Charakterisierung von Zytomegalieviren mit mutierten G-Protein gekoppelten Rezeptoren
Auch gedruckt in der BibliothekZ: J-H 11.336 ; W: W-H 9.446
Ressourcen- / MedientypDissertation, Text
Datum der Freischaltung2007-01-05
The genome of the human cytomegalovirus encodes 4 G-protein coupled receptors with sequence homology to human chemokine receptors(GPCR; UL33,UL78,US27,US28). US28 has been shown to bind and sequester several chemokines from the environment of infected cells. Signalling activity and endocytosis of US28 is ligand-independent. Only a small portion of the receptor is located on the cell surface. The major amount is found in intracellular membranes of transfected cells. Until now there was no suitable antibody directed against US28 in infected cells.In infected fibroblasts fusion of GFP or TAP-tags to the c-terminus of the receptor US28 leads to dramatically reduced binding capacity to RANTES as well as reduced levels of IP3. In order to generate antibodies raised against US28 we constructed an artificial gene (US28e) that only contains the coding regions of the extracellular parts of the protein, lacking all transmembrane domains and intracellular loops. This artificial protein was overexpressed in E. coli and purified. The purified US28e protein was used to immunize rabbits. The anti-US28e serum could also detect the whole US28 protein in Western Blot and immunofluorescence in transfected HEK293 cells. The antiserum was able to detect US28 in infected cells after cleavage of the Fc portion of the antibody. US28 shows a location close to the nucleus. To learn more about the spatiotemporal distribution of US28 during infection in different systems we generated an antiserum against US28e. It works in transfected and infected cells, but the cellular localization has to be further investigated. During viral infection US28 activates the promoter of VEGF, which was observed before in US28 transfected cells. Transfection of US28 also led to increased VEGF protein levels. However no increase in VEGF protein levels could be observed during infection, suggesting that other viral proteins may have influence on pathways leading to VEGF expression. This needs further investigation.
LizenzStandard (Fassung vom 03.05.2003)
US28 receptor, cytomegalovirus
Vascular endothelial Growth Factor