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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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US27 co-localizes with Golgi and endosome markers. A stable p3XFLAG-US27 HEK293 cell line was stained with anti-FLAG antibodies and antibodies to the indicated organelles followed by FITC conjugated anti-mouse and TRITC-conjugated anti-rabbit secondary antibodies. Blue represents the DAPI-stained nuclei, green represents US27, and red represents organelles. The merged images result from imaging that compiles all three color images into one. Areas where green and red co-localize appear yellow. Scale bars = 10 μm.
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Figure 5: US27 co-localizes with Golgi and endosome markers. A stable p3XFLAG-US27 HEK293 cell line was stained with anti-FLAG antibodies and antibodies to the indicated organelles followed by FITC conjugated anti-mouse and TRITC-conjugated anti-rabbit secondary antibodies. Blue represents the DAPI-stained nuclei, green represents US27, and red represents organelles. The merged images result from imaging that compiles all three color images into one. Areas where green and red co-localize appear yellow. Scale bars = 10 μm.

Mentions: To more precisely determine the subcellular location of wild type pUS27, HEK293 cells that stably expressed FLAG-tagged US27 proteins were analyzed by immunofluorescence microscopy. The US27 protein was found in compartments adjacent to the nucleus. These same compartments appeared red when labeled with an antibody specific for GMP130, a marker for cis-medial Golgi (Figure 5). A merged image revealed extensive co-localization of pUS27 with the Golgi apparatus, which is consistent with the observation that this protein is heavily glycosylated [38]. In addition, there was evidence that pUS27 was also present in the endosomes, which were stained with antibody to EEA1, early endosomal antigen 1. The distribution pattern was specific for the Golgi apparatus and endosomal compartments, as pUS27 did not significantly co-localize with a marker for the endoplasmic reticulum (calreticulin) or the lysosomes (LAMP2, lysosomal associated membrane protein 2). The results demonstrate that intracellular pUS27 is found mainly in the Golgi apparatus and endosomes.


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)

US27 co-localizes with Golgi and endosome markers. A stable p3XFLAG-US27 HEK293 cell line was stained with anti-FLAG antibodies and antibodies to the indicated organelles followed by FITC conjugated anti-mouse and TRITC-conjugated anti-rabbit secondary antibodies. Blue represents the DAPI-stained nuclei, green represents US27, and red represents organelles. The merged images result from imaging that compiles all three color images into one. Areas where green and red co-localize appear yellow. Scale bars = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: US27 co-localizes with Golgi and endosome markers. A stable p3XFLAG-US27 HEK293 cell line was stained with anti-FLAG antibodies and antibodies to the indicated organelles followed by FITC conjugated anti-mouse and TRITC-conjugated anti-rabbit secondary antibodies. Blue represents the DAPI-stained nuclei, green represents US27, and red represents organelles. The merged images result from imaging that compiles all three color images into one. Areas where green and red co-localize appear yellow. Scale bars = 10 μm.
Mentions: To more precisely determine the subcellular location of wild type pUS27, HEK293 cells that stably expressed FLAG-tagged US27 proteins were analyzed by immunofluorescence microscopy. The US27 protein was found in compartments adjacent to the nucleus. These same compartments appeared red when labeled with an antibody specific for GMP130, a marker for cis-medial Golgi (Figure 5). A merged image revealed extensive co-localization of pUS27 with the Golgi apparatus, which is consistent with the observation that this protein is heavily glycosylated [38]. In addition, there was evidence that pUS27 was also present in the endosomes, which were stained with antibody to EEA1, early endosomal antigen 1. The distribution pattern was specific for the Golgi apparatus and endosomal compartments, as pUS27 did not significantly co-localize with a marker for the endoplasmic reticulum (calreticulin) or the lysosomes (LAMP2, lysosomal associated membrane protein 2). The results demonstrate that intracellular pUS27 is found mainly in the Golgi apparatus and endosomes.

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