Complex formation facilitates endocytosis of the varicella-zoster virus gE:gI Fc receptor

Abstract

Open reading frames within the unique short segment of alphaherpesvirus genomes participate in egress and cell-to-cell spread. The case of varicella-zoster virus (VZV) is of particular interest not only because the virus is highly cell associated but also because its most prominent cell surface protein, gE, bears semblance to the mammalian Fc receptor Fc gammaRII. A previous study demonstrated that when expressed alone in cells, VZV gE was endocytosed from the cell surface through a tyrosine localization motif in its cytoplasmic tail (J. K. Olson and C. Grose, J. Virol. 71:4042-4054, 1997). Since VZV gE is normally found in association with gI in the infected cell, the present study was directed at defining the trafficking of the VZV gE:gI protein complex. First, VZV gI underwent endocytosis and recycling when it was expressed alone in cells, and interestingly, VZV gI contained a methionine-leucine internalization motif in its cytoplasmic tail. Second, VZV gI was found by confocal microscopy to colocalize with VZV gE during endocytosis and recycling in cells. Third, by a quantitative internalization assay, VZV gE:gI was shown to undergo endocytosis more efficiently (steady state, 55 to 60%) than either gE alone (steady state, approximately 32%) or gI alone (steady state, approximately 45%). Further, examination of endocytosis-deficient mutant proteins demonstrated that VZV gI exerted a more pronounced effect than gE on internalization of the complex. Most importantly, therefore, these studies suggest that VZV gI behaves as an accessory component by facilitating the endocytosis of the major constituent gE and thereby modulating the trafficking of the entire cell surface gE:gI Fc receptor complex.

FIG. 1

Endocytosis of VZV gI. HeLa cells were transfected with the gI gene. Sixteen hours posttransfection, the cells were incubated with anti-gI MAb 6B5 at 4°C for 30 min. The cells were returned to 37°C with fresh medium for 0 (A), 15 (B), 30 (C), 45 (D), or 60 (E) min. The cells were fixed and permeabilized and then incubated with secondary antibody for 1 h. The images shown represent the central section from the cells analyzed by laser scanning confocal microscopy. Panel F shows HeLa cells mock transfected and stained as a negative control.

FIG. 2

Colocalization of VZV gE and gI during endocytosis. HeLa cells were transfected with the gE gene and the gI gene. The cells were incubated with MAb 6B5 and polyclonal antiserum for gE at 4°C for 30 min. The cells were then returned to 37°C for 0 (A, B, and C), 15 (D, E, and F), 30 (G, H, and I), or 60 (J, K, and L) min. The cells were fixed and permeabilized and then incubated with goat anti-mouse–Texas red conjugate and goat anti-rabbit–FITC conjugate for 1 h. The cells were analyzed by laser scanning confocal microscopy with gE (green stain) (A, D, G, and J) and gI (red stain) (B, E, H, and K); the merged images (yellow stain) are also shown (C, F, I, and L).

FIG. 3

Colocalization of VZV gE and gI during recycling. HeLa cell monolayers were singly transfected with the gI gene (A, B, and C) or dually transfected with the gE and gI genes (D to I). Subsequently, monolayers were incubated with MAb 6B5 (A to C) or with both MAb 6B5 and polyclonal antiserum for gE (D to I) while on ice. Some monolayers were incubated at 37°C for 30 min to allow internalization and then treated with trypsin to remove surface proteins. The cells were returned to 37°C with fresh medium containing trypsin inhibitor for 0 (B, E, and H) or 30 (C, F, and I) min. Transfected cells that were neither trypsin treated nor returned to 37°C represented positive controls (A, D, and G). At the given time points, the cells were fixed and stained with goat anti-mouse–FITC conjugate (A to C) or both goat anti-mouse–Texas red conjugate and goat anti-rabbit–FITC conjugate (D to I). Singly transfected monolayers were analyzed by confocal microscopy for gI in panels A to C; cotransfected monolayers were probed for gI in panels D to F and for gE in panels G to I.

FIG. 4

Analysis of internalized VZV gE (A) and gI (B). (A) The graph of internalized gE was derived from gE either expressed alone or with gI. This graph is representative of four separate experiments (Table 1). (B) The graph of internalized gI was derived from gI either expressed alone or with gE. This graph is representative of four separate experiments (Table 1).

FIG. 5

Endocytosis of VZV gI and endocytosis mutant gE. HeLa cells were transfected with the gE-Y582G gene (A, B, and C) or with both the gE-Y582G gene and the wild-type gI gene (D to I). The cells were incubated with MAb 3B3 (A to F) or with MAb 6B5 and polyclonal antiserum for gE (G, H, and I) at 4°C for 30 min. The cells were returned to 37°C for 0 (A, D, and G), 30 (B, E, and H), or 60 (C, F, and I) min. After the incubations, the cells were fixed and permeabilized prior to incubation with goat anti-mouse–FITC conjugate (A to F) or both goat anti-mouse–Texas red conjugate and goat anti-rabbit–FITC conjugate (G, H, and I) for 1 h. The cells were analyzed by confocal microscopy, with gE-Y582G staining green in all panels and the merged images with gI (red) staining yellow in panels G, H, and I.

FIG. 6

Analysis of VZV gI with endocytosis mutant gE. HeLa cells were transfected with the gE-Y582G gene or with both the gE-Y582G and gI genes and radiolabeled with [³⁵S]methionine-cysteine. After 16 h, the cells were incubated with MAb 3B3 or MAb 6B5, respectively, at 4°C for 30 min. A quantitative internalization assay was performed. The percentage of protein internalized was calculated for gE-Y582G when expressed alone or with gI. This graph is representative of four experiments.

FIG. 7

Diminished endocytosis of VZV gI with a mutated internalization motif. HeLa cells were transfected with the mutant gI-AA gene (A and B) or the wild-type gI gene (C and D). The cells were incubated with MAb 6B5 at 4°C for 30 min. The cells were returned to 37°C with fresh medium for 0 (A and C) or 30 (B and D) min. After the incubation at 37°C, the cells were fixed and permeabilized prior to incubation with goat anti-mouse–FITC conjugate for 1 h. The cells were analyzed by laser scanning confocal microscopy.

FIG. 8

Analysis of VZV gE with endocytosis-mutant gI. HeLa cells were transfected with the gI-AA gene or with both the gI-AA gene and the wild-type gE gene and then radiolabeled with [³⁵S]methionine-cysteine. The cells were incubated with MAb 6B5 or MAb 3B3, respectively, at 4°C for 30 min. A quantitative internalization assay was performed. The percentage of protein internalized was calculated for gI-AA when expressed alone or with gI. This graph is representative of four experiments.

FIG. 9

Schema for trafficking of the VZV gE:gI complex. The VZV gE:gI complex is represented by adjacent black and white ovals. The different trafficking patterns are described in detail in the Discussion. Besides the results in this report, this schema is based on data collated from references , , , , , and . ER, endoplasmic reticulum.