The herpesvirus glycoproteins B and H.L are sequentially recruited to the receptor-bound gD to effect membrane fusion at virus entry

Abstract

Four glycoproteins (gD, gB, gH, and gL) are required for herpes simplex virus entry into the cell or for cell-cell fusion in transfected cells. gD serves as the receptor-binding glycoprotein and as the trigger of fusion; the other three execute fusion between the viral envelope and the plasma and endocytic membranes or the membranes of adjacent cells and are highly conserved among members of the herpesvirus family. Details of the interaction of gD with gB, gH, and gL were not known. Here, we report that the four glycoproteins assemble into a complex initiated by the interaction of gD with its cellular receptor. gB is recruited to the gD-receptor complex next, even in the absence of gH.gL. gH.gL is recruited next, but only to the receptor-gD-gB ensemble. A complex with the composition receptor-gD-gB-gH.gL is assembled transiently with a life span of 15-30 min in cells exposed to virus but can also be found in infected cells and in cells committed to form polykaryocytes after transfection of the glycoprotein quartet. The results indicate that the complex assembly is a critical step in the process of virus entry and fusion, and that no viral protein other than those that participate in the complex itself is required for complex assembly. These findings imply critical protein-protein interactions among the quartet as herpes simplex virions enter the cells and at cell-cell fusion, define a specific order of recruitment, and place gH.gL as the last link in the process of glycoprotein recruitment to the complex.

Fig. 1.

Electrophoretic and WB analysis of proteins coimmunoprecipitated by Abs to gD or to HVEM from lysates of HSV-1(F)-infected HEp-2 or 293 cells. gD or HVEM were immunoprecipitated (IP) with the Abs indicated in parentheses from lysates of HSV-1(F)-infected HEp-2 (HSV-HEp) (A) or 293 (HSV-293) (B) cells, or uninfected HEp-2 (HEp) (A). Coimmunoprecipitated proteins were separated by using SDS/PAGE and reacted by WB with Abs to the indicated glycoproteins. In A, the blot was reacted sequentially with Abs to gH, gD, and gB. In B Left, the blots were reacted with Abs to gB, gH, gC, and gD, and a parallel blot for gL. The proteins present in the lysate and unbound to the HVEM immunocomplex (HVEM-unbound fraction) were analyzed in parallel. B Right was developed for a much sorter time than B Left.

Fig. 2.

WB analysis of proteins coimmunoprecipitated by Abs to HVEM or to nectin 1. HVEM or nectin 1 (Nec1) were immunoprecipitated (IP) with Abs indicated in parentheses from HSV-1(F)-infected 293 cells. Coimmunoprecipitated proteins were separated by using SDS/PAGE and reacted with Abs to gB and gD. Both HVEM and nectin 1 coimmunoprecipitated gB and gD.

Fig. 3.

WB analysis of proteins coimmunoprecipitated by Abs to HVEM or gD from cells incubated with virions. The 293 or CHO cells were incubated with HSV-1(F) virions (wt) (50 pfu per cell) or gD^−/− virions (50 pfu-equivalents per cell) for 120 min, virus inoculum was removed, and cells were rinsed twice, shifted to 37°C in a water bath for the indicated times (in minutes), and lysed. HVEM or gD were immunoprecipitated (IP), and the coimmunoprecipitated proteins were identified by WB.

Fig. 4.

Time course of glycoprotein complex formation in transfected cells. 293 cells were transfected with expression plasmids for gD, gB, and gH·gL and twice the amount of HVEM plasmid. At the indicated hour p.t., cells were harvested and HVEM or gD were immunoprecipitated (IP). Immuno- and coimmunoprecipitated proteins were analyzed by WB to gD and gB, and, for the 24-h lane, to gH and gL. Complex formation was detectable at 18 and 24 h p.t. but not at 36 h p.t.

Fig. 5.

Glycoprotein complex assembly requires a gD receptor. The receptor-negative CHO cells were transfected with expression plasmids for gD, gB, and gH·gL with HVEM (+HVEM), with nectin 1 (+Nec), or with no receptor (+No R). gD was immunoprecipitated with pAb R8. Immuno- and coimmunoprecipitated proteins were analyzed by WB, as indicated. It can be seen that in the absence of receptor, no gB was coimmunoprecipitated.

Fig. 6.

Analysis of immuno- and coimmunoprecipitated proteins from cells infected with deletion viruses or transfected with the indicated mixtures. (A) HEp-2 cells. (B–D) 293 cells. (A and B) Cells infected with WT virions (wt) or ΔgD, ΔgB, ΔgH, and ΔgL viruses as gD^−/+, gB^−/+, gH^−/+, and gL^−/+ virions. (C and D) 293 cells transfected with the indicated mixtures containing the plasmids encoding for gD, gB, gH·gL, gC, and twice the amount of HVEM plasmid. DBHL, gD, gB, gH, gL; BHL, gB, gH, gL; DBL, gD, gB, gL; DBH, gD, gB, gH; DHL, gD, gH, gL; DBHLC, gD, gB, gH, gL, gC. In all panels, gD was immunoprecipitated with pAb R8 to gD. Immuno- and coimmunoprecipitated proteins were analyzed by WB. (D) Analysis of cell lysates from the experiment shown in C.