Plasmids expressing hybrid forms of gD were constructed (Table ). These hybrids had the first 66 or 98 amino acids from gD1 or gD2 and the remainder from gD2 or gD1, respectively. Plasmids expressing the hybrid proteins were used in cell fusion assays, along with plasmids expressing the wild-type forms of gD1 or gD2, in combination with the other HSV-1 or HSV-2 glycoproteins. The results presented in Fig. show that the gD2/1 hybrid having the first 66 amino acids from HSV-2 resembled gD2 in its enhancement of fusion with target cells expressing the CHO receptor only or also nectin-2 when combined with the HSV-1 glycoproteins. The activity of the gD2/1 hybrid was ca. 60 to 80% that observed with gD2 regardless of whether the other glycoproteins were from HSV-1 or HSV-2 and regardless of receptor (except that the gD2/1 hybrid had activity comparable to that of gD2 when the receptors were nectin-1 or HVEM and the other glycoproteins were from HSV-2). Also, the converse gD1/2 hybrid resembled gD1 in its poor cell fusion activity with target cells expressing the CHO receptor only or also nectin-2 when combined with either the HSV-1 or HSV-2 glycoproteins. The gD1/2 hybrid was slightly more active than the gD2/1 hybrid with the other HSV-1 glycoproteins and slightly less active with the other HSV-2 glycoproteins when the receptors were nectin-1 and HVEM. Results similar to those shown in Fig. for CHO cells were also obtained with hybrids in which the first 98 amino acids were switched. We conclude from these results (i) that the hybrids had no gross conformational abnormalities and had fusion activities comparable to those of gD1 and gD2, at least when tested with gB2 and gH2/gL2 and the fusion receptors, nectin-1 and HVEM, and (ii) that the superior activity of gD2 in fusion activity with the CHO receptor and nectin-2 is largely due to amino acid differences between gD2 and gD1 within the first 66 amino acids.
FIG. 2. Cell fusion induced by gD2/1 or gD1/2 hybrid molecules in combination with the other HSV-1 or HSV-2 glycoproteins. These hybrids have the first 66 amino acids from gD1 or gD2 and the remainder from gD2 or gD1, respectively. The CHO effector cells were (more ...)
Figure shows an alignment of the amino acid sequences of gD1 and gD2 in the first 66 amino acids. There are seven differences in sequence, five of which are also noted on a backbone trace of the structure of gD1 (1
). For the five positions most likely to affect secondary and tertiary structure (i.e., positions 7, 21, 42, 43 and 45), gD1 was mutated by substituting the amino acid present in gD1 for the one present in gD2. These substitutions were made individually and in pairs (A7P, A7P/P45E, A7P/D21N, A42P/G43S, and P45E). When these mutant forms of gD1 were tested for ability to induce the fusion of target cells expressing the CHO receptor only or also nectin-2, none exhibited as high as 50% of the activity observed with gD2, using either the HSV-1 or HSV-2 forms of gB and gH/gL, except in the case of the HSV-1 set of glycoproteins tested with target cells expressing only the CHO receptor. Only the gD1 mutants carrying the A7P substitution, either alone or in combination with P45E or D21N, exhibited enhanced cell fusion activity (50 to 60% that of gD2) under these conditions (data not shown). None of the gD1 mutants tested with either HSV-1 or HSV-2 gB and gH/gL differed significantly from wild-type gD1 in cell fusion activity when the receptors were nectin-1 or HVEM (data not shown). We conclude that the A7P substitution in gD2 contributes to the higher level of fusion activity of gD2 but that multiple substitutions within the first 53 amino acids of gD2 (the domain containing all of the differences between gD1 and gD2 in the first 66 amino acids), including A7P, are required for the gD2-like levels of cell fusion activity with the CHO receptor and nectin-2.
FIG. 3. (A) Alignment of the 66 N-terminal amino acids of HSV-1(KOS) gD and HSV-2(333) gD. Dots indicate identity. The positions where the amino acids differ are indicated. (B) Backbone trace of the crystal structure of gD1 bound to HVEM (1). HVEM is shown in (more ...)
The levels of gD expressed on the surfaces of glycoprotein-expressing cells used in the cell fusion assays were assessed by an immunoassay, in which the live cells in the monolayer were incubated with a type-common anti-gD rabbit serum, R#7, and then fixed and incubated with a detection system as previously described (6
). Comparable levels of cell surface expression were consistently noted for gD1, gD2, all of the gD1/gD2 hybrids, and all HSV-1 mutants (data not shown), indicating that the differences in cell fusion observed could not be explained by expression levels of gD.
The results presented here and elsewhere show that the amino acid sequence in the N-terminal region of gD, outside of the Ig-fold, influences whether human nectin-2 and an endogenous CHO receptor can serve as entry/fusion receptors. We showed here (i) that gD2 is more active than gD1 in inducing the fusion of cells expressing human nectin-2 or only an endogenous CHO receptor, regardless of whether the viral glycoproteins gB, gH, and gL were from HSV-1 or HSV-2 and consistent with the greater activity of these receptors for HSV-2 entry (16
), and (ii) that as many as 7 amino acid differences between gD1 and gD2 in the first 53 amino acids of the N terminus are largely responsible for the greater activity of gD2. It was previously shown that specific amino acid substitutions in the N terminus of gD1 enable functional interactions with human nectin-2. Substitutions Q27P or Q27R in gD1 confer the ability of HSV-1 to use nectin-2 as an entry or fusion receptor (15
). Also, substitution L25P in gD1 enables HSV-1 to use nectin-2 as an entry receptor (11
). Interestingly, the mutations at position 27 were also shown to enhance, by about 10-fold, the limited ability of HSV-1 strain KOS to infect CHO cells (3
). Other effects of the mutations at position 27 in gD1 include enhanced affinity of isolated soluble mutant gD1 for nectin-1 (10
) and significantly reduced ability of HSV-1 to use HVEM as an entry or fusion receptor (14
), a finding consistent with the location of amino acid 27 in a contact region identified in the X-ray structures (1
). It should be noted that L25 and Q27, and in fact all of the positions in the contact region from positions 24 to 32, are identical in gD1 and gD2. Thus, substitutions at positions 25 or 27 enable gD1 to interact functionally with nectin-2 but are not necessary for gD2 to interact with nectin-2.
Attempts were made to isolate the endogenous CHO receptor, without success. Oligonucleotides matching conserved sequences in nectin-1 and nectin-2 were tested for the ability to prime amplification of homologous sequences from reverse transcripts of RNA extracted from CHO cells or from plasmids extracted from a library of CHO expression clones. Antibodies raised against human or mouse nectin-2 were tested for their ability to bind to CHO cells by immunofluorescence. Identification of the endogenous CHO receptor would probably require expression cloning, which would in turn require a cell line with a high level of resistance to HSV-2 entry. Some pilot experiments done with the B78H1 cell line were not satisfactory. We abandoned the effort to identify the receptor because the results obtained in the present study were so similar for human nectin-2 and the endogenous CHO receptor, suggesting that the endogenous CHO receptor could be the Chinese hamster homolog of nectin-2 despite our inability to amplify nectin-2 gene sequences by PCR. Human nectin-2 and the endogenous CHO receptor are distinct because certain mutations in gD2 can inhibit cell fusion to a much greater extent with control CHO cells than with CHO cells expressing nectin-2 (22
Although the results summarized here indicate that the N-terminal regions of gD1 and gD2 influence entry and cell fusion with nectin-2 or the endogenous CHO receptor, further studies are required to determine whether the N terminus makes direct contact with these receptors, influences the conformation of other regions in gD that are involved in direct contacts, or influences interactions with the other viral glycoproteins that are required for cell fusion.