Figure 1.
FACS analysis of Fcγ and IgG binding.
A: Binding of Fcγ to RL13 in permeabilized cells. HEK293T cells transfected with empty vector or expression vectors for myc-tagged gB, RL10, RL11, RL12 or RL13 were fixed, permeabilized and stained using FITC-conjugated anti-myc and 25 µg/ml DyLight 649-conjugated human IgG Fc fragment (Fcγ). FITC positive cells were compared to mock transfected cells for their ability to bind Fcγ. B: Binding of Fcγ to surface-exposed RL13. HEK293T cells transfected with empty vector or expression vectors for myc-tagged gB, RL10, RL11, RL12 or RL13 were first stained at 4°C with DyLight 649-conjugated human IgG Fc fragment (25 µg/ml). Excess of probe was removed by washing in PBS and then cells were fixed and stained with FITC-conjugated anti-myc. FITC positive cells were compared to mock transfected cells for their ability to bind Fcγ. C: Binding specificity of RL10-RL13 towards different human immunoglobulin subclasses. HEK293T cells were transiently transfected with myc-tagged RL11 (blue), RL12 (green), RL13 (brown) or with empty vector (magenta). Cells were fixed, permeabilized and stained with FITC-conjugated anti-myc together with 10 µg/ml of human Ig of the different subclasses. Alexa fluor 647-conjugated goat anti-human was used as secondary antibody. FITC positive cells were compared to mock transfected cells for their ability to bind Fcγ.
Table 1.
Ig-binding specificity of Fc-binding proteins RL11, RL12 and RL13.
Figure 2.
Predicted structure of the RL13 protein and schematic representation of TR RL13 ectodomain mutants.
A: Schematic representation of the HCMV TR RL13 full length protein. The signal peptide at the N-terminus (SP), the Ig-like domain, the transmembrane domain (Tm), the tyrosine-based motif YxxL (Ym) sorting signal, 11 potential N-linked glycosylation sites (diamonds), and the O-linked glycosylation region (closed circles) are indicated. B: Multiple sequence alignment of the predicted RL13 Ig-like domain from the indicated strains of HCMV. The residues are colored according to the conservation level (red for higher conservation). Asterisks (*) below the alignments represent conserved amino acid in all sequences; colons (:) represent residues with similar physicochemical properties; dots (.) semi-conserved residues. The black arrows represent the positions of predicted β strands along the sequence. C: Graphic representation of TR RL13 and the ectodomain mutants used in this study. The amino-terminal signal peptide, the carboxy-terminal transmembrane (gray boxes) and the Ig-like domain (white box) are indicated.
Figure 3.
Fcγ-binding by RL13 ectodomain mutants.
A: Cytofluorometric analysis of Fcγ binding by RL13 ectodomain mutants. HEK293T cells transfected with the indicated constructs were permeabilized and stained with DyLight 649-conjugated human IgG Fc fragment (Fcγ, 12.5 µg/ml) and Alexa Fluor 488-conjugated mouse anti-myc. Signals from 10,000 myc-positive cells are shown in each graph. The percentage of cells in each quadrant is indicated. B: Quantitative analysis of the FACS data. Each histogram shows the percentage of Fcγ positive cells relative to the RL13 wild type positive population (RL13). Values and error bars represent the mean and range of three independent experiments.
Figure 4.
Localization of RL13 in transfected cells.
A: Co-localization of RL13 with organelle markers and Fcγ in ARPE-19 cells. ARPE-19 epithelial cells were transfected with RL13-YFP fusion protein (green color, central column) and treated for confocal analysis 48 h later. Cells were fixed, permeabilized and stained with antibodies against either GM130, TGN46 or EEA1 intracellular markers (red color, second column) and with 20 µg/ml of DyLight 649-conjugated human IgG Fc fragment (magenta color, fourth column). The merged panels on the far left show co-localization between RL13 and, from top to bottom, markers of Golgi (GM130), trans-Golgi (TGN46) and early endosomes (EEA1) respectively (co-localization in yellow). The merged panels on the far right show co-localization of Fcγ with RL13 (co-localization in white). B: Flow cytometry analysis of RL13 surface-expression. HEK293T cells were transiently transfected with vector coding for YFP (green line) or RL13-YFP fusion protein (red and blue lines). Cells were allowed to recover for 48 h and then surface-exposed RL13 was stained either with 5 µg/ml of mouse monoclonal antibody directed against RL13 ectodomain (clone 5H3/B10, red and green lines) or isotype control at the same concentration (isotype, blue line) on ice. Alexa fluor 647 conjugated goat anti-mouse was used as secondary antibody. YFP positive cells were compared to empty vector transfected cells for their ability to bind 5H3/B10 antibody. C: Immunofluorescence analysis of RL13 surface expression on ARPE-19 cells. ARPE-19 cells expressing RL13-YFP fusion protein (red color) were stained on ice with 12.5 µg/ml of 5H3/B10 monoclonal antibody (green color) and 20 µg/ml of DyLight 649-conjugated human IgG Fc fragment (magenta color) without permeabilization. Merge panels show co-localization between 5H3/B10 and either RL13-YFP or Fcγ signals (yellow color in the far left panel and white color in the far right panel, respectively).
Figure 5.
RL13 binds and internalizes Fcγ.
ARPE-19 epithelial cells were transfected with RL13-YFP (green) and allowed to recover for 48 h. Cells were placed on ice 5 min before adding DyLight 649-conjugated human IgG Fc fragment (red) and incubated on ice for 30 min. Then, cells were washed and A: immediately fixed, B: shifted to 37°C for 30 min, or C: shifted to 37°C for 90 min before proceeding to fixation and confocal analysis. Rab5 (purple) was revealed by specific antibodies after permeabilization of the samples. Examples of co-localization between signals are indicated by white arrows. Orthogonal projections of the optical sections acquired from Z-stack are shown. White arrows indicate examples of co-localized spots. Fluorescent Fcγ was absent on the membrane of ARPE-19 cells transfected with empty expression vector used as control and no staining was detected following 30 min switch at 37°C (data not shown).
Figure 6.
RL13 C-terminal YxxL motif is required for internalization.
A: Local multiple sequence alignment of the RL13 C-terminus. The gray box indicates the transmembrane domain, the black box the YxxL internalization motif. Darker color indicates higher conservation. Asterisks (*) below the alignments represent conserved amino acid in all sequences; colons (:) represent residues with similar physicochemical properties; dots (.) semi-conserved residues. B: Graphical representation of RL13 mutations used in this study. RL13 TR cytoplasmic tail sequence is shown with mutated amino acids in bold. Amino acids unchanged from the wild-type sequence are represented by a dot. Deleted amino acids are represented by a |. C: Internalization efficiency of RL13 variants. HEK293T cells were transfected with the indicated plasmids coding for wild type and mutated RL13. 48 h post-transfection, cells were detached and incubated on ice for 60 min with human Fcγ fragment. Cells were then washed and incubated in medium at 37°C for 30 min to allow for endocytosis (T37). The control sample remained on ice (T0). Following incubation, cells were cooled quickly by rinsing twice with cold PBS; Fcγ that remained on the cell surface after endocytosis was stained with Alexa Fluor 647-conjugated goat anti-human IgG and analyzed by flow cytometry. The % of internalization was calculated from the mean fluorescence intensities of transfected cells with the following formula: (T0–T37)/T0×100%. Value retrieved from RL13 wild type was set to 100%. Values are the mean and range of three independent experiments. Significant differences, P<0,001 (two-tailed unpaired Student's t-test), compared to RL13 are indicated by *.
Figure 7.
Fcγ binding is a conserved function for recombinant RL13 from Merlin and TR clinical strains.
A: HEK293T cells were transfected with plasmids coding for myc-His tagged RL13 from TR (red), Merlin (blue) strains and empty vector (green). 48 h post-transfection cells were collected, permeabilized and stained with FITC-conjugated mouse anti-myc and 25 µg/ml of DyLight 649-conjugated human IgG Fc fragment. 10,000 FITC-positive cells were compared to empty vector transfected cells. B: HEK293T cells were transfected with empty vector or plasmids coding for myc-His tagged RL13 from TR and Merlin strains. 48 h post-transfection cells were collected, lysed and subjected to immunoprecipitation with biotinylated Fcγ fragment. Total lysates and elution fractions were subjected to western blot analysis using HRP-conjugated anti-his tag (α-his) and anti-human IgG (α-h) antibodies. TR: sample expressing myc-His tagged RL13 from TR strain; Merlin: sample expressing myc-His tagged RL13 from Merlin strain; Empty: empty vector transfected cells.