Complexes between herpes simplex virus glycoproteins gD, gB, and gH detected in cells by complementation of split enhanced green fluorescent protein

Abstract

The interactions between herpes simplex virus gD and its nectin1 receptor or between gD, gB, and gH were analyzed by complementation of the N and C portions of split enhanced green fluorescent protein (EGFP) fused to the glycoproteins. The gD(N)-Nect(C) complex was readily detected; the gD(N)-gC(C) complex was undetectable, highlighting the specificity of the assay. Split EGFP complementation was detected between proteins designated gD(N)+gH(C), gD(N)+gB(C), and gH(N)+gB(C)+wtgD (gB was deleted of endocytosis motifs), both in cells transfected with two-tree glycoproteins and in syncytia. The in situ assay provides evidence that gD interacts with gH and gB independently of each other and supports a model whereby gH and gB in complex exert their activities to gD.

FIG. 1.

Electrophoretic mobility of proteins fused to N- or C-EGFP portions, or their wild-type alleles, expressed in COS or 293T cells. Numbers on the left indicate the migration positions of molecular mass markers (in kilodaltons).

FIG. 2.

EGFP CA between gD_N and Nect_C or between gH_N+gHc, and lack of complementation between gD_N and gC_C. COS or 239T cells were transfected with the indicated plasmids, gD_N (A, E, K, and O), Nect_C (B, F, L, and P), gD_N+Nect_C (C, G, H, M, Q, and R), gD_N+gC_c (D, I, J, N, S, and T), and gH_N+gH_c (U to Z). (A to D, K to N, U, and X) CA. (E to J, O to T, V, W, Y, and Z) IFA. Green, gD; red, all others. Antibodies used were R8 to gD, R1.302 to nectin1, H633 to gC, and 53S to gH (reacts to a gL-dependent epitope).

FIG. 3.

EGFP CA between HSV glycoproteins. COS or 239T cells were transfected with the indicated plasmids, gD_N+gB_C (A, D, G, and J), gD_N+gH_C (B, E, H, and K), gD_N+gC_C (C, F, I, and L), gH_N+gBc (M and O), and gH_N+gB_c+gD (N and P). Cells transfected with three glycoproteins (gps) received, in addition, plasmids encoding wtgL or epidermal growth factor receptor, as appropriate. Cells transfected with five glycoproteins received gD, gB_Δ867, gH, gL, and gC as the wild type or EGFP chimeras, as indicated.