The US27 gene product of human cytomegalovirus enhances signaling of host chemokine receptor CXCR4

Abstract

Human cytomegalovirus (HCMV) is a member of the Herpesviridae family that manipulates host immune responses and establishes life-long latent infection, in part through mimicry of cytokines, chemokines, and chemokine receptors. The HCMV US27 gene product is a putative chemokine receptor with no known ligands. We generated a stable US27 cell line to screen for chemokine ligands but unexpectedly found that US27 potentiated the activity of an endogenous human chemokine receptor, CXCR4. Cells expressing both US27 and CXCR4 exhibited greater calcium mobilization and enhanced chemotaxis in response to CXCL12/SDF-1α than controls. Quantitative RT-PCR revealed a significant increase in CXCR4 expression when US27 was present, and elevated CXCR4 receptor levels were detected via flow cytometry, western blot, and immunofluorescence microscopy. Potentiation of CXCR4 signaling by US27 could represent a novel strategy by which HCMV targets virus-infected cells to the bone marrow in order to expand the reservoir of latently infected cells.

Figure 1. Increased calcium mobilization in cells expressing CXCR4 and HCMV US27

HEK293 cells and stable 293-US27, 293-US28, and 293-CXCR3 cell lines were loaded with Fluo-4 dye, treated with 100 μg/ml CXCL12/SDF-1α in a volume of 10 μl, and fluorescence intensity monitored over time. An equal volume of PBS was added as a negative control and 1 mg/ml ionomycin in a volume of 5 μl served as a positive control. These results are representative of four independent experiments.

Figure 2. Treatment with AMD-3100 completely inhibits CXCL12/SDF-1α-induced calcium mobilization

Cells were loaded with Fluo-4 dye, and then relative fluorescence intensity was monitored over time. A) 293-US27 cells were pre treated with either 100 μM AMD-3100 or an equal volume of PBS for 10 minutes before treatment with 100 μg/ml CXCL12/SDF-1α or 1 μg/ml ionomycin. B) 293-CXCR3 cells were pre-treated with AMD-3100 or PBS before treatment with 100 μg/ml CXCL12/SDF-1α or CXCL11/ITAC. These results are representative of three independent experiments.

Figure 3. CXCR4 calcium responses in cells expressing US27 mutants are not enhanced

Cells were loaded with Fluo-4 dye, and then relative fluorescence intensity was monitored over time. HEK293, 293-US27, 293-CXCR3, 293-US27/CXCR3-CT, and 293-US27-DAY cells were treated with 10 μl of CXCL12/SDF-1α (100ug/ml) or 5 μl ionomycin (1 mg/ml). These results are representative of two independent experiments.

Figure 4. US27 preferentially increases CXCR4, but not CXCR3, calcium signaling

HEK293 and 293-US27 cells were transfected with p3XFLAG-CXCR3, loaded with Fluo-4 dye, and then relative fluorescence intensity monitored over time. Cells were stimulated with 10 μl of CXCL11/ITAC or CXCL12/SDF-1α (100ug/ml) as indicated. These results are representative of two independent experiments.

Figure 5. Increased migration of 293-US27 cells toward CXCL12/SDF-1α

HEK293, 293-US27 or 293-CXCR3 cells were seeded at a density of 2 x 10⁵ cells in a total volume of 0.1 ml in the upper chamber of an 8 μm trans-well filter. Migration media containing the indicated concentrations of CXCL12/SDF-1α was placed in the lower chamber. After four hours, cells in the lower chamber were harvested and quantified by the addition of CellTiter-Glo reagent and measurement of luminescence. Error bars represent standard error. Statistical analysis was performed using the two-tailed student’s t-test and * indicates p < 0.05; ** indicates p < 0.01. Results are representative of three independent experiments.

Figure 6. Surface expression of CXCR4 is higher in 293-US27 cells

Cells were stained with phycoerythrin-conjugated anti-CXCR4 antibody (solid line) or IgG_2b-PE isotype control (dotted line) and fluorescence intensity measured via flow cytometry. The lower right panel is an overlay of all three cell types stained with anti-CXCR4 antibody.

Figure 7. 293-US27 cells have elevated CXCR4 expression

RNA was isolated from each cell type, reverse transcribed, then gene specific primers were used to amplify either CXCR4 or β-actin (A). For qPCR (B), data was normalized to the β-actin control and expressed as fold change relative to CXCR4 levels from HEK293 cells. For Western blotting (C), cell lysates were probed with either anti-CXCR4 antibody or anti-MAPK antibody as indicated. In the lower panel, 293-US27 cell lysates were analyzed either undiluted (neat) or diluted as indicated. For immunofluorescence microscopy (D), cells were seeded onto glass coverslips in 6-well plates at a density of 2 x 10⁵ cells/well, and then stained with an anti-CXCR4 antibody followed by TRITC-conjugated secondary antibody (red). Nuclei appear blue due to DAPI staining. Co-localization of US27 and CXCR4 (E) is shown in cells that were fixed, permeabilized and stained with anti-FLAG (US27) and anti-CXCR4 antibodies followed by appropriate FITC- or TRITC-conjugated secondary antibodies. The merged image results from a compilation of the three colored images into one image where areas of red (CXCR4) and green (US27) overlap appear as discrete yellow spots indicated by white arrows.