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. 2015 Jul 1;195(1):227-36.
doi: 10.4049/jimmunol.1400291. Epub 2015 May 18.

Novel Human Cytomegalovirus Viral Chemokines, vCXCL-1s, Display Functional Selectivity for Neutrophil Signaling and Function

Jinho Heo  1 , Pranay Dogra  1 , Tom J Masi  1 , Elisabeth A Pitt  1 , Petra de Kruijf  2 , Martine J Smit  2 , Tim E Sparer  3
Affiliations

Novel Human Cytomegalovirus Viral Chemokines, vCXCL-1s, Display Functional Selectivity for Neutrophil Signaling and Function

Jinho Heo et al. J Immunol. .

Abstract

Human CMV (HCMV) uses members of the hematopoietic system including neutrophils for dissemination throughout the body. HCMV encodes a viral chemokine, vCXCL-1, that is postulated to attract neutrophils for dissemination within the host. The gene encoding vCXCL-1, UL146, is one of the most variable genes in the HCMV genome. Why HCMV has evolved this hypervariability and how this affects the virus' dissemination and pathogenesis is unknown. Because the vCXCL-1 hypervariability maps to important binding and activation domains, we hypothesized that vCXCL-1s differentially activate neutrophils, which could contribute to HCMV dissemination, pathogenesis, or both. To test whether these viral chemokines affect neutrophil function, we generated vCXCL-1 proteins from 11 different clades from clinical isolates from infants infected congenitally with HCMV. All vCXCL-1s were able to induce calcium flux at a concentration of 100 nM and integrin expression on human peripheral blood neutrophils, despite differences in affinity for the CXCR1 and CXCR2 receptors. In fact, their affinity for CXCR1 or CXCR2 did not correlate directly with chemotaxis, G protein-dependent and independent (β-arrestin-2) activation, or secondary chemokine (CCL22) expression. Our data suggest that vCXCL-1 polymorphisms affect the binding affinity, receptor usage, and differential peripheral blood neutrophil activation that could contribute to HCMV dissemination and pathogenesis.

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Figures

Figure 1
Figure 1. Amino-acid alignment of the mature forms of recombinant vCXCL-1s and the host chemokines CXCL1 and CXCL8 with vCXCL-1Toledo
Seven amino acid residues that are 100% conserved are indicated with an *. The important ELR, N-loop region, and 30s and 40s loops are indicated at the top.
Figure 2
Figure 2. CXCL8, CXCL1, and the different vCXCL-1s induce intracellular calcium mobilization on human PBNs
Changes in fluorescence were measured over time after exposure to different concentrations of chemokines (after 20 seconds at baseline as indicated with an arrow). Data shown are representative figures of three independent experiments.
Figure 3
Figure 3. vCXCL-1s elicit changes in surface expression of CD11a and CD11b
PBNs were incubated with 100 nM viral or host chemokine for 2 hours. The shaded curve represents expression levels of integrins on unstimulated PBNs. Table below lists the percentage change in mean fluorescence intensity (chemokine stimulated mean fluorescence intensity/unstimulated mean fluorescence intensity x 100). Graphs are representative of three independent experiments.
Figure 4
Figure 4. Differential chemotaxis of PBNs to vCXCL-1s partially correlates with affinity
Chemotactic response of human PBNs to 500 nM and 100nM of CXCL8, CXCL1, or vCXCL-1s. The chemotactic response was measured as fluorescence intensity of migrated PBN labeled with CalceinAM. Background chemotaxis was subtracted from all samples. Data shown are representative data of three independent experiments performed in triplicate.
Figure 5
Figure 5. vCXCL-1s have different binding affinities for human CXCR1 or CXCR2
Displacement of 125I-CXCL8 binding to HEK293 cells stably expressing human CXCR2 (A) or CXCR1 (B). Cells were incubated with indicated concentration of vCXCL-1s and 200 pM 125I-CXCL8 for one hour at room temperature. For simplicity, curves for TX11, C956, 102410, and C954 are not shown. (C) The average IC50 +/− standard error for all vCXCL-1s for either CXCR2 or CXCR1 (n=3–12). Those with incomplete competition curves are indicated with a > of a predicted IC50. Those chemokines with no competition at all concentrations tested are listed as >1000 IC50.
Figure 6
Figure 6. G protein activation and β-arrestin2 signaling correlates with CXCR2 affinity
vCXCL-1 chemokine induction of 35S-GTPγS binding to HEK293T membranes expressing CXCR2 (A–B). Data are corrected for basal 35S-GTPyS binding (n=3–4). vCXCL-1 β-arrestin2 recruitment in PathHunter™ indicator cells (C–D). Data are expressed as percentage of β-galactosidase activity, in which the response to 1 μM CXCL8 is set to 100% (n=3–4).
Figure 7
Figure 7. vCXCL-1s differentially induce CCL22 expression via CXCR2
Neutrophil-like HL60 T2 cells were incubated in the presence of 100 nM of the indicated vCXCL-1s or host chemokine. Each bar represents the average from three separate experiments of the fold change in CCL22 mRNA expression levels (stimulated/unstimulated cells +/− SEM). All data are normalized to β-actin mRNA expression levels. A ratio of 1, indicated with a gray line, represents no change in expression compared to unstimulated cells.
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References

    1. Mocarski ES., Jr Immunomodulation by cytomegaloviruses: manipulative strategies beyond evasion. Trends Microbiol. 2002;10:332–339. - PubMed
    1. Miller-Kittrell M, Sparer TE. Feeling manipulated: cytomegalovirus immune manipulation. Virol J. 2009;6:4. - PMC - PubMed
    1. McSharry BP, Avdic S, Slobedman B. Human cytomegalovirus encoded homologs of cytokines, chemokines and their receptors: roles in immunomodulation. Viruses. 2012;4:2448–2470. - PMC - PubMed
    1. Hansen SG, Powers CJ, Richards R, Ventura AB, Ford JC, Siess D, Axthelm MK, Nelson JA, Jarvis MA, Picker LJ, Fruh K. Evasion of CD8+ T cells is critical for superinfection by cytomegalovirus. Science. 2010;328:102–106. - PMC - PubMed
    1. Cha TA, Tom E, Kemble GW, Duke GM, Mocarski ES, Spaete RR. Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J Virol. 1996;70:78–83. - PMC - PubMed

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