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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
Results
Introduction
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, lifethreatening disease can occur in immune compromised
individuals, such as transplant recipients, AIDS patients, and
infants.
More CXCR4 is present on the surface of cells expressing US27
CXCR4 responds with a greater calcium response when
US27 is present
Isotype Control
SDF-1
25
HEK293CXCR3
HEK293US27
HEK293-CXCR3
CXCR4
Relative fluorescence Units
HEK293-US27
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.
20
15
HEK293-US27
β-actin
10
HEK293-CXCR3
5
Fluorescence Intensity
0
0
20
40
60
80
100
120
140
Time (Seconds)
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.
CXCR4 gene expression is higher in cells expressing US27
Figure 1. Calcium flux in response to SDF-1. HEK293-US27 and HEK293CXCR3 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.
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.
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 genespecific primers to amplify CXCR4. β-actin is a housekeeping gene that
serves as a control to ensure equal amounts of cDNA were used.
Conclusions
CXCR4 receptor levels are significantly elevated throughout
cells expressing US27
Cells expressing US27 exhibit more robust migration
towards SDF-1
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.
250000
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.
HEK293-CXCR3
Relative Light Units
200000
HEK293-US27
150000
100000
50000
0
0
0.01
1
1000
ng/ml SDF-1
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.
Figure 2. Cell migration towards SDF-1. Cells were seeded at a density
of 2 x 105 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.
HEK293-CXCR3
HEK293-US27
Figure 4. Immunofluorescence microscopy of CXCR4 receptor
expression. Cells were seeded onto glass coverslips at a density of 2 x 105
cells/well, stained with a fluorochrome conjugated antibody specific for
CXCR4 (red). Nuclei appear blue due to DAPI staining.
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.
Acknowledgements
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.
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