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. 2018 Feb 12;92(5):e01981-17.
doi: 10.1128/JVI.01981-17. Print 2018 Mar 1.

Human Cytomegalovirus UL111A and US27 Gene Products Enhance the CXCL12/CXCR4 Signaling Axis via Distinct Mechanisms

Carolyn C Tu  1 , Kathleen L Arnolds  1 , Christine M O'Connor  2 , Juliet V Spencer  3
Affiliations

Human Cytomegalovirus UL111A and US27 Gene Products Enhance the CXCL12/CXCR4 Signaling Axis via Distinct Mechanisms

Carolyn C Tu et al. J Virol. .

Abstract

Human cytomegalovirus (HCMV) is a prevalent pathogen that establishes lifelong infection in the host. Virus persistence is aided by extensive manipulation of the host immune system, particularly cytokine and chemokine signaling pathways. The HCMV UL111A gene encodes cmvIL-10, an ortholog of human interleukin-10 that has many immunomodulatory effects. We found that cmvIL-10 increased signaling outcomes from human CXCR4, a chemokine receptor with essential roles in hematopoiesis and immune cell trafficking, in response to its natural ligand CXCL12. Calcium flux and chemotaxis to CXCL12 were significantly greater in the presence of cmvIL-10 in monocytes, epithelial cells, and fibroblasts that express CXCR4. cmvIL-10 effects on CXCL12/CXCR4 signaling required the IL-10 receptor and Stat3 activation. Heightened signaling occurred both in HCMV-infected cells and in uninfected bystander cells, suggesting that cmvIL-10 may broadly influence chemokine networks by paracrine signaling during infection. Moreover, CXCL12/CXCR4 signaling was amplified in HCMV-infected cells compared to mock-infected cells even in the absence of cmvIL-10. Enhanced CXCL12/CXCR4 outcomes were associated with expression of the virally encoded chemokine receptor US27, and CXCL12/CXCR4 activation was reduced in cells infected with a deletion mutant lacking US27 (TB40/E-mCherry-US27Δ). US27 effects were Stat3 independent but required close proximity to CXCR4 in cell membranes of either HCMV-infected or US27-transfected cells. Thus, HCMV encodes two proteins, cmvIL-10 and US27, that exhibit distinct mechanisms for enhancing CXCR4 signaling. Either individually or in combination, cmvIL-10 and US27 may enable HCMV to exquisitely manipulate CXCR4 signaling to alter host immune responses and modify cell trafficking patterns during infection.IMPORTANCE The human chemokine system plays a central role in host defense, as evidenced by the many strategies devised by viruses for manipulating it. Human cytomegalovirus (HCMV) is widespread in the human population, but infection rarely causes disease except in immunocompromised hosts. We found that two different HCMV proteins, cmvIL-10 and US27, act through distinct mechanisms to upregulate the signaling activity of a cellular chemokine receptor, CXCR4. cmvIL-10 is a secreted viral cytokine that affects CXCR4 signaling in both infected and uninfected cells, while US27 is a component of the virus particle and impacts CXCR4 activity only in infected cells. Both cmvIL-10 and US27 promote increased intracellular calcium signaling and cell migration in response to chemokine CXCL12 binding to CXCR4. Our results demonstrate that HCMV exerts fine control over the CXCL12/CXCR4 pathway, which could lead to enhanced virus dissemination, altered immune cell trafficking, and serious health implications for HCMV patients.

Keywords: CMV; CXCL12; CXCR4; US27; chemokine; chemokine receptors; cmvIL-10; cytokine; cytomegalovirus.

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Figures

FIG 1
FIG 1
cmvIL-10 enhances CXCL12/CXCR4-mediated calcium flux and cell migration. (A and B) HEK293 (A) or THP-1 (B) cells loaded with Fluo-4 AM were stimulated with 100 ng/ml CXCL12 in the presence or absence of 100 ng/ml cmvIL-10, and fluorescence intensity was measured by flow cytometry over time. Arrows indicate stimulus addition. RFU, relative fluorescence units. (C) Peak calcium flux in HEK293 cells was measured in response to CXCL12 alone or in combination with 100 ng/ml cmvIL-10. (D and E) Transwell migration of HEK293 (D) or THP-1 (E) cells toward CXCL12 in the lower chamber ± 100 ng/ml cmvIL-10 after 4 h. Cell numbers were quantified by Cell Titer Glo assay. RLU, relative light units. Error bars, standard error for 3 replicates; *, P < 0.01 by paired Student's t test.
FIG 2
FIG 2
Increased CXCL12-CXCR4 signaling with cmvIL-10 and hIL-10. (A) HEK293 cells were loaded with Fluo-4AM calcium indicator dye and then stimulated with 100 ng/ml CXCL12 alone or in the presence of cmvIL-10 or hIL-10. The arrow indicates stimulus addition; data for addition of hIL-10 and cmvIL-10 alone are shown by dashed lines on the baseline. (B) Transwell migration assay with HEK293 cells in the upper chamber and various doses of CXCL12 in the presence or absence of cmvIL-10 or hIL-10 (100 ng/ml) in the lower chamber. Cells were quantified by Cell Titer Glo after 4 h. Error bars, standard error for 3 replicates, *, P < 0.01 by paired Student's t test. (C) HEK293 cells were treated with 100 ng/ml CXCL12, hIL-10, and/or cmvIL-10 as indicated for 15 min, and then total cell lysates were harvested and immunoblotted with the indicated antibodies.
FIG 3
FIG 3
cmvIL-10 does not impact CXCR4 gene expression or protein levels. HEK293 cells were treated with CXCL12 (100 ng/ml) and/or cmvIL-10 (100 ng/ml) for 2 h or 24 h. (A) Total RNA was harvested for qPCR analysis. Fold change was calculated relative to the mock-treated control (Con) cells. Error bars, standard error for 3 replicates. (B) The cDNA from the experiment whose data are shown in panel A was used to amplify CXCR4 or β-actin by PCR. The resulting products were visualized via agarose gel electrophoresis. (C) Cells were treated as described above and then lysed, and total protein was harvested and immunoblotted for CXCR4 or total MAPK (protein loading control).
FIG 4
FIG 4
cmvIL-10 requires IL-10R and Stat3 to enhance CXCR4 signaling. (A and B) Calcium response in Fluo-4 AM-loaded HEK293 cells stimulated with 100 ng/ml CXCL12 ± 100 ng/ml cmvIL-10 and either IL-10R neutralizing antibody (NAb) (A), or IL-10R siRNA (B). Relative fluorescence units (RFU) measured by flow cytometry; the arrow indicates stimulus addition. (C) Lysates were harvested from siRNA-transfected cells and assessed for IL-10R and MAPK expression by immunoblotting. (D and E) Calcium response in Fluo-4 AM-loaded HEK293 cells stimulated with 100 ng/ml CXCL12 ± 100 ng/ml cmvIL-10 and Stat3 inhibitor (D) or Stat3 siRNA (E). (F) Lysates were harvested from siRNA-transfected cells and probed with antibodies to Stat3 or MAPK by immunoblotting. (G) Transwell migration of HEK293 cells toward CXCL12 in the lower chamber ± 100 ng/ml cmvIL-10 with 10 μM Stat3 inhibitor or DMSO. Error bars, standard error for 3 replicates; *, P < 0.01 by paired Student's t test versus CXCL12 alone (gray) and CXCL12 + cmvIL-10 + Stat3 Inhib (stripes).
FIG 5
FIG 5
cmvIL-10 enhances CXCR4 signaling in HCMV-infected cells. (A) NuFF cells were infected with TB40/E-mCherry (MOI = 1). Total RNA was harvested from mock- or HCMV-infected NuFFs at 72 h postinfection (hpi) and reverse transcribed, and UL123 (IE1) or β-actin genes were amplified. The resulting bands were visualized via agarose gel electrophoresis. (B) Mock- or HCMV-infected NuFF cells were stained with IL-10R-FITC or isotype control antibody and analyzed by flow cytometry. (C) Flow cytometric comparison of mock- or HCMV-infected NuFF cells for mCherry fluorescence as a measure of infection (left) or IL-10R stained as described above (right). (D) Calcium response was evaluated in Fluo-4 AM-loaded mock- or HCMV-infected cells stimulated with 100 ng/ml CXCL12 ± 100 ng/ml cmvIL-10 at 72 hpi. Relative fluorescence units (RFU) were measured by flow cytometry; the arrow indicates stimulus addition. (E) Transwell migration of mock-infected (gray bars) or HCMV-infected (black bars) NuFF cells toward CXCL12 in the lower chamber ± 100 ng/ml cmvIL-10 (striped bars, mock infected; white bars, HCMV infected) was measured at 72 hpi. Error bars, standard error for 3 replicates; *, P < 0.01 by paired Student's t test.
FIG 6
FIG 6
HCMV US27 and cmvIL-10 enhance CXCR4 signaling via distinct mechanisms. (A) NuFF cells were infected with HCMV TB40/E-mCherry (WT) or US27Δ (MOI = 1, 72 h). Total RNA was harvested, RT-PCR was performed with the indicated gene specific primers, and resulting bands were visualized via agarose gel electrophoresis. (B) Calcium release was measured in mock-infected or infected cells loaded with Fluo-4. Peak fluorescence intensity levels following stimulation with 100 ng/ml CXCL12 are shown. (C) Calcium release measured as described for panel B, with 100 ng/ml cmvIL-10. (D) Migration of cells through a transwell was measured using 5 × 104 mock-infected or infected cells in the upper chamber and 0.1 ng/ml CXCL12 ± 100 ng/ml cmvIL-10 in the lower chamber. Cells in the lower chamber were quantified after 4 h via Cell Titer Glo. (E and F) HEK293 cells expressing US27 or US28 were evaluated for transwell migration (E) and calcium flux (F) as described above. (G and H) Calcium flux in HEK293 or HEK293-US27 cells ± Stat3 inhibitor (S31-201) or DMSO (G) or in the presence of additional unlabeled (UN) HEK293 or HEK293-US27 cells (H). Error bars represent standard error; *, P < 0.01; ns, not significant by paired Student's t test.
FIG 7
FIG 7
cmvIL-10 enhances CXCR4 signaling in primary monocytes. (A) CD14+ monocytes were infected with HCMV TB40/E-mCherry (MOI = 5) for 24 h and then analyzed by flow cytometry. (B) Transwell migration of monocytes to the indicated doses of CXCL12 ± 100 ng/ml cmvIL-10 in the lower chamber. Cells in the lower chamber were quantified after 4 h via Cell Titer Glo. (C) Monocytes were stained with antibodies directed against CXCR4, IL-10R, or the appropriate isotype control and analyzed via flow cytometry at 24 hpi.
FIG 8
FIG 8
US27 is found in close proximity to CXCR4. (A) Immunofluorescence microscopy of HEK293-US27 or HEK293-US28 cells stained with anti-FLAG (green) and anti-CXCR4 (red) followed by goat anti-mouse FITC and donkey anti-goat TRITC secondary antibodies, respectively. Nuclei were visualized with DAPI (blue). Bar, 10 μm. (B) HEK293-US27 or HEK293-US28 cells were stained with anti-FLAG and anti-CXCR4 followed by oligonucleotide-conjugated secondary antibodies and amplification using the DuoLink kit for the proximity ligation assay (PLA). Red PLA spots indicate receptors in close proximity. (C) NuFF cells were infected with AD169-GFP (MOI = 1, 72 hpi) and stained with antibodies against CXCR4 and US27 (left) or CXCR4 and US28 (right) followed by DuoLink PLA. Infected cells are shown in green, and red indicates PLA spots. Images were captured using a Zeiss LSM700 laser scanning confocal microscope, and representative images are shown. For PLA (B, C), images are maximum-intensity projections from z-stacks.
FIG 9
FIG 9
Effects of US27 and cmvIL-10 on CXCR4 signaling during HCMV infection. (A) US27 is present in cell membranes immediately following virus fusion and entry. US27 can boost signaling output resulting from CXCL12 engagement of CXCR4, triggering higher levels of calcium release and enhanced chemotaxis to stromal cells expressing CXCL12. Infected cells ultimately produce cmvIL-10, which is secreted into the extracellular environment. cmvIL-10 can act through IL-10R and Stat3 to increase CXCL12/CXCR4 signaling in infected cells (autocrine signaling) but can also act in a paracrine manner on other bystander cells (B) which may be recruited to aid in processes such as virus dissemination or immune evasion. N, nucleus; C, cytoplasm. Dashed arrows indicate signaling events; solid arrows indicate functional outcomes.
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