Artikel
Combinatory Effects of HHV-8-Encoded Proteins: High Throughput Analysis for the Discovery of Pathogenic Factors in the Development of Kaposi’s Sarcoma
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Veröffentlicht: | 20. März 2006 |
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Gliederung
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Human herpesvirus-8 (HHV-8) is the etiological agent of the Kaposi’s sarcoma (KS), a tumor of endothelial origin and a prominent inflammatory component. HHV-8 encodes for about 85 genes. Little is known which genes may contribute to KS pathogenesis. In addition most experiments so far analyzed the tumorigenic effects of single viral genes. However, for KS development the cooperation between several different viral genes may be required. Here we present a high throughput approach (cell chips, reverse transfection arrays) for the determination of combinatory effects of HHV-8-encoding genes on the nuclear factor-kappa B (NF-κB) signal transduction pathway. NF-κB is an important mediator of inflammation and cell activation. At present 80 open reading frames (ORF) of HHV-8 were cloned in expression vectors. All of these ORFs were sequenced and aligned with the published sequences of HHV-8. Expression of the respective viral genes was analyzed by western blotting and immunofluorescence. The effect of these genes, single and in combinations of two, on a GFP-reporter gene under the control of a NF-κB inducible response element was investigated in reverse transfection experiments with HEK 293T cells. Two genes of HHV-8 were found to interact synergistically on the activation of NF-κB in this screen. This finding was confirmed in standard transfection experiments. Further studies are ongoing to determine the molecular mechanisms of this interaction. These results show that the method of reverse transfection is a powerful approach to detect combinatory effects of viral genes on signal transduction pathways.