Bimolecular complementation defines functional regions of Herpes simplex virus gB that are involved with gH/gL as a necessary step leading to cell fusion

Abstract

Herpes simplex virus (HSV) entry into cells requires four membrane glycoproteins: gD is the receptor binding protein, and gB and gH/gL constitute the core fusion machinery. Crystal structures of gD and its receptors have provided a basis for understanding the initial triggering steps, but how the core fusion proteins function remains unknown. The gB crystal structure shows that it is a class III fusion protein, yet unlike other class members, gB itself does not cause fusion. Bimolecular complementation (BiMC) studies have shown that gD-receptor binding triggers an interaction between gB and gH/gL and concurrently triggers fusion. Left unanswered was whether BiMC led to fusion or was a by-product of it. We used gB monoclonal antibodies (MAbs) to block different aspects of these events. Non-virus-neutralizing MAbs to gB failed to block BiMC or fusion. In contrast, gB MAbs that neutralize virus blocked fusion. These MAbs map to three functional regions (FR) of gB. MAbs to FR1, which contains the fusion loops, and FR2 blocked both BiMC and fusion. In contrast, MAbs to FR3, a region involved in receptor binding, blocked fusion but not BiMC. Thus, FR3 MAbs separate the BiMC interaction from fusion, suggesting that BiMC occurs prior to fusion. When substituted for wild-type (wt) gB, fusion loop mutants blocked fusion and BiMC, suggesting that loop insertion precedes BiMC. Thus, we postulate that each of the gB FRs are involved in different aspects of the path leading to fusion. Upon triggering by gD, gB fusion loops are inserted into target lipid membranes. gB then interacts with gH/gL, and this interaction is eventually followed by fusion.

FIG. 1.

Crystal structure of gB. (a) Ribbon representation of a gB trimer. Functional regions were defined based on antibody mapping (6). Functional region 1 (FR1) comprises domains I (blue) and the C terminus of domain V (red). FR2 contains domain II (green). FR3 comprises amino acids located between domain III (yellow) and domain IV (orange). Color code for the structural domains is as originally published (19). Monoclonal antibodies used in this study representative of each FR are indicated. (b) Close view of fusion loops region with fusion loop mutants used (cyan).

FIG. 2.

gB-gH/gL interaction can be blocked with gB-neutralizing antibodies from FR1. Immunofluorescence of nectin-expressing cells transfected with gL and EYFP-tagged gB (Bc) and gH (Hn). Twenty hours posttransfection, cells were incubated 1 h with 100 μg/ml MAb. Fusion was triggered with 250 μg/ml soluble gD306. Cells were fixed and examined with a confocal microscope at 100× magnification. In the absence of gD, no fusion and no BiMC was detected. Soluble gD306 triggers fusion and BiMC in the absence (b) or in the presence (c) of nonneutralizing antibody DL21. Neutralizing antibodies (d and e) block EYFP restoration and fusion. N, number of syncytia from a representative experiment; numbers are from one coverslip per sample. (f) Quantification of fusion (luciferase assay). No ab, no antibodies.

FIG. 3.

Characterization of EYFP-tagged fusion loop mutants. (a) Surface expression of EYFP-tagged gB fusion loop mutants in fixed, nonpermeabilized cells. (b) Quantification of fusion levels in cells triggered with soluble gD306 (luciferase assay). (c) gB fusion loop mutants and wild-type gB do not interact with gH/gL in the absence of gD. (d) Wild-type gB interacts with gH/gL, an interaction that leads to fusion. (e to g) Fusion loop mutants are negative for both BiMC and fusion.

FIG. 4.

gB-gH/gL interaction can be blocked with gB-neutralizing antibodies from FR2. Immunofluorescence of nectin-expressing cells transfected with gL and EYFP-tagged gB (Bc) and gH (Hn) and stained with R137 gH/gL polyclonal antibodies. In the absence of gD, no BiMC and no fusion occurs (a). In the presence of neutralizing MAbs (c to e), gD306 does not induce fusion and gB does not interact with gH/gL. N, number of syncytia found on each coverslip (one coverslip per sample). (f) Quantification of fusion by luciferase assay.

FIG. 5.

Antibodies from FR3 do not interfere with the interaction between gB and gH/gL, but they block cell-cell fusion. Nectin-1 cells transfected with gL and EYFP-tagged gB and gH/gL do not show BiMC in the absence of gD306 (a). In the presence of gD (b) and nonneutralizing MAbs (A22), BiMC and fusion occur (c). Neutralizing MAbs (d and e) do not affect the gB-gH/gL interaction. N, number of syncytia found on each coverslip (one coverslip per sample). (f) Syncytia formation quantification by luciferase assay. The extent of fusion was calculated as the percentage of no-antibody sample.

FIG. 6.

Working model of events that lead to fusion. Cartoon representation of gB trimer and gD dimer with domains color coded as in the crystal structures previously published (19) and Fig. 1. Receptor and receptor bound to gD (HVEM is depicted) are shown in purple. Heterocomplex gH/gL is shown in blue, nonfluorescent YFPN and YFPC halves in white, and reconstituted YFP in yellow. CM, cell membrane. Arrowheads point to extended gB fusion loops (step 2).