Internalization of pseudorabies virus glycoprotein B is mediated by an interaction between the YQRL motif in its cytoplasmic domain and the clathrin-associated AP-2 adaptor complex

Abstract

The cytoplasmic domain of pseudorabies virus (PRV) glycoprotein B (gB) contains three putative internalization motifs. Previously, we demonstrated that the tyrosine-based YQRL motif at positions 902 to 905, but not the YMSI motif at positions 864 to 867 or the LL doublet at positions 887 and 888, is required for correct functioning of gB during antibody-mediated internalization of PRV cell surface-bound glycoproteins. In the present study, we demonstrate that the YQRL motif is also crucial to allow spontaneous internalization of PRV gB, and thus, that spontaneous and antibody-mediated internalizations of PRV gB occur through closely related mechanisms. Furthermore, we found that PRV gB colocalizes with the cellular clathrin-associated AP-2 adaptor complex and that this colocalization depends on the YQRL motif. In addition, by coimmunoprecipitation assays, we found that during both spontaneous and antibody-dependent internalization, PRV gB physically interacts with AP-2, and that efficient interaction between gB and AP-2 required an intact YQRL motif. Collectively, these findings demonstrate for the first time that during internalization of an alphaherpesvirus envelope protein, i.e., PRV gB, a specific amino acid sequence in the cytoplasmic tail of the protein interacts with AP-2 and may constitute a common AP-2-mediated mechanism of internalization of alphaherpesvirus envelope proteins.

FIG. 1.

Spontaneous (A) and antibody-dependent (B) internalization of PRV gB are both dependent on an intact YQRL motif. Confluent monolayers of PK15 cells were infected at an MOI of 10 with either PRV Be or PRV Y902A. At 6 (left column) and 10 (right column) h p.i., the efficiency of spontaneous internalization was studied by determining the levels of gB remaining at the cell surface (A), whereas antibody-dependent internalization was studied using an internalization assay based on FITC-labeled anti-gB antibodies, as described in Materials and Methods (B). The cells were analyzed by confocal microscopy. Bar, 10 μm.

FIG. 2.

Colocalization between gB and α-adaptin in PRV-infected cells. PK15 cells were infected at an MOI of 10 with either PRV Be, PRV Y864A, or PRV Y902A. All infected cells were processed for an antibody-dependent internalization assay and stained for gB (left column) as described in Materials and Methods. Internalization was allowed to proceed for 0 or 90 min at 37°C. Thereafter, the cells were costained for α-adaptin (middle column). To this end, the cells were incubated with monoclonal anti-α-adaptin IgG1 antibodies (clone AP-6), biotinylated rabbit anti-mouse IgG1-specific antibodies, and streptavidin-Texas Red. Merged images (right column), obtained by superposition of the green (gB) and red (α-adaptin) stainings, indicate areas of colocalization (yellow) between PRV gB and α-adaptin. The arrows indicate colocalization between wild-type or Y864A gB and α-adaptin in cytoplasmic internalization vesicles or at the cell surface, whereas the arrowheads indicate colocalization between Y902 gB and α-adaptin at the cell surface. Bar, 10 μm.

FIG. 3.

Colocalization between gB and α-adaptin in gB-transfected cells. PK15 cells were transiently transfected with either Be gB (pGVM3), Y864A gB (pGVM16), Y902A gB (pGVM17), or gB truncated (pGVM50). At 24 h posttransfection, the cells were incubated with FITC-labeled monoclonal anti-gB antibodies at 4°C for 1 h. Subsequently, the cells were shifted for 60 min to 37°C to allow internalization of gB (right column). Thereafter, the cells were fixed, permeabilized, and costained for α-adaptin (middle column) by the use of monoclonal anti-α-adaptin IgG1 antibodies, biotinylated IgG1-specific antibodies, and streptavidin-Texas Red. Merged images (right column), obtained by superposition of the green (gB) and red (α-adaptin) stainings, indicate areas of colocalization (yellow) between gB and α-adaptin. The arrows indicate colocalization between wild-type or Y864A gB and α-adaptin in cytoplasmic internalization vesicles. Bar, 10 μm.

FIG. 4.

PRV gB interacts with α-adaptin, and mutation of the Y902 residue strongly reduces this interaction. (A) Coimmunoprecipitation of α-adaptin with PRV gB during spontaneous gB internalization. At 6 h p.i., PK15 cells infected with either PRV Be, PRV Y864A, PRV Y902A, PRV LL887RR, PRV Y864A/Y902A, or PRV Y864A/Y902A/LL887RR were lysed and immunoprecipitated with monoclonal anti-gB antibodies, and the immunoprecipitates were analyzed for the presence of gB (upper row) and α-adaptin (lower row). (B) Coimmunoprecipitation of α-adaptin with PRV gB during antibody-mediated gB internalization. At 10 h p.i., PK15 cells infected with PRV Be, PRV LL887RR, PRV Y902A, or PRV Y864A/Y902A/LL887RR were incubated for 15 min at 37°C with monoclonal anti-gB antibodies to induce internalization. Subsequently, gB was immunoprecipitated as described for panel A, and the immunoprecipitates were analyzed for gB (upper row) and α-adaptin (lower row). (C) Coimmunoprecipitation of PRV gB with α-adaptin during spontaneous internalization of gB. At 6 h p.i., PK15 cells infected with either PRV Be or PRV Y902A were lysed and immunoprecipitated with monoclonal anti-α-adaptin antibodies, and the immunoprecipitates were analyzed for the presence of α-adaptin (upper row) and gB (lower row). (D) Densitometric quantification of coimmunoprecipitation (IP) of α-adaptin with wild-type or Y902A gB (left) and of wild-type or Y902A gB with α-adaptin (right) (both relative to wild-type gB band intensity).