Date of Award

8-2014

Degree Type

Thesis

Degree Name

Master of Science

Department

Biology

Program

Biology

First Advisor/Chairperson

Dr. Robert J. Winn, Ph.D.

Second Advisor

Dr. Robert J. Belton

Third Advisor

Dr. Erich N. Ottem

Abstract

Human Cytomegalovirus (HCMV) encodes the G-protein coupled receptor US28. Using a mouse model system, US28 was previously found to be oncomodulatory, increasing proliferation, inducing anchorage independent growth and loss of contact inhibition. Similarly, in the human glioblastoma cell line U373, US28 activated VEGF expression. To determine if US28 is oncomodulatory in normal human cells, we engineered the human fibroblast cell line MSU1.1 to express US28 via lentivirus infection. MSU1.1 cells were transduced with pHAGE-US28-ZsGreen. Fluorescent confocal microscopy was utilized to detect the ZsGreen tag and confirmed the successful transduction of MSU1.1 cells with the US28 vector or empty pHAGE vector. MSU1.1 cells expressing US28 did not exhibit loss of contact inhibition nor anchorage independent growth in focus formation and soft agar assays, respectively. Using the AlamarBlue assay, US28-expressing cells showed a 2.303 fold increase in proliferation compared to wild type MSU1.1 cells and a 2.201 fold increase compared to mock transduced cells. US28 expression in MSU1.1 cell line increased proliferation but did not trigger a transformed phenotype. This study reveals potential cell-type and species-specific differences from previous findings assessing US28 expression in NIH-3T3 (mouse fibroblast) and U373 (malignant glioma) cell lines. The reported prevalence of US28 in GBM and confirmation of oncomodulation via increased proliferation in MSU1.1 cells makes US28 a potential target warranting further understanding of the virus’ role in GBM.

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