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.

Capripoxvirus G-protein-coupled chemokine receptor: a host-range gene suitable for virus animal origin discrimination

“…This is in clear contrast with the predicted structure of the prototype monkey CCR8 with its 7-TM domains (Case et al, 2008;Oppermann, 2004;Schoneberg et al, 1999). Although shorter than in other known GPCRs, the C-tail of CaPV GPCR is still rich in serine residues, which, upon phosphorylation, is involved in the clathrin-mediated endocytosis of the receptor (Fraile-Ramos et al, 2001). In the prediction, the four conserved cysteine residues putatively involved in disulfide bond formation remain in the extracellular compartment, which is in agreement with a proper 3D cylindrical folding model of the receptor (Cabrera-Vera et al, 2003), although the exact conformation of the CaPV GPCR still remains to be established.…”

Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63

“…The mouse monoclonal anti-anxA2 antibody HH7 (1:1,000) has been described previously 31 . Mouse monoclonal antibodies used for the detection of LBPA (clone 6C4; 1:50) 21 were kindly provided by Jean Gruenberg (University of Geneva, Geneva, Switzerland) and mouse monoclonal antibodies used for the visualization of internalized CD63 (clone 1B5; 1:300; 1:100 for surface staining) 32 were a kind gift from Mark Marsh (University College London). Rat UZ4 monoclonal antibodies used for the detection of surface E-selectin (1:2) were kindly provided by Rupert Hallmann (University Hospital Münster, Germany) 33 .…”

Constitutive Endocytic Cycle of the CB1 Cannabinoid Receptor

“…tonic) endocytosis in the absence of ligand has also been observed for other wild type GPCRs such as chemokine CXCR4 receptor (44), thyrotropin receptor (25), M2 muscarinic receptor (45), thrombin receptor (46), and thromboxane A2 receptor (47). However, these studies do not address the relationship between constitutive activity and tonic endocytosis.…”