Coexpression of UL20p and gK inhibits cell-cell fusion mediated by herpes simplex virus glycoproteins gD, gH-gL, and wild-type gB or an endocytosis-defective gB mutant and downmodulates their cell surface expression

Abstract

Syncytium formation in cells that express herpes simplex virus glycoprotein B (gB), gD, gH, and gL is blocked by gK (E. Avitabile, G. Lombardi, and G. Campadelli-Fiume, J. Virol. 77:6836-6844, 2003). Here, we report the results of two series of experiments. First, UL20 protein (UL20p) expression weakly inhibited cell-cell fusion. Coexpression of UL20p and gK drastically reduced fusion in a cell-line-dependent manner, with the highest inhibition in BHK cells. Singly expressed UL20p and gK localized at the endoplasmic reticulum and nuclear membranes. When they were coexpressed, both proteins relocalized to the Golgi apparatus. Remarkably, in cells that coexpressed UL20p and gK, the antifusion activity correlated with a downmodulation of gD, gB, gH, and gL cell surface expression. Second, gB(Delta867) has a partial deletion in the cytoplasmic tail that removed endocytosis motifs. Whereas wild-type (wt) gB was internalized in vesicles lined with the endosomal marker Rab5, gB(delta867) was not internalized, exhibited enhanced cell surface expression, and was more efficient in mediating cell-cell fusion than wt gB. The antifusion activity of UL20p and gK was also exerted when gB(delta867) replaced wt gB in the cell fusion assay. These studies show that the gB C tail carries a functional endocytosis motif(s) and that the removal of the motif correlated with increased gB surface expression and increased fusion activity. We conclude that cell-cell fusion in wt-virus-infected cells is negatively controlled by at least two mechanisms. The novel mechanism described here involves the concerted action of UL20p and gK and correlates with a moderate but consistent reduction in the cell surface expression of the fusion glycoproteins. This mechanism is independent of the one exerted through endocytosis-mediated downmodulation of gB from the plasma membrane.

FIG.1.

Intracellular localization of UL20p and gK detected by immunofluorescence. (A to D) BHK cells transduced with baculovirus-UL20 (Bac20), baculovirus-gK (BacK), or both and stained with PAb to UL20p (A and C) or PAb to myc (B and D). (E to X) Two paired images, one green and one red, are shown for the same photographic field, and the paired panels are E and F, G and H, I and J, K and L, M and N, O and P, Q and R, S and T, U and V, and W and X. (E to H) COS cells transfected with pUL20-pcDNA and stained with PAb to UL20p with (F) and without (E) Ab to giantin and with (H) and without (G) ConA-FITC. (I and J) COS cells transfected with pgK and stained with anti-myc Ab with (J) or without (I) Ab to calnexin. (K to N) COS cells cotransfected with pUL20-pcDNA and pgK and stained with Ab to UL20p (K and M), myc (L), or giantin (N). (O and P) Vero cells transfected with pgK and stained with Abs to myc (O) or calnexin (P). (Q to T) Vero cells transfected with pUL20-pcDNA and stained with Ab to UL20p (Q and S), anti-giantin (T), or ConA-FITC (R). (U to X) Vero cells cotransfected with pUL20-pcDNA and pgK and stained with Ab to UL20p (U and W), myc (V), or giantin (X). Cells were transfected with 250 ng of each plasmid. Cells transfected with a single plasmid received an additional 250 ng of pMTS. The protein indicated after the slash identifies the stained protein in double immunofluorescence.

FIG. 2.

Quantification of cell-cell fusion in BHK cells cotransfected with plasmids encoding only gB, gD, gH, and gL (BDHL) (40 ng of each) or gB, gD, gH, and gL plus pUL20-pcDNA or pgK at the indicated amounts and the pcDNA 3.1(−) Myc-His/Lac vector (80 ng). The amounts of DNA in the transfection mixtures were made equal by addition of pMTS1, as appropriate. Syncytia were stained with X-Gal at 48 h. A digital micrograph of the entire coverslip was taken. The blue areas corresponding to syncytia were quantified with the Histogram software of Photoshop. Samples were run three times. The bars indicate standard errors (SE).

FIG. 3.

Quantification of cell-cell fusion in BHK cells cotransfected with expression plasmids encoding wt gB, gD, gH, and gL (40 ng of each) (A) or gB_Δ867, gD, gH, and gL (20 ng of each) (B) plus pUL20-pcDNA and/or pgK (160 ng of each) and the pcDNA 3.1(−) Myc-His/Lac vector (80 ng). BDHL, gB, gD, gH, and gL. The amounts of DNA in the transfection mixtures were made equal by addition of pMTS1, as appropriate. Syncytium staining and quantification were performed as described in the legend to Fig. 2. The bars indicate SE.

FIG. 4.

(A) Quantification of cell-cell fusion in COS cells cotransfected for 48 h with expression plasmids encoding wt gB, gD, gH, and gL (40 ng of each) (wtgB) or gB_Δ867, gD, gH, and gL (20 ng of each) (gB_Δ867) plus pUL20-pcDNA and/or pgK (160 ng each). (B) Quantification of cell-cell fusion in COS cells cotransfected for 48 h with expression plasmids encoding wt gB, gD, gH, and gL (20 or 40 ng of each) plus fourfold-higher amounts of pUL20-pcDNA and pgK or EGFR2Δ. BDHL, gB, gD, gH, and gL. The transfection mixtures contained the pcDNA 3.1(−) Myc-His/Lac vector (40 ng), and amounts of DNA in the transfection mixtures were made equal by addition of pMTS1, as appropriate. Syncytium staining and quantification were performed as described in the legend to Fig. 2. The bars indicate SE.

FIG. 5.

Quantification of cell-cell fusion in Vero cells cotransfected for 48 h with expression plasmids encoding wt gB, gD, gH, and gL (80 ng of each) or gB_Δ867, gD, gH, and gL (80 ng of each) plus pUL20-pcDNA and/or pgK (160 ng of each) and the pcDNA 3.1(−) Myc-His/Lac vector (240 ng). BDHL, gB, gD, gH, and gL. The amounts of DNA in the transfection mixtures were made equal by addition of pMTS1, as appropriate. Syncytium staining and quantification were performed as described in the legend to Fig. 2. The bars indicate SE.

FIG. 6.

Micrographs of COS cells transfected with pgB-MTS (encoding wt gB) (A, B, E, F, G, H, K, L, O, P, and Q) or pgB_Δ867-MTS (encoding gB_Δ867) (C, D, I, J, M, and N) and reacted with MAb to gB (A, C, E, G, I, K, M, and O) or MAb to Rab5a (B, D, F, H, J, L, N, and P). (A to J) Endocytosis assays. Cells were reacted with MAb to gB at 4°C and then shifted to 37°C for the indicated times (in minutes), fixed, permeabilized, and reacted with secondary antibodies. Arrows indicate vesicles lined with gB and Rab5a. Filled arrowheads point to gB-labeled cell surfaces. Open arrowheads point to a lack of gB on cell surfaces. (O and P) Higher magnification of panels K and L, respectively. (Q) Merged image of panels O and P shows colocalization of Rab5a and gB.

FIG. 7.

Comparison of the levels of cell-cell fusion induced by wt gB and gB_Δ867. BHK, COS, and Vero cells cotransfected with expression plasmids encoding wt gB, gD, gH, and gL (wtBDHL) or gB_Δ867, gD, gH, and gL (B_Δ867DHL) (40 ng of each in BHK and COS cells; 80 ng of each in Vero cells) plus the pcDNA 3.1(−) Myc-His/Lac vector (40 ng in BHK and COS cells; 240 ng in Vero cells). The amounts of DNA in all transfection mixtures were made equal by addition of pMTS1, as appropriate. Syncytium staining and quantification were performed as described in the legend to Fig. 2. The bars indicate SE.

FIG. 8.

Immunostaining with MAb to gD of COS cells cotransfected with plasmids encoding gB, gD, gH, and gL alone (BDHL) (80 ng of each) (A and C) or gB, gD, gH, and gL plus pUL20-pcDNA and pgK (160 ng of each) (B and D). The amounts of DNA in all transfection mixtures were made equal by addition of pMTS1, as appropriate. Filled arrowheads indicate gD-labeled cell surfaces. Open arrowheads indicate a lack of gD on cell surfaces.