Human herpesvirus 8 glycoprotein B (gB), gH, and gL can mediate cell fusion

Abstract

Herpesvirus entry into cells and herpesvirus-induced cell fusion are related processes in that virus penetration proceeds by fusion of the viral envelope and cell membrane. To characterize the human herpesvirus 8 (HHV-8) glycoproteins that can mediate cell fusion, a luciferase reporter gene activation assay was used. Chinese hamster ovary (CHO) cells expressing the HHV-8 glycoproteins of interest along with a luciferase reporter gene under the control of the T7 promoter were cocultivated with human cells transfected with T7 RNA polymerase. Because HHV-8 glycoprotein B (gB) expressed in CHO cells localizes to the perinuclear region, a truncated form of gB (designated gB(MUT)) that lacks putative endocytosis signals was constructed by deletion of the distal 58 amino acids of the cytoplasmic tail. HHV-8 gB(MUT) was expressed efficiently on the surface of CHO cells. HHV-8 gB, gH, and gL could mediate the fusion of CHO cells with two different human cell types, embryonic kidney cells and B lymphocytes. Substituting gB(MUT) for gB significantly enhanced the fusion of CHO cells with human embryonic kidney cells but not B lymphocytes. Thus, two human cell types known to be susceptible to HHV-8 entry were also suitable targets for cell fusion induced by HHV-8 gB, gH, and gL. For human embryonic kidney cells and B cells at least, optimal fusion was noted with the expression of all three HHV-8 glycoproteins.

FIG. 1.

Immunoprecipitation of HHV-8 gB, gH, and gL. CHO cells were transfected with plasmids expressing the indicated HHV-8 glycoproteins. The cells were radiolabeled 24 h after transfection and then lysed before immunoprecipitation with the indicated mouse polyclonal sera. In the left panel (lanes 1 and 2), radiolabeled proteins were immunoprecipitated with anti-HHV-8 gB sera. In the right panel (lanes 3 to 6), proteins were immunoprecipitated with anti-HHV-8 gH-gL sera. Two bands (designated with an asterisk) were consistently visualized in all six lanes on the original autoradiographs at between approximately 70 and 80 kDa, although the intensities varied.

FIG. 2.

Amino acid sequence of the final 120 amino acids of HHV-8 gB. The amino acid sequence of HHV-8 gB was derived from the nucleotide sequence obtained from published reports (53, 60). The predicted transmembrane domain is in bold letters. The Yxxφ (YKPL) and dileucine endocytosis motifs are boxed. The 58 amino acids deleted in the truncated form of gB (designated gB^MUT) are underlined.

FIG. 3.

Enhanced cell surface expression of HHV-8 gB^MUT. CHO cells were transfected with plasmids expressing HHV-8 gB or gB^MUT, replated on 96-well dishes, and incubated for 18 h. (A) For detection of cell surface glycoprotein expression, cells were first incubated with primary antibody and then fixed. (B) For detection of intracellular and cell surface glycoprotein expression, cells were fixed, permeabilized, and then incubated with primary antibody. Subsequently, cells were incubated with biotinylated goat anti-mouse IgG and streptavidin-horseradish peroxidase conjugate. After the addition of substrate, OD readings at 370 nm were obtained. Shown are the means and standard deviations for four replicate samples. In panel A, the difference between the OD readings obtained for cells transfected with a vector alone and cells transfected with gB^MUT was statistically significant (P < 0.001; two-tailed t test), while the difference between the readings obtained with the vector alone and gB was not (P = 0.155). In panel B, the differences between the readings obtained with the vector alone and gB and gB^MUT were both statistically significant (P < 0.001 and P = 0.013, respectively).

FIG. 4.

Detection of gH and gL on the cell surface. The detection of gH and gL on the cell surface was optimal when both glycoproteins were coexpressed. CHO cells were transfected with plasmids expressing the indicated HHV-8 glycoproteins. Detection of cell surface (A) and total (B) glycoprotein expression was done by using anti-gH-gL sera as described in the legend to Fig. 3. Shown are the means and standard deviations for three to eight replicate samples. In panel A, the differences between the OD readings obtained for cells transfected with a vector alone and cells transfected with gH or with gH plus gL were both statistically significant (P < 0.001; two-tailed t test), as was the difference between the readings obtained with gH and with gH plus gL (P < 0.001). The difference between the readings obtained with the vector and with gL was not statistically significant (P = 0.774). In panel B, the differences between the readings obtained with the vector and those obtained with gH, gL, or gH plus gL were all statistically significant (P = 0.015, P = 0.007, and P = 0.002, respectively). The difference between the readings obtained with gH and with gH plus gL was not statistically significant (P = 0.632).

FIG. 5.

Cell surface expression of HHV-8 glycoproteins. CHO cells were transfected with plasmids expressing the indicated HHV-8 glycoproteins. After cells were replated on 96-well dishes and incubated for 18 h, they were incubated with primary antibody. Subsequently, cells were fixed and then incubated with biotinylated goat anti-mouse IgG and streptavidin-horseradish peroxidase conjugate. After the addition of substrate, OD readings at 370 nm were obtained. Shown are the means and standard deviations for four replicate samples. In the left panel, the differences between the OD readings obtained for cells transfected with a vector alone and those obtained for cells transfected with gB^MUT alone or with gB^MUT, gH, and gL were both statistically significant (P < 0.001 or P = 0.005, respectively; two-tailed t test), while the differences between readings obtained with the vector and those obtained with gH plus gL or with a combination of gB, gH, and gL were not (P = 1.0). In the right panel, the differences between the readings obtained with the vector and those obtained with gH plus gL, a combination of gB, gH, and gL, or a combination of gB^MUT, gH, and gL were all statistically significant (P < 0.001). The difference between the readings obtained with the vector and with gB^MUT was not statistically significant (P = 0.986).

FIG. 6.

Kinetics of cell fusion as measured by luciferase production. Luciferase activity was measured at the indicated times after cocultivation of effector and target cells. CHO effector cells were transfected with plasmids expressing the indicated glycoproteins or a control vector and the luciferase reporter plasmid. 293T target cells were transfected with T7 RNA polymerase. Shown are mean relative luminosity units (RLU) and standard deviations for six replicate samples obtained on two separate occasions.

FIG. 7.

Efficient cell fusion requires HHV-8 gB and gH-gL. CHO effector cells were transfected with plasmids expressing the indicated glycoproteins or a control vector and the luciferase reporter plasmid. 293T target cells were transfected with T7 RNA polymerase. After cocultivation for 24 h, luciferase activity was measured. Shown are the mean relative luminosity units (RLU) and standard deviations for six replicate samples obtained on two separate occasions. Mean differences between values for the vector and those for gB^MUT, gH plus gL, a combination of gB, gH, and gL, or a combination of gB^MUT, gH, and gL were all statistically significant (P < 0.001; two-tailed t test). The difference between the vector and gB values was also statistically significant (P = 0.012). Additional experiments done independently resulted in similar findings (data not shown). For comparison, cocultivation of 293T cells with CHO effector cells expressing HSV-1 glycoproteins gB, gD, and gH-gL resulted in a mean RLU of 226.2.

FIG. 8.

Visualization of cells that express luciferase. CHO effector cells transfected with plasmids expressing the indicated glycoproteins along with the luciferase reporter plasmid were cocultivated for 24 h with 293T target cells transfected with T7 RNA polymerase. After cells were fixed and permeabilized, detection of luciferase expression was performed by immunostaining with an antiluciferase antibody. For comparison, results obtained after cocultivation of 293T cells with effector cells expressing HSV-1 glycoproteins gB, gD, and gH-gL are shown.

FIG. 9.

HHV-8 gB and gH-gL can mediate fusion of CHO and B cells. CHO effector cells were transfected with plasmids expressing the indicated glycoproteins or a control vector and the luciferase reporter plasmid. Target B cells were electroporated with T7 RNA polymerase. Luciferase activity was measured 24 h after cocultivation of effector and target cells. Shown are the mean relative luminosity units (RLU) and standard deviations for three replicate samples. In the left panel, differences between values for the vector and those for a combination of gB, gH, and gL or a combination of gB^MUT, gH, and gL were both statistically significant (P < 0.001; two-tailed t test), while the difference between values for gB, gH, and gL and for gB^MUT, gH, and gL was not (P = 0.219). In the right panel, differences between values for the vector and those for a combination of gB, gH, and gL or for a combination of gB^MUT, gH, and gL were both statistically significant (P = 0.002; two-tailed t test), while the difference between the values for gB, gH, and gL and for gB^MUT, gH, and gL was not (P = 0.920). Similar results were obtained in additional experiments done independently (data not shown).

FIG. 10.

Fusion of various cell lines mediated by HHV-8 glycoproteins. CHO effector cells were transfected with plasmids expressing the indicated glycoproteins or a control vector and the luciferase reporter plasmid. CHO or Vero target cells were transfected with T7 RNA polymerase. After cocultivation of the effector and target cells, luciferase activity was measured. Shown are the means and standard deviations for calculated fold increases for three replicate samples. The fold increases (or relative luciferase activities) were calculated as the ratio of values obtained with effector cells expressing the indicated glycoproteins to values obtained with effector cells transfected with the vector. For comparison, relative luciferase activities were calculated from the results presented in Fig. 7 and 9 when 293T and BJAB cells were used as target cells. Using the unadjusted mean luciferase values, the differences obtained with CHO target cells between values for the vector and those for a combination of gB, gH, and gL or a combination of gB^MUT, gH, and gL were both statistically significant (P < 0.001; two-tailed t test). The differences obtained with Vero target cells between values for the vector and those for a combination of gB, gH, and gL or a combination of gB^MUT, gH, and gL were also both statistically significant (P = 0.002 and P < 0.001, respectively). The differences between values for gB, gH, and gL and those for gB^MUT, gH, and gL with both CHO and Vero target cells were not statistically significant (P = 0.871 and P = 1.0, respectively).