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Receptor chimeras demonstrate that the C-terminal domain of the human cytomegalovirus US27 gene product is necessary and sufficient for intracellular receptor localization.

Stapleton LK, Arnolds KL, Lares AP, Devito TM, Spencer JV - Virol. J. (2012)

Bottom Line: However, swapping of the C-terminal intracellular domains resulted in a significant change in receptor distribution.When the C-terminal domain of US27 was fused to CXCR3, this chimeric receptor (CXCR3/US27-CT) was found in the same intracellular pattern as wild-type US27.Co-localization with organelle-specific markers revealed that wild-type US27 was found predominantly in the Golgi apparatus and in endosomal compartments, whereas the US27/CXCR3-CT chimera, US27ΔCT and US27Δ348 mutants were not localized to endosomal compartments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of San Francisco, CA 94117, USA.

ABSTRACT

Background: Human cytomegalovirus (HCMV) is ubiquitous in the population but generally causes only mild or asymptomatic infection except in immune suppressed individuals. HCMV employs numerous strategies for manipulating infected cells, including mimicry of G-protein coupled receptors (GPCRs). The HCMV US27 gene product is a putative GPCR, yet no ligand or signaling has been identified for this receptor. In the present study, immunofluorescence microscopy was used to examine the cellular distribution of wild type US27, as well as US27 deletion mutants and chimeric receptors.

Results: In transiently transfected cells, wild type US27 was found primarily in intracellular compartments, in striking contrast to the cell surface distribution seen for the human cellular chemokine receptor CXCR3. When the N-terminal extracellular domains of the two receptors were swapped, no change in protein localization was observed. However, swapping of the C-terminal intracellular domains resulted in a significant change in receptor distribution. A chimera that contained US27 fused to the C-terminal intracellular tail of CXCR3 exhibited surface distribution similar to that of wild-type CXCR3. When the C-terminal domain of US27 was fused to CXCR3, this chimeric receptor (CXCR3/US27-CT) was found in the same intracellular pattern as wild-type US27. In addition, a US27 mutant lacking the C-terminus (US27ΔCT) failed to accumulate inside the cell and exhibited cell surface distribution. Co-localization with organelle-specific markers revealed that wild-type US27 was found predominantly in the Golgi apparatus and in endosomal compartments, whereas the US27/CXCR3-CT chimera, US27ΔCT and US27Δ348 mutants were not localized to endosomal compartments.

Conclusions: The results indicate that the C-terminal domain of the HCMV US27 protein, which contains a di-leucine endocytic sorting motif, is both necessary and sufficient for intracellular localization, which may also help explain why no cellular ligands have yet been identified for this viral receptor.

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Immunofluorescence images of cells expressing US27 and US27 chimeras. HEK293 cells were seeded onto glass coverslips, transfected with the indicated pcDNA constructs, and then fixed and stained with anti-myc antibodies. After treatment with FITC-conjugated anti-mouse secondary antibodies, the coverslips were mounted with Prolong Gold containing DAPI. Line drawings beside each photo depict each 7TM receptor and the chimeras that were made using domains of US27 (black) and CXCR3 (red). Scale bars = 10 μm.
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Figure 1: Immunofluorescence images of cells expressing US27 and US27 chimeras. HEK293 cells were seeded onto glass coverslips, transfected with the indicated pcDNA constructs, and then fixed and stained with anti-myc antibodies. After treatment with FITC-conjugated anti-mouse secondary antibodies, the coverslips were mounted with Prolong Gold containing DAPI. Line drawings beside each photo depict each 7TM receptor and the chimeras that were made using domains of US27 (black) and CXCR3 (red). Scale bars = 10 μm.

Mentions: In order to characterize the biological activities of the HCMV US27 receptor, we first constructed an expression plasmid encoding US27. The US27 gene from strain AD169 was cloned into the pcDNA3.1 vector, allowing for expression of the US27 protein as a fusion with a C-terminal myc/HIS epitope tag. US27 protein expression was investigated via immunofluorescence microscopy using human embryonic kidney cells (HEK293) grown on glass cover slips and transiently transfected with pcDNA-US27. After 48 hours, the cells were fixed, permeabilized, and stained with an anti-myc monoclonal antibody followed by a FITC-conjugated goat anti-mouse secondary antibody. The US27 protein (pUS27) was expressed in transfected cells and was found in a predominantly intracellular pattern (Figure 1A). Nuclei appeared blue due to DAPI staining, and the green fluorescence representing pUS27 was concentrated around the nucleus and in adjacent compartments. The epitope tag is unlikely to have altered distribution of pUS27, as transfection of HEK293 cells with either p3XFLAG-US27, which enabled expression of the protein with an N-terminal FLAG tag (Figures 2 and 3), or pEGFP-US27 (data not shown) resulted in the same intracellular localization pattern. These results are consistent with the perinuclear localization for pUS27 previously described in both transfected and infected cells by Fraile-Ramos et al. [42].


Receptor chimeras demonstrate that the C-terminal domain of the human cytomegalovirus US27 gene product is necessary and sufficient for intracellular receptor localization.

Stapleton LK, Arnolds KL, Lares AP, Devito TM, Spencer JV - Virol. J. (2012)

Immunofluorescence images of cells expressing US27 and US27 chimeras. HEK293 cells were seeded onto glass coverslips, transfected with the indicated pcDNA constructs, and then fixed and stained with anti-myc antibodies. After treatment with FITC-conjugated anti-mouse secondary antibodies, the coverslips were mounted with Prolong Gold containing DAPI. Line drawings beside each photo depict each 7TM receptor and the chimeras that were made using domains of US27 (black) and CXCR3 (red). Scale bars = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298792&req=5

Figure 1: Immunofluorescence images of cells expressing US27 and US27 chimeras. HEK293 cells were seeded onto glass coverslips, transfected with the indicated pcDNA constructs, and then fixed and stained with anti-myc antibodies. After treatment with FITC-conjugated anti-mouse secondary antibodies, the coverslips were mounted with Prolong Gold containing DAPI. Line drawings beside each photo depict each 7TM receptor and the chimeras that were made using domains of US27 (black) and CXCR3 (red). Scale bars = 10 μm.
Mentions: In order to characterize the biological activities of the HCMV US27 receptor, we first constructed an expression plasmid encoding US27. The US27 gene from strain AD169 was cloned into the pcDNA3.1 vector, allowing for expression of the US27 protein as a fusion with a C-terminal myc/HIS epitope tag. US27 protein expression was investigated via immunofluorescence microscopy using human embryonic kidney cells (HEK293) grown on glass cover slips and transiently transfected with pcDNA-US27. After 48 hours, the cells were fixed, permeabilized, and stained with an anti-myc monoclonal antibody followed by a FITC-conjugated goat anti-mouse secondary antibody. The US27 protein (pUS27) was expressed in transfected cells and was found in a predominantly intracellular pattern (Figure 1A). Nuclei appeared blue due to DAPI staining, and the green fluorescence representing pUS27 was concentrated around the nucleus and in adjacent compartments. The epitope tag is unlikely to have altered distribution of pUS27, as transfection of HEK293 cells with either p3XFLAG-US27, which enabled expression of the protein with an N-terminal FLAG tag (Figures 2 and 3), or pEGFP-US27 (data not shown) resulted in the same intracellular localization pattern. These results are consistent with the perinuclear localization for pUS27 previously described in both transfected and infected cells by Fraile-Ramos et al. [42].

Bottom Line: However, swapping of the C-terminal intracellular domains resulted in a significant change in receptor distribution.When the C-terminal domain of US27 was fused to CXCR3, this chimeric receptor (CXCR3/US27-CT) was found in the same intracellular pattern as wild-type US27.Co-localization with organelle-specific markers revealed that wild-type US27 was found predominantly in the Golgi apparatus and in endosomal compartments, whereas the US27/CXCR3-CT chimera, US27ΔCT and US27Δ348 mutants were not localized to endosomal compartments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of San Francisco, CA 94117, USA.

ABSTRACT

Background: Human cytomegalovirus (HCMV) is ubiquitous in the population but generally causes only mild or asymptomatic infection except in immune suppressed individuals. HCMV employs numerous strategies for manipulating infected cells, including mimicry of G-protein coupled receptors (GPCRs). The HCMV US27 gene product is a putative GPCR, yet no ligand or signaling has been identified for this receptor. In the present study, immunofluorescence microscopy was used to examine the cellular distribution of wild type US27, as well as US27 deletion mutants and chimeric receptors.

Results: In transiently transfected cells, wild type US27 was found primarily in intracellular compartments, in striking contrast to the cell surface distribution seen for the human cellular chemokine receptor CXCR3. When the N-terminal extracellular domains of the two receptors were swapped, no change in protein localization was observed. However, swapping of the C-terminal intracellular domains resulted in a significant change in receptor distribution. A chimera that contained US27 fused to the C-terminal intracellular tail of CXCR3 exhibited surface distribution similar to that of wild-type CXCR3. When the C-terminal domain of US27 was fused to CXCR3, this chimeric receptor (CXCR3/US27-CT) was found in the same intracellular pattern as wild-type US27. In addition, a US27 mutant lacking the C-terminus (US27ΔCT) failed to accumulate inside the cell and exhibited cell surface distribution. Co-localization with organelle-specific markers revealed that wild-type US27 was found predominantly in the Golgi apparatus and in endosomal compartments, whereas the US27/CXCR3-CT chimera, US27ΔCT and US27Δ348 mutants were not localized to endosomal compartments.

Conclusions: The results indicate that the C-terminal domain of the HCMV US27 protein, which contains a di-leucine endocytic sorting motif, is both necessary and sufficient for intracellular localization, which may also help explain why no cellular ligands have yet been identified for this viral receptor.

Show MeSH
Related in: MedlinePlus