Glycoprotein D actively induces rapid internalization of two nectin-1 isoforms during herpes simplex virus entry

Abstract

Entry of herpes simplex virus (HSV) occurs either by fusion at the plasma membrane or by endocytosis and fusion with an endosome. Binding of glycoprotein D (gD) to a receptor such as nectin-1 is essential in both cases. We show that virion gD triggered the rapid down-regulation of nectin-1 with kinetics similar to those of virus entry. In contrast, nectin-1 was not constitutively recycled from the surface of uninfected cells. Both the nectin-1alpha and beta isoforms were internalized in response to gD despite having different cytoplasmic tails. However, deletion of the nectin-1 cytoplasmic tail slowed down-regulation of nectin-1 and internalization of virions. These data suggest that nectin-1 interaction with a cytoplasmic protein is not required for its down-regulation. Overall, this study shows that gD binding actively induces the rapid internalization of various forms of nectin-1. We suggest that HSV activates a nectin-1 internalization pathway to use for endocytic entry.

Figure 1

gD on HSV virions down-regulates nectin-1. Virus was added to NGC12 cells for 45 min at 4°C before shifting to 37°C for indicated time. Cells were then stained with anti-GFP pAb ab290 followed by anti-rabbit Ig-PE to detect GFP-nectin-1α and analyzed by FACS. The level of GFP-nectin-1α detected on the cell surface when no virus was added was set to 100%. Average of 3 experiments +/− SE. (A) HSV KOS MOI=50 or KOSgDβ equivalent of MOI=50 was added to cells for 30 min. (B) Various MOIs of HSV KOS were added cells for 30 min. (C) MOI=50 of HSV KOS was added to cells for various time points. Average of 3 experiments +/− SE. (D) Rate of HSV entry. HSV KOS (100 pfu/well) was added to cell monolayers and extracellular virus was acid inactivated at various timepoints. Cells were then overlaid and plaques were allowed to form (Highlander et al., 1989; Huang and Wagner, 1964; Milne et al., 2005; Milne et al., 2003). A representative experiment of 3 is shown. (E) Soluble gD down-regulates nectin-1. Soluble gD285t (Rux et al., 1998) (100 μg/ml) was added to NGC12 cells for 30 min at 37°C. Cells were stained for FACS as in A-C. The level of GFP-nectin-1α detected on the cell surface in the absence of gD was set to 100%. Data represent the average of 4 experiments +/− SE.

Figure 2

HSV and nectin-1 colocalize inside the cell. Virus was added to NGC12 cells for 45 min at 4°C before shifting to 37°C for 15 min. Cells were then fixed and permeabilized. GFP-nectin-1 is shown in green and virus stained with anti-gD mAb MC5 and Alexa 594 is shown in red. White arrowheads point to spots of colocalization (yellow) between nectin-1 and gD staining. A single confocal slice from a representative experiment of two is shown.

Figure 3

Characterization of nectin-1α and β cell lines. (A) Diagram of nectin-1α and β constructs. Glycosylation sites are represented by lollipops. Cells line marked with * were previously described by Krummenacher et al. (2003). ‘G’ in the cell line name indicates a GFP tag. (B) Surface expression of nectin-1 was detected by FACS using mAb CK41-PE. Histogram of PE fluorescence intensity is shown. B78 are the nectin-1 negative parental cells.

Figure 4

HSV is endocytosed during entry into nectin-1 cell lines. Virus was allowed to attach to cells at 4°C before infection was allowed to proceed for 15 min at 37°C. Cells were then treated with proteinase K (+) or mock digested (−). Cells were lysed in the presence of protease inhibitors and gB was detected by immunoprecipitation with mAb DL16 and western blot (pAb R69). The presence of gB in lane 3 indicates virion endocytosis. PK means proteinase K and the bar on the right indicates the position of the 115 kDa molecular weight marker.

Figure 5

Decreased expression of nectin-1α and β after co-culture with gD expressing cells. Cells expressing GFP tagged forms of nectin-1α (CG23, NGC12, NGC-389t) or nectin-1β (N1BG, N1BG-373t) were co-cultured with B78 cells (no gD) (A) or wild type gD expressing cells (B). After 16 hr of co-culture, cells were fixed and permeabilized. Nectin-1-GFP is shown in green and nuclei are stained with DAPI (blue). A representative experiment is shown. (C) Down-regulation of nectin-1 detected by FACS. Target cells were labeled with Qtracker655 and mixed with effector cells expressing the indicated forms of gD or with B78 cells at a target:effector ratio of 1:2. After overnight co-culture, cells were stained with mAb CK41-PE to detect nectin-1. Target nectin-1 cells were positively selected based on Qtracker655 fluorescence while unlabelled effector (gD expressing) cells were excluded. Bar graphs represent PE fluorescence of Qtracker655-positive target nectin-1 expressing cells as a percent of the PE fluorescence in co-cultures with B78 cells from the average of 3 experiments +/− SE.

Figure 6

Rate of nectin-1 down-regulation from the cell surface. Target cells were labeled with Qtracker655 and co-cultured with effector cells expressing the indicated forms of gD or with B78 cells (no gD) at a target:effector ratio of 1:2. At each time point, cells were stained with mAb CK41-PE to detect nectin-1 and analyzed by FACS. Target nectin-1 cells were positively selected based on Qtracker655 fluorescence. The level of nectin-1 surface expression (PE fluorescence) on cells co-cultured with B78 cells was set at 100% as in figure 5. For each target cell line, we normalized the level of fluorescence to that at time 0 for comparison between effector cells. An average of 3 experiments is shown +/− SE.

Figure 7

Nectin-1 internalization is triggered by gD. (A) Steps of nectin-1 internalization assay. (B) NGC12 cells were biotinylated and co-cultured with B78 (left panel) or B78-gD(wt) (middle panel) cells for various times before treatment with Proteinase K. Cells were lysed in the presence of protease inhibitors and internalized biotinylated GFP-nectin-1α was detected by pull-down with neutravidin beads and western blot (mAb CK8). Right panel shows control conditions: biotinylated, mock digested (lane 1); biotinylated, held at 4°C, digested with Proteinase K (lane 2); not biotinylated (lane 3). Arrowhead points to GFP-nectin-1α and the bar on the left indicates the 115 kDa molecular weight marker.