Bimolecular complementation reveals that glycoproteins gB and gH/gL of herpes simplex virus interact with each other during cell fusion

Abstract

Herpes simplex virus entry into cells requires four glycoproteins, gB, gD, gH, and gL. Binding of gD to one of its receptors triggers steps requiring the core fusion proteins, gB and the gH/gL heterodimer. There is evidence that gH/gL initiates hemifusion of cells, but whether this complex interacts physically with gB to cause complete fusion is unknown. We used bimolecular complementation (BiMC) of enhanced yellow fluorescent protein (EYFP) to detect glycoprotein interactions during cell-cell fusion. The N- or C-terminal half of EYFP was fused to the C terminus of gD, gB, and gH to form six chimeric proteins (Dn, Dc, Bn, Bc, Hn, and Hc). BiMC was detected by confocal microscopy. Receptor-bearing (C10) cells cotransfected with Dn and Bc or Dn, Hc, and untagged gL exhibited EYFP fluorescence, indicative of interactions between gD and gB and between gD and gH/gL. EYFP complementation did not occur in cells transfected with gL, Bc, and Hn. However, when gD was coexpressed with these other three proteins, cell-cell fusion occurred and the syncytia exhibited bright EYFP fluorescence. To separate glycoprotein expression from fusion, we transfected C10 cells with gL, Bc, and Hn for 20 h and then added soluble gD to trigger fusion. We detected fluorescent syncytia within 10 min, and both their number and size increased with exposure time to gD. Thus, when gD binds its receptor, the core fusion machinery is triggered to form a multiprotein complex as a step in fusion and possibly virus entry.

Fig. 1.

Construction and analysis of EYFP constructs. (A–C) Glycoprotein-EYFP chimeras used in this study. (D and E) Western blot analysis. C10 cells were transfected with EYFP-tagged or WT HSV proteins. Cell lysates were analyzed by using R68 anti-gB (D), R8 anti-gD (E), or R137 anti-gH/gL (F) antibodies. (G–I) Giemsa staining of syncytia. C10 cells were transfected with plasmids expressing gB, gD, gH, and gL; in each case, a pair of plasmids for the EYPF-tagged version were used in place of the WT version: G, Bn-Bc; H, Dn-Dc; I, Hn-Hc. Arrows denote syncytia.

Fig. 2.

Glycoprotein homooligomerization. C10 cells were transfected, fixed, and stained with the appropriate antibodies (red): anti-gB (A, B, and D), anti-gD (C and E), or anti-gH (F). Fluorescent images were captured at ×20 (A and B) or ×100 (C–F) magnification. The same camera settings were used for green and red fluorescence.

Fig. 3.

BiMC detects heterologous interactions between glycoproteins. C10 cells were transfected with the indicated DNAs for 20 h, fixed, and stained with anti-gD mAbs (A, B, D, and E) or fixed, permeabilized, and stained with anti-gB mAbs (C). All were analyzed by immunofluorescent assay for protein (red) and YFP (green). Confocal images were captured at ×40 (A–C) or ×100 (D and E) magnification. All images were captured by using the same camera setting. In a parallel experiment (D and E), cells were stained with Giemsa and examined at ×20 magnification. Arrows indicate syncytia.

Fig. 4.

Synchronization of fusion with soluble gD. (A) C10 cells were transfected with Bc+Hn+gL plasmids for 20 h, fixed, permeabilized, stained with anti-gB mAbs (red), and also examined for YFP in the green channel. (B) Same as in A, except that at 20 h posttransfection, 250 μg/ml gD306t was added and cells were incubated for an additional 4 h before fixation. Images were captured at ×40 (A) or ×100 (B) magnification. The same camera setting was used to capture red, green, and merged images. In the merged image, the green fluorescence is very bright (yellow) or less bright (orange). An arrow indicates an incompletely fused cell.

Fig. 5.

Time course of fusion triggered by gD306t. C10 cells were transfected with Bc+Hn+gL plasmids for 20 h. Soluble gD306t was added for 0 (A), 10 (B), 20 (C), 30 (D), or 60 min (E). Cells were fixed, permeabilized (A), and stained with anti-gB mAbs (red). EYFP was examined in green channel. Confocal images were captured at ×40 (A) or ×100 (B–E) magnification. The numbers of syncytia per coverslip (one experiment) are indicated by n.