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. 2009 Oct;83(19):10016-27.
doi: 10.1128/JVI.00354-09. Epub 2009 Jul 15.

The carboxy-terminal tail of human cytomegalovirus (HCMV) US28 regulates both chemokine-independent and chemokine-dependent signaling in HCMV-infected cells

Melissa P Stropes  1 , Olivia D Schneider, William A Zagorski, Jeanette L C Miller, William E Miller
Affiliations

The carboxy-terminal tail of human cytomegalovirus (HCMV) US28 regulates both chemokine-independent and chemokine-dependent signaling in HCMV-infected cells

Melissa P Stropes et al. J Virol. 2009 Oct.

Abstract

The human cytomegalovirus (HCMV)-encoded G-protein-coupled receptor (GPCR) US28 is a potent activator of a number of signaling pathways in HCMV-infected cells. The intracellular carboxy-terminal domain of US28 contains residues critical for the regulation of US28 signaling in heterologous expression systems; however, the role that this domain plays during HCMV infection remains unknown. For this study, we constructed an HCMV recombinant virus encoding a carboxy-terminal domain truncation mutant of US28, FLAG-US28/1-314, to investigate the role that this domain plays in US28 signaling. We demonstrate that US28/1-314 exhibits a more potent phospholipase C-beta (PLC-beta) signal than does wild-type US28, indicating that the carboxy-terminal domain plays an important role in regulating agonist-independent signaling in infected cells. Moreover, HMCV-infected cells expressing the US28/1-314 mutant exhibit a prolonged calcium signal in response to CCL5, indicating that the US28 carboxy-terminal domain also regulates agonist-dependent signaling. Finally, while the chemokine CX3CL1 behaves as an inverse agonist or inhibitor of constitutive US28 signaling to PLC-beta, we demonstrate that CX3CL1 functions as an agonist with regard to US28-stimulated calcium release. This study is the first to demonstrate that the carboxy terminus of US28 controls US28 signaling in the context of HCMV infection and indicates that chemokines such as CX3CL1 can decrease constitutive US28 signals and yet simultaneously promote nonconstitutive US28 signals.

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Figures

FIG. 1.
FIG. 1.
Strategy for construction of the HCMV recombinant FLAG-US28/1-314 in HCMV FIX-BAC. (A) Schematic of US28 depicting the Ser/Thr-rich carboxy-terminal regulatory region. Amino acids 315 to 354 that are deleted in the US28/1-314 mutant are expanded underneath the diagram. (B) Schematic representation of the recombination strategy used to manipulate US28 and construct the US28/1-314 mutant.
FIG. 2.
FIG. 2.
Construction and analyses of the FLAG-US28/1-314 HCMV recombinant in infected cells. (A) FIX-BAC DNAs isolated from E. coli were used as templates in all PCRs. Primers with homology to 5′ and 3′ sequences flanking the US28 coding region were used to PCR amplify the US28 locus (upper panel). The primer with homology to the 5′ flanking sequence was used in combination with a FLAG-specific primer to verify the addition of an N-terminal FLAG epitope (middle panel). Amplification of the UL146 gene serves as a positive PCR control (lower panel). (B) Immunoprecipitation followed by Western blotting with FLAG-specific antibodies was performed to detect expression of the various forms of FLAG-US28 encoded by HCMV ΔUS28, HCMV FLAG-US28/WT, and HCMV FLAG-US28/1-314 viruses at 48 h postinfection (upper panel). Whole-cell lysates from the same samples were subjected to Western blotting with an α-IE1/IE2 or α-UL44 antibodies (lower panels). The results shown are representative of six independent experiments.
FIG. 3.
FIG. 3.
US28/WT and US28/1-314 proteins exhibit similar cell surface expression and ligand-binding activity in infected cells. (A) HFFs were infected with HCMV ΔUS28 or HCMV FLAG-US28/WT (top panel) and with HCMVΔUS28 or HCMV FLAG-US28/1-314 (bottom panel) viruses for 48 h. Surface expression of FLAG-US28 on infected cells was detected by staining with FLAG-specific M2-biotin, followed by streptavidin-PE and analyzed by FACS. The histograms shown are representative of six independent experiments performed in duplicate. (B) Infected HFFs were incubated with 28 pM [125I]CCL5 in the absence or presence of 14 nM unlabeled RANTES to discriminate between specific and nonspecific binding. The data shown represent specific binding of [125I]CCL5, as assessed by liquid scintillation chromatography, and are derived from six independent experiments performed in duplicate and represent the means ± the standard errors of the mean (SEM).
FIG. 4.
FIG. 4.
Endocytic properties of US28/WT and US28/1-314 proteins in HCMV-infected cells. HFFs were infected with the HCMV FLAG-US28/WT or HCMV FLAG-US28/1-314 viruses for 48 h. Infected cells were then labeled at 4°C with [125I]CCL5, washed, and then warmed to 37°C for various time to allow for endocytosis of the US28 proteins. Internalized ligand was assessed by acid washing to remove surface-bound ligand and is presented as the percentage of total ligand bound. The data shown are derived from six independent experiments performed in duplicate and represent the means ± the SEM.
FIG. 5.
FIG. 5.
US28/1-314 exhibits increased levels of constitutive PLC-β signaling in HCMV-infected cells. HFFs were infected with increasing MOIs (0.03 to 0.3 PFU/cell) using HCMV FLAG-US28/WT or HCMV FLAG-US28/1-314 viruses. Medium containing 1 μCi of [3H]myoinositol/ml was added at 24 h postinfection, and the accumulated inositol phosphates were determined at 48 h postinfection by using anion-exchange chromatography. The data shown are derived from 10 independent experiments performed in duplicate and represent the means ± the SEM.
FIG. 6.
FIG. 6.
US28/1-314 exhibits prolonged levels of calcium signaling in response to CCL5/RANTES. (A) HFFs were infected with HCMV ΔUS28, HCMV FLAG-US28/WT, or HCMV FLAG-US28/1-314 viruses at an MOI of 0.1. Medium containing 1μCi of [3H]myoinositol/ml was added 24 h postinfection. At 45 h postinfection, the cells were left untreated or were stimulated with 10 nM CCL5 for 3 h in the presence of LiCl. Accumulated inositol phosphates were determined at 48 h postinfection by using anion-exchange chromatography as described above. The data shown are derived from seven independent experiments performed in duplicate and represent the means ± the SEM. (B) HFFs were infected with the above-described viruses at an MOI of 3. At 48 h postinfection, the effects of CCL5 was measured by labeling cells with Fluo-4 AM and analyzing calcium signaling after addition of 10 nM CCL5 using a FlexStation II fluorometer. The calcium traces are representative of at least three independent experiments performed in duplicate. (C) The peak calcium response after the addition of CCL5 is displayed graphically. The data shown are derived from eight independent experiments performed in duplicate and represent the means ± the SEM. n.s., not significant.
FIG. 7.
FIG. 7.
CX3CL1/Fractalkine exhibits both agonist and inverse agonist properties toward US28 in HCMV-infected cells. (A) HFFs were infected with HCMV ΔUS28, HCMV FLAG-US28/WT, or HCMV FLAG-US28/1-314 virus at an MOI of 0.1. Medium containing 1 μCi of [3H]myoinositol/ml was added 24 h postinfection. At 45 h postinfection, the cells were left untreated or stimulated with 10 nM CX3CL1 for 3 h in the presence of LiCl (left panel). HFFs similarly infected with FLAG-US28/WT HCMV were incubated with 10 nM CX3CL1 in the presence or absence of 50 nM CCL5 for 3 h in the presence of LiCl (right panel). Accumulated inositol phosphates were determined at 48 h postinfection by using anion-exchange chromatography as described above. The data shown are derived from at least four independent experiments performed in duplicate and represent the means ± the SEM. (B) HFFs were infected with the above-described viruses at an MOI of 3. At 48 h postinfection, the effects of CX3CL1 were measured by labeling cells with Fluo-4 AM and analyzing calcium signaling after addition of 10 nM CX3CL1 using a FlexStation II fluorometer. The calcium traces are representative of at least three independent experiments performed in duplicate. (C) The peak calcium response after the addition of CX3CL1 is displayed graphically. The data shown are derived from eight independent experiments performed in duplicate and represent the means ± the SEM.
FIG. 8.
FIG. 8.
CCL5 and CX3CL1 binding to US28 triggers calcium release by activating PTx-insensitive Gq proteins. (A) HFFs were infected with HCMV FLAG-US28/WT or HCMV FLAG-US28/1-314 viruses at an MOI of 3 and left either untreated (solid symbols) or treated overnight with 200 ng of PTx/ml (open symbols). At 48 h postinfection, cells were labeled with Fluo-4 AM and stimulated with 10 nM CCL5 (top panel) or 10 nM CX3CL1 (lower panel), and the calcium flux was measured by using a FlexStation II fluorometer. (B) To control for the effects of PTx, uninfected HFFs left untreated (closed symbols) or treated with 200 ng of PTx/ml (open symbols) were stimulated with 10 nM LPA, and the calcium flux was measured as described above. The calcium traces are representative of at least four independent experiments performed in duplicate.
FIG. 9.
FIG. 9.
CCL5 and CX3CL1 binding to US28 triggers calcium release via a PLC-β/IP3 signaling pathway. HFFs were infected with HCMV FLAG-US28/WT or HCMV FLAG-US28/1-314 viruses at an MOI of 3. (A) At 48 h postinfection, cells were labeled with Fluo-4 AM in the absence (closed symbols) or presence (open symbols) of the PLC-β inhibitor U73122. Cells were then stimulated with 10 nM CCL5 (upper panel) or 10 nM CX3CL1 (lower panel) and analyzed by using a FlexStation II fluorometer. (B) At 48 h postinfection, cells were labeled with Fluo-4 AM in the absence (closed symbols) or presence (open symbols) of the IP3 channel blocker 2-ABP. Cells were then stimulated with 10 nM CCL5 (upper panel) or 10 nM CX3CL1 (lower panel) and analyzed by using a FlexStation II fluorometer. The calcium traces shown are representative of at least four independent experiments performed in duplicate.
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