Regulation of Herpes Simplex Virus Glycoprotein-Induced Cascade of Events Governing Cell-Cell Fusion

Abstract

Receptor-dependent herpes simplex virus (HSV)-induced cell-cell fusion requires glycoproteins gD, gH/gL, and gB. Our current model posits that during fusion, receptor-activated conformational changes in gD activate gH/gL, which subsequently triggers the transformation of the prefusion form of gB into a fusogenic state. To examine the role of each glycoprotein in receptor-dependent cell-cell fusion, we took advantage of our discovery that fusion by wild-type herpes simplex virus 2 (HSV-2) glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we established that fusion speed was governed by gH/gL, with gH being the main contributor. While the mutant forms of gB fuse at distinct rates that are dictated by their molecular structure, these restrictions can be overcome by gH/gL of HSV-2 (gH₂/gL₂), thereby enhancing their activity. We also found that deregulated forms of gD of HSV-1 (gD₁) and gH₂/gL₂ can alter the fusogenic potential of gB, promoting cell fusion in the absence of a cellular receptor, and that deregulated forms of gB can drive the fusion machinery to even higher levels. Low pH enhanced fusion by affecting the structure of both gB and gH/gL mutants. Together, our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process, and the critical role of gH/gL in regulating HSV-induced fusion. IMPORTANCE Cell-cell fusion mediated by HSV glycoproteins requires gD, gH/gL, gB, and a gD receptor. Here, we show that fusion by wild-type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we found that the fusion process was controlled by gH/gL. Restrictions imposed on the gB structure by mutations could be overcome by gH₂/gL₂, enhancing the activity of the mutants. Under low-pH conditions or when using deregulated forms of gD₁ and gH₂/gL₂, the fusogenic potential of gB could only be increased in the absence of receptor, underlining the exquisite regulation that occurs in the presence of receptor. Our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process, and the critical role of gH/gL in regulating HSV-induced fusion.

FIG 1

Split luciferase assay (SLA) and surface expression of all-HSV-1 (all type 1) versus all-HSV-2 (all type 2) glycoproteins. (A) Kinetics of fusion. Cells were transfected with B₁D₁H₁L₁ or B₂D₂ H₂ L₂, and the rate of fusion was determined by SLA. Data are normalized to the results for all type 1 glycoproteins. (B) Results of CELISA. Cells transfected with all type 1 or all type 2 constructs were used to determine surface expression of gB₁ and gB₂ (R68 PAb); replica wells were used to determine the expression of gD₁ and gD₂ (R7 PAb). (C) Surface expression of gH₂/gL₂ (CELISA). Cells were transfected with various amounts of gH₂/gL₂ DNA, while DNA of the other two glycoproteins was maintained at 125 ng. The overall DNA concentration in all samples was maintained constant by using pCAGGS empty vector. Data were normalized to the expression of gH₂/gL₂ when cells were transfected with 125 ng of gH₂/gL₂ DNA (black bar). (D) Rate of fusion as a function of gH₂/gL₂ DNA concentration. Cells were transfected as described for panel C. Fusion was monitored for 8 h. Data were normalized to fusion levels measured when cells were transfected with 125 ng of gH₂/gL₂ DNA (black bar).

FIG 2

gH/gL governs the rates of fusion of type 1 and type 2 glycoproteins. (A) Cells were transfected with type 1 glycoproteins (control, black). Each type 1 form was replaced with the type 2 form (gB₂, red; gD₂, green; gH₂/gL₂, purple). Fusion was measured over an 8-h period. (B) Cells were transfected with type 2 glycoproteins (control, black). Each type 2 form was replaced with its type 1 counterpart (gB₁, red; gD₁, green; gH₁/gL₁, purple). (C, D) The rates of fusion of glycoproteins with substitutions as described for panels A and B were determined over a 15-min period to examine initiation. Each experiment was done multiple times, in duplicate. The results of representative experiments are shown.

FIG 3

The early stages of fusion are controlled by gH. Cells were transfected with all type 1 glycoproteins (control, black) or a combination of gB₁, gD₁, and either gH₂/gL₂ (purple), gH₁/gL₂ (blue), or gH₂/gL₁ (pink) and fusion measured over 8 h (A) or 30 min (C). Cells were transfected with all type 2 glycoproteins (control, black) or a combination of gB₂, gD₂, and either gH₁/gL₁ (purple), gH₁/gL₂ (pink), or gH₂/gL₁ (blue) and fusion measured over 8 h (B) or 30 min (D).

FIG 4

Determining the relative fusogenic potential of gB. (A, B) The rates of fusion of mutants with select mutations in FR1 controlled by gH₁/gL₁ (A) or gH₂/gL₂ (B). (D, E) Rates of fusion of hypofusogenic gB₁ FR3 mutants controlled by gH₁/gL₁ (D) or gH₂/gL₂ (E). (G, H) Rates of fusion of hyperfusogenic gB₁ mutants with mutations in the crown (H657R) and the cytoplasmic tail (LL871AA) controlled by gH₁/gL₁ (G) or gH₂/gL₂ (H). Data are shown as percentages of B₁D₁H₁L₁ fusion. (C, F, I) Data presented in panels B, E, and H were replotted as percentages of the results for B₁D₁H₂L₂ to show the relative fusion activities of gB mutants when combined with gH₂/gL₂.

FIG 5

Testing the regulation of fusion in the presence of receptor. (A) In the presence of receptor, fusion by gD₁^V231W, gH₂Δ48/gL₂, or the combination is the same as for wt gD and gH/gL. (B) Treating cells transfected with wt glycoproteins with a low-pH solution does not alter the kinetics of fusion compared to that of an untreated sample.

FIG 6

Effects of mutant forms of gD and gH/gL on fusion in the absence of receptor. (A) Fusion in the absence of receptor by gB₁ is enhanced by gD₁^V231W (D₁²³¹; light gray) or gH₂Δ48/gL₂ (dark gray) compared to the results for wt gD and gH/gL (black bar). The combination of gD₁^V231W and gH₂Δ48/gL₂ (white bar) represents the maximum fusogenic potential of gB₁ under these conditions. (B, C) Compared to the results for the wt, hyperfusogenic gB₁ mutants gB^H657R (B) or gB^LL871AA (C) show increased fusion levels when combined with gD₁^V231W (light gray), gH₂Δ48/gL₂ (dark gray), or both (white bar). (D, E, F) Low-pH treatment increases the fusion levels in the absence of receptor by wt gB₁ (D), gB^H657R (E), or gB^LL871AA (F). Data were normalized to fusion levels by wt gB₁, gD₁, and gH₂/gL₂ in nectin-1-expressing C10 cells (dashed line).