In addition, the extracellular domain of US

Ding for many GPCRs [28,36,37]. In addition, the extracellular domain of US27 has been shown to be heavily glycosylated [38], a common characteristic among receptors that haveStapleton et al. Virology Journal 2012, 9:42 http://www.virologyj.com/content/9/1/Page 3 ofsmall peptide or chemokine ligands [39]. Although neither ligands nor any ligand-independent constitutive signaling [22] have been identified for this receptor so far, these observations suggest that HCMV US27 may be involved in the modification of host chemokine signaling. The US27 gene product exhibits greatest amino acid sequence identity with US28, followed closely by human chemokine receptors CCR1 and CCR4 (29 sequence identity for each). US27 is expressed late in infection [40], and the 2.9 kb transcript was found to be spliced within the 5‘ untranslated region, a possible indication that post-transcriptional regulation of US27 gene expression occurs [41]. Both UL33 and US27 have been shown to be present in the virion particle and in endocytic 2-Bromo-1,3-difluoro-4-nitrobenzene membrane compartments of infected cells, suggesting that they may play a role during virus infection [24,38,42]. Viral mutants that lack the US27 gene are replication competent [14]; however, a slight growth defect was observed at low multiplicities of infection compared to wild type virus [43]. While the US27 deficient virus could spread directly from cell to cell, the mutant failed to spread by an extracellular route. Interestingly, no replication deficiency PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/20618955 was noted in a virus lacking both US27 and US28 [14]. Although US27 is not required for lytic virus infection in vitro, this does not preclude an important role for this receptor in subverting the host immune response in vivo. In this study, the US27 protein was expressed in mammalian cells. Due to the predominantly intracellular localization noted here and by others [42], a series of 4-Bromopicolinaldehyde deletion mutants and chimeric receptors were created in order to identify specific protein domains required for the subcellular distribution. We found that the C-terminal intracellular domain contained sequences that were both necessary and sufficient for directing US27 to the endosomes.Results 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 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13867361 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 (Figure1A). 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.