Human cytomegalovirus (HCMV) is a member of the herpesvirus family. About 70-90% of the population is infected with HCMV. In healthy individuals there are often no symptoms, and the virus is maintained as a lifelong latent infection. However, life- threatening disease can occur in immune compromised individuals, such as transplant recipients, AIDS patients, and infants. HCMV maintains a peaceful co-existence with its host through the action of viral genes that modify the host immune system. In this study, we focused on the effects of one viral gene, US27. The US27 gene is of interest because it has similarity to cellular chemokine receptors. Chemokines and their receptors can promote cell growth and also play a crucial role in directing immune cells to sites of infection. In a previous study, we investigated whether any cellular chemokine might be a ligand for US27. While no chemokine ligands were identified, the results indicated that US27 might enhance the signaling of human chemokine receptor CXCR4. In this study, the effect of US27 on CXCR4 function was further examined. The results show that US27 potentiates CXCR4 functional activity and also increases CXCR4 protein and gene expression. Conclusions The US27 protein of human cytomegalovirus enhances signaling by human chemokine receptor CXCR4 Kathleen L. Arnolds and Juliet V. Spencer Herpes Virus Research Lab ~ Department of Biology ~ University of San Francisco Introduction Results CXCR4 responds with a greater calcium response when US27 is present Figure 1. Calcium flux in response to SDF-1. HEK293-US27 and HEK293- CXCR3 cells were loaded with Fluo-4 calcium indicator dye, then treated with 500 ng/ml SDF-1, the natural ligand for CXCR4, where indicated. Fluorescence intensity was measured via flow cytometry. Cells expressing US27 exhibit more robust migration towards SDF-1 Figure 2. Cell migration towards SDF-1. Cells were seeded at a density of 2 x 10 5 cells in the upper chamber of an 8 um trans-well filter; media with SDF-1 was in the lower chamber. After 4 hours, cells that had migrated to the lower chamber were harvested and counted using Cell Titer-Glo luminescence. Error bars represent standard error. More CXCR4 is present on the surface of cells expressing US27 Figure 3. Surface expression of CXCR4. Cells were stained with fluorochrome-conjugated anti-CXCR4 antibody. CXCR4 expression was observed on the BD FACSCalibur flow cytometer. Data was analyzed using CellQuest Pro software. CXCR4 receptor levels are significantly elevated throughout cells expressing US27 Figure 4. Immunofluorescence microscopy of CXCR4 receptor expression. Cells were seeded onto glass coverslips at a density of 2 x 10 5 cells/well, stained with a fluorochrome conjugated antibody specific for CXCR4 (red). Nuclei appear blue due to DAPI staining. Thank you to Carolyn Tu for technical assistance. This work was supported by funding from the National Institutes of Health and USF Faculty Development Funds. HEK293-CXCR3HEK293-US27 Figure 5. RT-PCR analysis of CXCR4. RNA was harvested from each cell type and reverse transcribed (RT) into complementary DNA (cDNA). The polymerase chain reaction (PCR) was then performed with gene- specific primers to amplify CXCR4. β-actin is a housekeeping gene that serves as a control to ensure equal amounts of cDNA were used. Acknowledgements Fluorescence Intensity Isotype Control HEK293-CXCR3 HEK293-US27 HEK293-CXCR3 Figure 1. Depiction of stable cell lines used in this study. Human embryonic kidney cells (HEK293) cells were transfected with plasmid DNA encoding either the HCMV receptor US27 (green) or a control cellular receptor, CXCR3 (purple). After transfection, cells were cultured in selective antibiotic until all cells stably expressed the indicated receptor. HEK293 cells naturally express the human chemokine receptor CXCR4 (red). All three receptors exhibit the same basic structure with seven transmembrane domains. CXCR4 signaling in response to its natural ligand, SDF-1, is greatly increased in cells that also express HCMV US27. Both calcium flux and cell migration were increased when US27 was expressed in combination with CXCR4, indicating that US27 enhances CXCR4 functional activity. The increased functional activity correlates strongly with increased CXCR4 receptor expression. When US27 was present, more CXCR4 protein was found on the cell surface and throughout the cell. In addition, RT-PCR revealed that CXCR4 gene transcription was higher in cells expressing US27. The next step will be to analyze downstream signaling of CXCR4 in the presence of US27. This will aid in our understanding of how HCMV avoids the human immune system. CXCR4 is involved in immune responses, angiogenesis, fetal development, and formation of metastases by cancer cells. Therefore, modulation of CXCR4 by a viral gene could have many implications for human health. β-actin CXCR4 HEK293- CXCR3 HEK293- US27 HEK293-CXCR3 HEK293-US27 CXCR4 gene expression is higher in cells expressing US27