Abstract
BackgroundHuman 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.ResultsIn 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.ConclusionsThe 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.
Highlights
Human cytomegalovirus (HCMV) is ubiquitous in the population but generally causes only mild or asymptomatic infection except in immune suppressed individuals
In order to characterize the biological activities of the HCMV US27 receptor, we first constructed an expression plasmid encoding US27
A chimera containing amino acids 1-336 of CXCR3 and 304-362 of pUS27 (CXCR3/US27-CT) was found to exhibit intracellular localization similar to that of wild type pUS27 (Figure 1F). These findings strongly suggest that sequences within the C-terminal intracellular domain of HCMV US27 contribute to the intracellular receptor distribution
Summary
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 UL18 gene product is a homolog of the MHC class I protein that is postulated to act as a decoy on the cell surface to assist in the evasion of natural killer cells [6]. The HCMV genome encodes four putative GPCRs containing seven transmembrane domains and similarity to human chemokine receptors: US27, US28, UL33, and UL78 [10]. Chemokines are a class of cytokines important in their regulation of motility and activation of immune cells, and the existence of viral homologs of chemokine receptors indicates additional mechanisms for manipulation of the host immune response
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