Document Type Master's Dissertation Author Subramoney, Preya email@example.com URN etd-06222011-170730 Document Title The role of human cytomegalovirus encoded viral G protein-coupled receptors in onco-modulatory signalling Degree MSc Department Pharmacology Supervisor
Advisor Name Title Dr M Siderius Co-Supervisor Dr A D Cromarty Supervisor Keywords
- interleukin 6 IL-6
- signal transducer
- activator of transcription STAT3
- G protein-coupled receptors GPCR
- human cytomegalovirus HCMV
Date 2011-04-08 Availability unrestricted AbstractHuman cytomegalovirus (HCMV) is a ubiquitous virus of the herpes type that infects a high percentage of some populations. One of the most researched genes expressed by HCMV with close homology to human chemokine receptors is the US28 G protein-coupled receptor. Study design: This study was initiated to elucidate the intracellular signalling pathways of an inflammatory factor (IL-6) and an angiogenic factor (STAT3) triggered by the viral US28 oncogene and the presence of US28 in the HCMV viral particle. These pathways were observed by introducing the US28 gene into two human cell lines by infection with a HCMV strain that expresses the US28 gene (wild type), and two HCMV strains where the US28 gene was deleted (ÄUS28 and ÄUS28/UL33). Special attention was directed at the expression of IL-6 after promotion of the US28 gene and subsequent phosphorolation of STAT3. A new US28 antibody was validated and a method developed in an attempt to determine US28 on the viral particle. The following techniques were applied: Cell culture work, two mammalian cell lines were used, HFF’s and U373 MG. Virus stock titre determination to determine the multiplicity of infection. Protein quantitation to determine very small quantities of protein for Western blot analysis. ELISA for the quantitative determination of IL-6. Western blotting for phospho- STAT3 determination and validation of the US28 antibody. Immunocytochemistry was used for back titrations of virally infected cells. Immunofluorescence assay and use of confocal microscopic techniques was used for the location of the US28 gene in the virion and for tSTAT3 translocation to the nucleus. Conclusion: A clear increase in IL-6 secretion (495% ± 1%) was seen, and this was after only an hour in HCMV WT infected cells. From the increase in IL-6 secretion a subsequent increase in STAT3 phosphorylation was detected in the same samples. A clear link has been established between IL-6 and STAT3. A method to determine whether US28 was present in the HCMV viral particle was designed and preliminary results obtained. The results were inclusive.
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Please cite as follows:
Subramoney, P 2011, The role of human cytomegalovirus encoded viral G protein-coupled receptors in onco-modulatory signalling, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-06222011-170730/ >
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