die Rolle der Proteinkinase RIP1 bei Infektionen durch das humane Cytomegalievirus

In this thesis, the importance of the cellular serine/threonine-proteinkinase RIP1 was verified during HCMV infection. RIP1 was upregulated during HCMV infection in HFF-cells, as it was already described in literature. It is demonstrated here that forced expression of RIP K45R - a kinase inactive RIP1 mutant - could potently block HCMV replication in HFF cells. RIP1 was up regulated during HCMV infection in HFF cells, as it has already been described in literature. Further HCMV replication assays using different RIP1 constructs show that not only kinase activity but also substrate binding to intermediate- and deathdomain of RIP1 seem to be essential for HCMV replication. The effect of RIP1 during HCMV replication seem to be cell type specific in HFF cells. Experiments with chemical inhibitors of RIP1 could also demonstrate the importance of RIP1 for the HCMV infection. Other possible explanations of the effect of RIP1, like interactions with other HCMV proteins, effects on physiological cell functions or activation of cellular signaling pathways (e.g. MAP kinase- and NFB-signaling pathways) has been excluded. By secretions-experiments it could be proved that supernatants of the RIP K45R transducted cells suppress the HCMV replication by 50%. Especially antiviral cytokines such as MIP-1, RANTES, Eotaxin, IL-6 and IL8 has been identified by cDNA arrays, PCR-analysis and cytokine EILSA assays. The transcription and secretion of these antiviral cytokines has been impeded by RIP1. IL-8 and RANTES could suppress the HCMV replication per se. With the model of the IL-8 promoter it could be demonstrated that expression and suppression of the cytokines might be impeded via interaction of the RIP1 kinase- and intermediate domain with their promotors. The assumption has been discussed that RIP K45R probably “activates” the expression of antiviral cytokines via a “dominant-negative” effect on endogenous RIP1 via a so far unexplained signaling pathway. The data collected during the thesis suggest that the up regulation of RIP1 during the HCMV infection impedes the antiviral immune response. These results indicate RIP1 to be a candidate for the development of specific inhibitors, which would be advantageous because they avoid formation of resistance.