This study investigated interactions between the human cytomegalovirus (HCMV) protein US27, the human interleukin-10 receptor (IL-10R), and the human chemokine receptor CXCR4 in human cells. Using a proximity ligation assay, the researchers were able to visualize that US27, IL-10R, and CXCR4 are in close proximity and likely forming a membrane protein complex. This interaction between the viral and host cell proteins likely increases intracellular signaling and helps explain how HCMV alters host immune responses.

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Human Cytomegalovirus (HCMV) is a widespread pathogen that causes lifelong
latent infection. HCMV rarely causes disease in healthy adults; however, immune-
compromised individuals like transplant recipients and AIDS patients can suffer from
life-threatening disease.
Many HCMV gene products manipulate cellular functions and immune responses.
Among these are the UL111A gene product, which encodes cmvIL-10, a homolog of
the human cytokine IL-10 (hIL-10). cmvIL-10 can bind to the same cellular IL-10
receptor and cause potent immune suppressive properties (1). Here, we wanted to
visualize interactions between cellular and viral proteins by using a highly sensitive
imaging technique called the Proximity Ligation Assay (PLA). This method uses two
antibodies that will each recognize one target antigen of interest, followed by PLA
probes that bind to the primary antibodies. If the target proteins are in close
proximity, within 15-30 nm, the probes interact, forming discrete fluorescent spots.
In this project, the two surface receptors of interest are human IL-10R and viral
US27. Both proteins have been shown to influence the human chemokine receptor
CXCR4 signaling pathway (2, 3). CXCR4 is an essential receptor that causes
immune cells to migrate to sites of injury and infection and also plays critical roles in
development and homeostasis. Our results show that all three proteins, CXCR4, IL-
10R, and HCMV US27 are forming a membrane complex.
Visualizing Virus-Host Interactions at the Cell Surface
Joy Kim, Carolyn Tu and Juliet Spencer
Herpesvirus Research Laboratory, Department of Biology, University of San Francisco
Introduction
Figure 2. Localization of viral US27, cellular IL-10R and CXCR4 in human cells. HEK293 cells were seeded onto glass
coverslips at a density of 2 x 105 cells/well. After 48 hours, cells were fixed, permeabilized, and co-stained with anti-US27 (green)
anti-IL-10Rα (red) and CXCR4 (magenta) antibodies, followed by staining with Alexa Fluor-conjugated secondary antibodies. Blue
represents the DAPI-stained nucleus.
Conclusions
• CXCR4 is a human chemokine receptor with important roles in immune surveillance,
development, and hematopoiesis.
• By using the Proximity Ligation Assay (PLA), we were able to visualize the
interaction between the CXCR4, IL-10R, and the viral receptor US27.
• We believe that interaction between the each receptor with CXCR4 increases the
overall signaling due to the formation of these heteromeric complexes.
• These results help clarify how HCMV alters host cellular functions and immune
responses.
References and Acknowledgements
Thank you to Dr. Spencer and Herpesvirus Lab members,
especially Carolyn Tu for working with me. This work was
supported by funding from the National Institutes of Health and
USF Faculty Development Funds.
1. Spencer et al., 2002. J Virol 76: 1285-1292.
2. Balabanian et al., 2012. Blood 99: 427-436.
3. Arnolds et al., 2013. Virology 439: 122-131.
Figure 4. Proximity Ligation Assay of US27, IL-10R, and CXCR4. A) The images show US27, IL-10R and CXCR4 (in HEK293 or
293-US27 cell lines) to be in close proximity with each other and likely forming heteromers. With the use of primary and secondary
antibodies, the oligonucleotide probes will ligate and result in a fluorescent amplicon. B) CXCR4 homodimers represent the positive
control, while oncostatin M was a negative control to show CXCR4 doesn’t interact with all membrane proteins in a cell.
Figure 6. Schematic image of US27 and CXCR4 heterodimer formation and receptor crosstalk with IL-
10R. All three receptors are in close proximity to each other, thereby increasing intracellular signaling.
IL-10R and CXCR4 form more complexes with US27 present
Figure 5. Graph detailing the PLA count per cell. From the analysis of the PLA images, the count of fluorescent spots was
greatest between IL10R and CXCR4 in US27-transfected cells. The amount of spots per cell decreases when one of the receptors
or US27 viral gene is taken out of the equation. This indicates that ligation occurs between all three receptors because they are
within 15-30nm of each other. An average of 10 cells were counted per sample. Error bars represent the standard deviation
between the cell counts of the different samples, further supporting the proximity and interactions of the three receptors of interest.
Ligation &
Amplification
- Probe+ Probe
Protein 2
Protein 1
15-30
nm
IL-10R & US27 CXCR4 & US27
IL-10R & CXCR4 IL-10R & CXCR4
0
30
60
90
PLAcount/cell
US27
CXCR4 & CXCR4
CXCR4 & CXCR4
OSM & IL10-R
OSM & IL10-R
OSM & CXCR4
HEK293
CXCR4 & OSM
US27 CXCR4 IL-10R MERGE
Figure 3. Schematic image of staining for Proximity Ligation Assay (PLA). Primary antibodies are used against proteins of
interest, oligonucleotide-conjugated secondary antibodies are used to act as plus and minus-sense probes. If probes are within 15-
30 nm of each other, they will ligate and get amplified, resulting in discrete fluorescent spots when viewed by confocal microscopy.
Figure 1. Schematic image of process for staining
in standard immunofluorescence microscopy. A
primary antibody is used to label the antigen of
interest. Next, a secondary antibody that’s chemically
conjugated to a fluorescent dye is used to directly
bind the primary antibody. This technique allows for
antigen(s) of interest to be detected through confocal
microscopy.
Proximity
Ligation Assay
(PLA)
Results
US27
US27
US27
HEK293
A
B
HEK293
US27