Human cytomegalovirus US28 found in glioblastoma promotes an invasive and angiogenic phenotype

Human cytomegalovirus (HCMV) infections are seen often in glioblastoma multiforme (GBM) tumors, but whether the virus contributes toGBMpathogenesis is unclear. In this study, we explored an oncogenic role for the G-protein-coupled receptor-like protein US28 encoded by HCMV that we found to be expressed widely in human GBMs. Immunohistochemical and reverse transcriptase PCR approaches established that US28 was expressed in approximately 60% of human GBM tissues and primary cultures examined. In either uninfected GBM cells or neural progenitor cells, thought to be the GBM precursor cells, HCMV infection or US28 overexpression was sufficient to promote secretion of biologically active VEGF and to activate multiple cellular kinases that promote glioma growth and invasion, including phosphorylated STAT3 (p-STAT3) and endothelial nitric oxide synthase (e-NOS). Consistent with these findings, US28 overexpression increased primary GBM cell invasion in Matrigel. Notably, this invasive phenotype was further enhanced by exposure to CCL5/RANTES, a US28 ligand, associated with poor patient outcome in GBM. Conversely, RNA interference-mediated knockdown of US28 in human glioma cells persistently infected with HCMV led to an inhibition in VEGF expression and glioma cell invasion in response to CCL5 stimulation. Analysis of clinical GBM specimens further revealed that US28 colocalized in situ with several markers of angiogenesis and inflammation, including VEGF, p-STAT3, COX2, and e-NOS. Taken together, our results indicate that US28 expression from HCMV contributes to GBM pathogenesis by inducing an invasive, angiogenic phenotype. In addition, these findings argue that US28-CCL5 paracrine signaling may contribute to glioma progression and suggest that targeting US28 may provide therapeutic benefits in GBM treatment.