Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Feb 16:9:42.
doi: 10.1186/1743-422X-9-42.

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

Lance K Stapleton  1 , Kathleen L Arnolds, Angela P Lares, Tori M Devito, Juliet V Spencer
Affiliations

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

Lance K Stapleton et al. Virol J. .

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.

PubMed Disclaimer

Figures

Figure 1
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.
Figure 2
Figure 2
Expression of US27 deletion mutants in transfected cells. HEK293 cells were seeded onto glass coverslips, transfected with the indicated p3XFLAG constructs, and then fixed and stained with permeabilization with anti-FLAG antibodies as described. Scale bars = 10 μm. The amino acid sequence of the C-terminal intracellular domain of US27 and each truncation mutant is shown.
Figure 3
Figure 3
US27 is found at the cell surface only transiently. Stably transfected HEK293 cells were grown on glass coverslips in culture medium containing anti-FLAG antibodies for one hour, then washed, fixed, permeabilized, and stained with the appropriate secondary antibody. Scale bars = 10 μm.
Figure 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.
Figure 6
Figure 6
US27 mutants do not co-localize with the endosome marker. HEK293 cells were transfected with p3XFLAG constructs expressing the chimeric receptor US27/CXCR3-CT, deletion mutant US27ΔCT, or US27Δ348. Cells were co-stained with anti-FLAG and antibodies to the early endosomal antigen as described. Endosomes appear red, nuclei blue, and US27 mutants green. Scale bars = 10 μm.
Figure 4
Figure 4
Cell surface staining of US27. A) HEK293 cells were transiently transfected with the indicated p3XFLAG constructs, harvested via gently scraping, staining with anti-FLAG antibodies, then examined via flow cytometry using a BD FACSCalibur. Data was analyzed using CellQuestPro. B) Transiently transfected cells grown on glass coverslips were fixed and stained in the absence of any detergents or permeabilization steps. After treatment with FITC-conjugated anti-mouse secondary antibodies, the coverslips were mounted with Prolong Gold containing DAPI. Scale bars = 10 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Staras SA, Dollard SC, Radford KW, Flanders WD, Pass RF, Cannon MJ. Seroprevalence of cytomegalovirus infection in the United States, 1988-1994. Clin Infect Dis. 2006;43:1143–1151. doi: 10.1086/508173. - DOI - PubMed
    1. de la Hoz RE, Stephens G, Sherlock C. Diagnosis and treatment approaches of CMV infections in adult patients. J Clin Virol. 2002;25:(Suppl 2):S1–12. - PubMed
    1. Damato EG, Winnen CW. Cytomegalovirus infection: perinatal implications. J Obstet Gynecol Neonatal Nurs. 2002;31:86–92. doi: 10.1111/j.1552-6909.2002.tb00026.x. - DOI - PubMed
    1. Hengel H, Brune W, Koszinowski UH. Immune evasion by cytomegalovirus-survival strategies of a highly adapted opportunist. Trends Microbiol. 1998;6:190–197. doi: 10.1016/S0966-842X(98)01255-4. - DOI - PubMed
    1. Ahn K, Angulo A, Ghazal P, Peterson PA, Yang Y, Fruh K. Human cytomegalovirus inhibits antigen presentation by a sequential multistep process. Proc Natl Acad Sci USA. 1996;93:10990–10995. doi: 10.1073/pnas.93.20.10990. - DOI - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources