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. 2012;7(11):e50524.
doi: 10.1371/journal.pone.0050524. Epub 2012 Nov 29.

US28 is a potent activator of phospholipase C during HCMV infection of clinically relevant target cells

William E Miller  1 , William A Zagorski, Joanna D Brenneman, Diana Avery, Jeanette L C Miller, Christine M O'Connor
Affiliations

US28 is a potent activator of phospholipase C during HCMV infection of clinically relevant target cells

William E Miller et al. PLoS One. 2012.

Abstract

Members of the cytomegalovirus family each encode two or more genes with significant homology to G-protein coupled receptors (GPCRs). In rodent models of pathogenesis, these viral encoded GPCRs play functionally significant roles, as their deletion results in crippled viruses that cannot traffic properly and/or replicate in virally important target cells. Of the four HCMV encoded GPCRs, US28 has garnered the most attention due to the fact that it exhibits both agonist-independent and agonist-dependent signaling activity and has been demonstrated to promote cellular migration and proliferation. Thus, it appears that the CMV GPCRs play important roles in viral replication in vivo as well as promote the development of virus-associated pathology. In the current study we have utilized a series of HCMV/US28 recombinants to investigate the expression profile and signaling activities of US28 in a number of cell types relevant to HCMV infection including smooth muscle cells, endothelial cells and cells derived from glioblastoma multiforme (GBM) tumors. The results indicate that US28 is expressed and exhibits constitutive agonist-independent signaling activity through PLC-β in all cell types tested. Moreover, while CCL5/RANTES and CX3CL1/Fractalkine both promote US28-dependent Ca(++) release in smooth muscle cells, this agonist-dependent effect appears to be cell-specific as we fail to detect US28 driven Ca(++) release in the GBM cells. We have also investigated the effects of US28 on signaling via endogenous GPCRs including those in the LPA receptor family. Our data indicate that US28 can enhance signaling via endogenous LPA receptors. Taken together, our results indicate that US28 induces a variety of signaling events in all cell types tested suggesting that US28 signaling likely plays a significant role during HCMV infection and dissemination in vivo.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. pUS28 is expressed in smooth muscle and glioblastoma cells.
A) HASMCs and U373MG were infected with FIX-US28Flag at a MOI of 3 and pUS28 expression was analyzed by immunoprecipitation/western blot at the indicated times post-infection (upper panels). IE1/IE2 expression was analyzed by western blot and is shown for reference (lower panels). B) HASMCs and U373MG were infected with the indicated viruses at a MOI of 3 and pUS28 expression was analyzed by immunoprecipitation/western blot (upper panels) and IE1/IE2 expression was analyzed by western blot (lower panels). In each case, pUS28 expression was detected by IP for the Flag epitope, followed by immunoblot with a Flag antibody.
Figure 2
Figure 2. US28 constitutively activates PLC-β in a ligand independent but G-protein coupling dependent manner in HCMV infected smooth muscle and glioblastoma cells.
A) HASMCs and B) U373MG were infected with FIX-US28Flag and each of the US28 mutant viruses at MOIs of 0.6 and 3.0. PLC-β activation was measured by labeling cells 24 hpi with 1 µCi/ml 3H-myoinositol followed by isolation of total inositol phosphates at 48 hpi. PLC-β activity is represented as the percent conversion of input myoinositol into inositol phosphates. The data presented are the results from at least three independent experiments performed in duplicate. The results are presented graphically and represent the mean+/−S.E. of four independent experiments performed in duplicate.
Figure 3
Figure 3. CCL5/RANTES or CX3CL1/Fractalkine binding to US28 triggers calcium release in HCMV infected smooth muscle but not glioblastoma cells.
A) HASMC or B) U373MG cells were infected with the indicated FIX-based viruses at a MOI of 3. At 48 hpi, cells were labeled with Fluo-4 AM and stimulated with 10 nM CCL5/RANTES (top panels) or 10 nM CX3CL1/Fractalkine (bottom panels). Calcium responses were measured using a FlexStation II fluorometer. The calcium traces represent change in relative fluorescence units (ΔRFU) for each condition tested and are representative of at least three independent experiments performed in duplicate.
Figure 4
Figure 4. US28-mediated calcium release in infected HASMCs is activated by PTx insensitive G proteins, PLC-β, and IP3.
To gain further insight into the agonist effects of CCL5 and CX3CL1 on US28-promoted Ca++ release, HASMCs were MOCK infected or infected with FIX-US28Flag virus. At 48 hpi cells were labeled with Fluo-4AM, treated with the Gαi inhibitor PTx (A), the PLC-β inhibitor U73122 (B), or the IP3 channel inhibitor 2ABP (C) and stimulated with 10 nM CCL5 or CX3CL1. Calcium responses were measured using a FlexStation fluorometer. The calcium traces represent change in relative fluorescence units (ΔRFU) for each condition tested and are representative of at least three independent experiments performed in duplicate.
Figure 5
Figure 5. US28 potentiates signaling via endogenous Gαi coupled LPA receptors in HCMV infected arterial smooth muscle cells.
A) HASMCs were infected with the indicated viruses at a MOI of 3. At 48 hpi, cells were labeled with Fluo-4 AM, stimulated with 1uM lysophosphatidic acid (LPA) and calcium flux was measured using a FlexStation II fluorometer. The results are also presented graphically (B) The results are presented graphically and represent the mean+/−S.E. of three to seven independent experiments performed in duplicate. C) To determine if the US28 effect was due to LPA signaling through endogenous Gαi proteins, we pretreated cells with the Gαi inhibitor PTx prior to stimulation with LPA. PTx completely abrogated the effects of US28 on LPA signaling indicating the involvement of Gαi in this process. The calcium traces represent change in relative fluorescence units (ΔRFU) for each condition tested and are representative of three to seven independent experiments performed in duplicate. **p<0.05..
Figure 6
Figure 6. Inhibition of viral replication in HCMV infected glioblastoma cells severely restricts pUS28 expression but only modestly affects the total magnitude of US28 signaling activity.
A) U373MG were mock infected or infected with FIX-US28Flag at a MOI of 3 and pUS28, IE1/IE2, and pUL44 expression was analyzed by immunoprecipitation/western blot (pUS28) or western blot (IE1/IE2 and pUL44) at the indicated times post-infection. To determine what effect inhibition of viral replication has on US28 expression in U373MG cells, cells were treated with or without the replication inhibitor phosphonoacetic acid (PAA) at a concentration of 2 mM, where indicated. B) US28 stimulated PLC-β activity was analyzed in the presence or absence of PAA. PLC-β activation was measured by labeling cells 24 hpi with 1 µCi/ml 3H-myoinositol followed by isolation of total inositol phosphates at 48 hpi. PLC-β activity is represented as the percent conversion of input myoinositol into inositol phosphates. The data presented are the results from three independent experiments performed in duplicate.
Figure 7
Figure 7. US28 is a potent activator of signaling even at very low expression levels.
A) Development of a HEK-based Tet-regulatable US28 expression system and analysis of DOX regulated changes in US28 protein levels. US28 expression was titrated using DOX concentrations ranging from 1 pg/ml to 1000 pg/ml. pUS28 expression was analyzed by western blot and the results indicate that 100 pg/ml DOX blocks US28 expression >90%, while 1000 pg/ml DOX blocks US28 expression >99%. Cellular GAPDH expression in whole cell lysates is shown for control purposes. B) US28 stimulated PLC-β activity was then analyzed over the range of pUS28 expression levels. Interestingly, while 100 pg/ml DOX blocks pUS28 expression >90%, the corresponding drop in US28 stimulated PLC-β was ∼45%. Similarly, while 1000 pg/ml blocks pUS28 expression >99%, the corresponding drop in US28 stimulated PLC-β was ∼75%. CCL5/RANTES was used at a concentration of 10 nM.
Figure 8
Figure 8. US28 expression and PLC-β signaling in fibroblasts and endothelial cells infected with TB40/E recombinant viruses.
A) HS68 HFFs infected at MOIs of 0.2, 1, and 5 (left panel) or HUVECs infected at MOIs of 0.6, 3, and 15 (right panel) were analyzed by western blot for IE1/2 or β-actin expression at 48 hpi. B) Extracts prepared from HFFs infected at a MOI of 5 (left panel) or HUVECs infected at a MOI of 15 (right panel) were analyzed by immunoprecipitation/western blot for pUS28 expression at 48 hpi. C) HS68 HFFs (left panel) or HUVECs (right panel) infected with increasing MOIs as described in panel A were analyzed for PLC-β activity at 48 hpi. In each cell type, PLC-β activation was measured by labeling cells 24 hpi with 1 µCi/ml 3H-myoinositol followed by isolation of total inositol phosphates at 48 hpi. PLC-β activity is represented as the percent conversion of input myoinositol into inositol phosphates.
Figure 9
Figure 9. Viral replication in fibroblasts and endothelial cells infected with TB40/E recombinant viruses.
A) To examine growth kinetics of wild-type and ΔUS28 viruses, HFFs (left panel, MOI = 0.01) or HUVECs (right panel, MOI = 0.1) were infected with TB40/Ewt-mCherry or the ΔUS28 recombinants, samples of tissue culture supernatant were collected at the indicated time points, and viral progeny was assayed by infecting fibroblasts and quantifying IE1-positive cells 24 h later by immunofluorescence. B) To examine growth kinetics of wild-type and ΔALL viruses, HFFs (left panel, MOI = 0.01) and HUVECs (right panel, MOI = 0.1) were infected with TB40/Ewt-mCherry or the ΔALL recombinants and viral progeny was assayed as in panel A. C) HFFs infected at a MOI of 3 (left panels) or HUVECs infected with an MOI of 15 (right panels) were analyzed by western blot for IE1/2, pUL44 or β-actin expression over a 96 hour time course following infection.
Figure 10
Figure 10. Summary of findings.
A summary of our findings in all cell types tested, included smooth muscle cells (HASMCs), GBM cells (U373MG), fibroblasts (HFFs), and endothelial cells (HUVECs). *Ca++ data and G-protein dependency in HFF cells was determined previously and published in Stropes et al. .
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