title: The human cytomegalovirus US28 protein is located in endocytic vesicles and undergoes constitutive endocytosis and recycling.
abstract: Genes encoding chemokine receptor-like proteins have been found in herpes and poxviruses and implicated in viral pathogenesis. Here we describe the cellular distribution and trafficking of a human cytomegalovirus (HCMV) chemokine receptor encoded by the US28 gene, after transient and stable expression in transfected HeLa and Cos cells. Immunofluorescence staining indicated that this viral protein accumulated intracellularly in vesicular structures in the perinuclear region of the cell and showed overlap with markers for endocytic organelles. By immunogold electron microscopy US28 was seen mostly to localize to multivesicular endosomes. A minor portion of the protein (at most 20%) was also expressed at the cell surface. Antibody-feeding experiments indicated that cell surface US28 undergoes constitutive ligand-independent endocytosis. Biochemical analysis with the use of iodinated ligands showed that US28 was rapidly internalized. The high-affinity ligand of US28, the CX(3)C-chemokine fractalkine, reduced the steady-state levels of US28 at the cell surface, apparently by inhibiting the recycling of internalized receptor. Endocytosis and cycling of HCMV US28 could play a role in the sequestration of host chemokines, thereby modulating antiviral immune responses. In addition, the distribution of US28 mainly on endosomal membranes may allow it to be incorporated into the viral envelope during HCMV assembly.
authors.affiliation: Medical Research Council Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, London WC1E 6BT, United Kingdom.
authors.forename: A
authors.initials: A
authors.lastname: Fraile-Ramos
authors.forename: T N
authors.initials: TN
authors.lastname: Kledal
authors.forename: A
authors.initials: A
authors.lastname: Pelchen-Matthews
authors.forename: K
authors.initials: K
authors.lastname: Bowers
authors.forename: T W
authors.initials: TW
authors.lastname: Schwartz
authors.forename: M
authors.initials: M
authors.lastname: Marsh
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C / Q000378:metabolism; D006224:Cricetinae; D003587:Cytomegalovirus / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism / Q000821:virology; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D019707:Receptors, Chemokine / Q000096:biosynthesis*; D013696:Temperature; D013997:Time Factors; D014764:Viral Proteins / Q000096:biosynthesis*
chemical_list: C515538:CX3CL1 protein, human; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C; D018076:DNA, Complementary; D008024:Ligands; D008565:Membrane Proteins; D019707:Receptors, Chemokine; C411191:US28 receptor, Cytomegalovirus; D014764:Viral Proteins