Contribution of endocytic motifs in the cytoplasmic tail of herpes simplex virus type 1 glycoprotein B to virus replication and cell-cell fusion

Abstract

The use of endocytic pathways by viral glycoproteins is thought to play various functions during viral infection. We previously showed in transfection assays that herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is transported from the cell surface back to the trans-Golgi network (TGN) and that two motifs of gB cytoplasmic tail, YTQV and LL, function distinctly in this process. To investigate the role of each of these gB trafficking signals in HSV-1 infection, we constructed recombinant viruses in which each motif was rendered nonfunctional by alanine mutagenesis. In infected cells, wild-type gB was internalized from the cell surface and concentrated in the TGN. Disruption of YTQV abolished internalization of gB during infection, whereas disruption of LL induced accumulation of internalized gB in early recycling endosomes and impaired its return to the TGN. The growth of both recombinants was moderately diminished. Moreover, the fusion phenotype of cells infected with the gB recombinants differed from that of cells infected with the wild-type virus. Cells infected with the YTQV-mutated virus displayed reduced cell-cell fusion, whereas giant syncytia were observed in cells infected with the LL-mutated virus. Furthermore, blocking gB internalization or impairing gB recycling to the cell surface, using drugs or a transdominant negative form of Rab11, significantly reduced cell-cell fusion. These results favor a role for endocytosis in virus replication and suggest that gB intracellular trafficking is involved in the regulation of cell-cell fusion.

FIG. 1.

Endocytosis of gB during infection assayed by biotinylation. (A) Biotinylation of gB was assayed in 143B cells with noncleavable biotin 8 h after infection with KOS. Samples were immunoprecipitated with a polyclonal anti-gB antibody and then separated by sodium dodecyl sulfate-7% polyacrylamide gel electrophoresis under nonreducing conditions, and biotinylated glycoproteins were revealed with streptavidin-HRP by Western blotting (lanes 1 to 4). After stripping, the membrane was revealed with a monoclonal anti-gB antibody (lanes 5 to 8). Lanes: 1, 2, 5, and 6, uninfected control cells; lanes 3, 4, 7, and 8, infected cells; lanes 1, 3, 5, and 7, nonbiotinylated; lanes 2, 4, 6, and 8, biotinylated. (B) 143B cells were biotinylated with Sulfo-NHS-SS-cleavable biotin 8 h after infection with KOS and left for 4 h for infection to proceed. After treatment of the cells with GSH, lysed samples were gB immunoprecipitated and visualized with streptavidin-HRP (lane 3). As controls, cells were gB immunoprecipitated immediately after the biotinylation step, without (lane 1) or after (lane 2) GSH treatment. Lane 4 shows the gB Western blot of lane 3 after stripping.

FIG. 2.

Confocal microscopy analysis of gB internalization in KOS-infected cells. 143B cells were infected with wild-type KOS virus at an MOI of 0.1. At 7 h postinfection, the cells were incubated with an anti-gB antibody at 4°C for 45 min and then placed at 37°C for 0 min (A, E, I, M, and Q), 15 min (B, F, J, N, and R), 30 min (C, G, K, O, and S), or 60 min (D, H, L, P, and T). Cells were fixed in methanol and labeled with anti-TGN 46 and anti-VP5 antibodies. Panels A to D correspond to phase-contrast images of infected cells, whereas panels E to P show indirect immunofluorescence from anti-VP5 and Cy2-labeled secondary antibodies (green) (E to H), from anti-TGN 46 and Cy5-labeled secondary antibodies (pseudo-colored in blue) (I to L), or from anti-gB and Cy3-labeled secondary antibodies (red) (M to P). (Q to T) Merged images. Fluorescence was visualized with a Leica TCS SP2 AOBS confocal microscope.

FIG. 3.

Internalization of gB in KgBY889A-infected cells. The same experiment as that described in Fig. 1 was reproduced in 143B cells infected with a virus expressing a mutated YTQV→ATQV gB protein. Subcellular localization of surface-stained gB was analyzed by confocal microscopy as described in the legend for Fig. 1. (A, E, I, M, and Q) Incubation for 0 min at 37°C; (B, F, J, N, and R) 15-min incubation at 37°C; (C, G, K, O, and S) 30-min incubation at 37°C; (D, H, L, P, and T) 60-min incubation at 37°C. Cells were fixed with methanol and then stained with anti-TGN 46 and anti-VP5 antibodies. Panels A to D show phase-contrast images of the different time intervals analyzed. Panels E to H show immunostaining with VP5 (green). Panels I to L show immunostaining with TGN 46 (blue). Panels M to P show immunostaining with anti-gB antibodies (red). (Q to T) Merged images.

FIG. 4.

Internalization of gB in KgBLL871AA-infected cells. Seven hours after infection of 143B cultures by a mutant virus in which the gB LL motif was replaced by AA, cells were processed as described in the legend of Fig. 1. (A, E, I, M, and Q) Incubation for 0 min at 37°C; (B, F, J, N, and R) 15-min incubation at 37°C; (C, G, K, O, and S) 30-min incubation at 37°C; (D, H, L, P, and T) 60-min incubation at 37°C. Panels A to D show phase-contrast images. Anti-VP5, anti-TGN 46, and anti-gB staining is shown in panels E to H, I to L, and M to P, respectively. (Q to T) Merged images.

FIG. 5.

Cointernalization of gB and transferrin receptor in infected cells. To characterize the vesicular compartment where gB was internalized, the same experiments as in Fig. 2, 3, and 4 were repeated except that the cells were incubated with a CD71-specific antibody, in addition to the anti-gB antibody, for 60 min. After fixation, cells were stained with an anti-TGN antibody. Merged images of the anti-TGN (blue), anti-CD71 (green), and anti-gB (red) stains are shown for KOS-, KgBY889A-, and KgBLL871AA-infected cells in panels D, H, and L, respectively.

FIG. 6.

Subcellular distribution of gB in infected cells. 143B cells were infected with KOS, KgBY889A, or KgBLL871AA at an MOI of 0.1, fixed 7 h postinfection; permeabilized; and stained with anti-TGN, anti-CD71, and anti-gB antibodies. Immunostaining of TGN (blue), CD71 (green), and gB (red) is shown in panels A to C, D to E, and F to H, respectively. (I to K) Merged images.

FIG. 7.

Virus growth. 143B cells were infected with KOS, KgBY889A, or KgBLL871AA, and the infectivity of the progeny viruses was analyzed at different times postinfection by titration on Vero cells. (A) Cells were infected at an MOI of 5, and extra- and intracellular infectivities were assayed at 6, 9, 12, 16, 20, and 24 h postinfection. (B) Multiple-step growth analysis. Cells were infected at an MOI of 0.001, and progeny viruses were harvested 24, 48, and 72 h postinfection. The data shown correspond to averages of two or three independent experiments. Vertical lines indicate the standard deviations.

FIG. 8.

Incorporation of gB in virions. Extracellular virus recovered from infected cell supernatants at 8 h postinfection were subjected to polyacrylamide gel electrophoresis under denaturing conditions and transferred onto nitrocellulose membrane. The membrane was treated with anti-gB and anti-ICP5 antibodies and then with HRP-coupled secondary antibodies visualized by enhanced chemiluminescence. Quantification analysis was performed with Fujifilm Multi-Gauge software.

FIG. 9.

Infection phenotypes of wild-type and gB-mutated viruses in Cos-7 cells. Confluent cell monolayers were infected with KOS (A), KgBY889A (B), and KgBLL871AA (C) at an MOI of 1. Live cells were visualized by phase-contrast microscopy at 20 h postinfection.

FIG. 10.

Effects of chlorpromazine on infection phenotypes. Confluent Cos-7 cell monolayers were infected with KOS (A and B), KgBY889A (C and D), or KgBLL871AA (E and F) or were mock infected (G and H). At 2 h after infection, cells were either treated with 5 μg of chlorpromazine/ml (A, C, E, and G) or left untreated (B, D, F, and H). Cells were observed at 20 h postinfection.

FIG. 11.

Internalization assay of bafilomycin A1-treated infected cells. The internalization assay was repeated in KOS- or KgBLL871AA-infected cells as described in Fig. 2 and 4, except that the cells were treated with bafilomycin A1 at 2 h postinfection. Images taken at the latest time point of the assay (60 min) correspond to the merge of the gB (red) and TGN (blue) stainings. (A and B) Control untreated cells; (C and D) treated cells.

FIG. 12.

Effects of bafilomycin A1 on infection phenotypes. Confluent Cos-7 cell monolayers were infected with KOS (A to D), KgBY889A (E to H), or KgBLL871AA (I to L) or were mock infected (M to O). At 2 h after infection, cells were either left untreated (A, E, I, and M) or treated with 1 μg of DMSO/ml (B, F, and J) or 250 nM bafilomycin A (C, D, G, H, K, L, N, and O). Live cells were observed 20 h postinfection by phase-contrast microscopy (A to C, E to G, I to K, and M and N). To check for infection, cells were fixed with methanol and then incubated successively with an anti-VP5 antibody, a biotin-coupled secondary antibody, and β-galactosidase-coupled streptavidin; the staining was revealed with X-Gal (D, H, L, and O).

FIG. 13.

Subcellular distribution of gB in Rab11S25N-transfected infected cells. Cells were transfected with the control or Rab11 mutant-expressing plasmids as for Fig. 13, infected at 0.1 PFU, fixed at 7 h postinfection, permeabilized, and stained with anti-gB antibody (red).

FIG. 14.

Phenotypes of infections in infected cells expressing a transdominant-negative form of Rab11. Cos-7 cells were transfected with plasmids expressing either ECFP (A to H) or an ECFP-tagged transdominant-negative Rab11 mutant, ECFP-Rab11S25N (I to P). Cells were infected 24 h after transfection with KOS (A, E, I, and M), KgBY889A (B, F, J, and N), or KgBLL871AA (C, G, K, and O) at an MOI of 2 or were mock infected (D, H, L, and P). Cells were fixed 20 h postinfection and visualized under phase-contrast and conventional fluorescence microscopy (E to H and M to P). (A to D and E to L) Merged images.