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.

Figure 1

Schematic representation and Western blot of US28 constructs. (A) GFP was linked to the C terminus of US28 (US28-GFP). The two N-terminal Ig domains of CD4 or a nine-amino acid epitope from the influenza virus HA were fused to the N-terminal ectodomain of US28 (CD4-US28 and HA-US28). (B) Western blots of lysates of HeLa cells transiently transfected with US28-GFP or CD4-US28. The CD4 moiety was detected with Q4120 and GFP with a rabbit antibody against GFP. Molecular weight standards (the masses are given in kDa) migrated as indicated on the left. The mass of the US28 moiety predicted from the amino acid sequence is ∼41 kDa; two domains of CD4 or the GFP moiety have predicted masses of 22 and 26 kDa, respectively.

Figure 2

Subcellular localization and quantification of US28. Localization of US28-GFP in transiently transfected HeLa cells (A). HeLa cells expressing CD4-US28 were stained with an antibody against CD4 either without (B) or after permeabilization with saponin (C). Scale bars = 10 μm. (D) ¹²⁵I-Q4120 was used to quantify CD4-US28 distribution. Intact HeLa CD4-US28 cells were incubated with ¹²⁵I-Q4120 to determine the protein at the cell surface, and cells permeabilized with saponin were used to determine the total protein present in the cell. HeLa cells, intact or permeabilized with saponin, were incubated with ¹²⁵I-Q4120 to determine the unspecific binding. The plots show the bound radioactivity as counts per minute per well after subtraction of the nonspecific binding.

Figure 3

Colocalization of US28 with markers for late endosomes and lysosomes. HeLa cells transfected with US28-GFP (left-hand panels and in green) were stained with an antibody against CD63 and LAMP-1 (A and B, respectively, middle panels and in red), markers for late endosomes and lysosomes. Vesicles positive for both markers are seen in yellow on the right, merged panels. Scale bars, 10 μm.

Figure 4

EM immunolocalization of US28. (A and B) Cryosections of HeLa cells transiently expressing US28-GFP were stained with an anti-GFP antibody and 10-nm protein A-gold particles (PAG₁₀). Most of the gold particles were found associated with multivesicular endosomes, both over the limiting membrane and over internal vesicles. Some gold particles were also found at the plasma membrane (including coated pits, asterisk in A) and in small tubular structures and vesicles that could correspond to endosomes (arrows in A). (C) Cryosections of HeLa cells stained with the anti-CD63 antibody 1B5 and PAG₁₀ also showed enriched labeling of multivesicular bodies. Scale bars, 200 nm.

Figure 5

Internalization of US28. HeLa cells stably expressing CD4-US28 were incubated with an antibody against CD4, Q4120, at 37°C for 1 h to allow internalization. After acid washing to remove the antibody bound to the cell surface the cells were fixed. Cells permeabilized with saponin (A) or intact (B, top and bottom show the same field) were stained with a rhodamine-conjugated secondary antibody to detect Q4120. Scale bars, 10 μm.

Figure 6

Endocytosis of US28. (A) HeLa CD4-US28 cells were labeled at 4°C with ¹²⁵I-Q4120 (□) or ¹²⁵I-RANTES (⋄), washed, and warmed to 37°C to allow endocytosis. The amount of internalized ligand was determined by acid washing as described in MATERIALS AND METHODS. (B) Mv-1-Lu cells expressing the hybrid CD4(2D)CXCR4 were labeled at 4°C with ¹²⁵I-Q4120 for 2 h and then warmed to 37°C in BM (□), BM containing 100 ng/ml phorbol myristate acetate (⋄), or 250 nM SDF-1 (○). At the indicated times the cells were cooled and the internalized antibody was determined. The plots show the acid-resistant radioactivity (internal) as a proportion of the total cell-associated activity. All data points show means and SDs for duplicate samples from a representative experiment.

Figure 7

Down-modulation of US28 and CCR5. (A) HeLa CD4-US28 cells were incubated in medium (□), or in medium containing 0.3 nM (⋄), or 2.5 nM (○) unlabeled Q4120 antibody at 37°C for the indicated times. The cells were acid washed and the cell surface CD4-US28 molecules were then detected by labeling with ¹²⁵I-Q4120 antibody for 2 h at 4°C. (B) HeLa CD4-US28 cells were incubated in medium (□), medium containing 100 nM RANTES (⋄), or 100 nM fractalkine (○) for up to 30 min at 37°C. At the indicated times the cells were cooled on ice, washed with cold medium, and labeled as in A with ¹²⁵I-Q4120 to detect cell surface CD4-US28. (C) CHO-CCR5 cells were incubated in medium (□) or medium containing 100 nM RANTES (⋄) for the indicated times at 37°C. The cells were cooled on ice, washed with cold medium, and then labeled with ¹²⁵I-MC-5 at 4°C to determine the cell surface levels of CCR5. Each point shows the mean and SD of triplicate samples from a representative experiment.

Figure 8

Recycling of US28. HeLa CD4-US28 cells were incubated with Q4120 antibody at 37°C for 1 h to allow antibody uptake, cooled on ice, and washed in acid medium to remove the Q4120 bound to the cell surface. (A) The cells were fixed, left intact or permeabilized with saponin (right and left, respectively), and then stained with a biotin-conjugated secondary antibody to detect Q4120 followed by fluorescein-streptavidin. (B) The cells were reincubated with a biotin-conjugated secondary antibody for 1 h at 37°C, cooled on ice, acid washed, fixed, and stained with fluorescein-streptavidin as in A. Scale bars, 10 μm. The righthand fields for each panel show Nomarski and fluorescence images of the same field.

Figure 9

Endocytosis of US28 in the presence of ligands. HeLa CD4-US28 cells were labeled with ¹²⁵I-Q4120 in medium (□), medium containing 100 nM RANTES (⋄), or 100 nM fractalkine (○) for 2 h at 4°C, washed, and then warmed to 37°C. The amount of internalized antibody was determined by acid wash. All data points show means and SDs for duplicate samples from a representative experiment.

Figure 10

Intracellular localization of US28 in HCMV-infected fibroblasts. Localization of US28-YFP in transiently transfected human foreskin fibroblasts, infected with the Towne strain of HCMV. Antibody against HCMV IE1 used to detect HCMV infected cells is seen as red nuclear staining. Hoechst staining of all nuclei is seen in blue. (A–C) US28-YFP (green) is seen as mainly punctate intracellular staining in both uninfected (A) and HCMV-infected fibroblasts at both 24 h (B) and 48 h (C) postinfection. Scale bar, 10 μm.