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The glycoproteins gE and gI of alphaherpesviruses form stable complexes, which have been implicated in multiple functions. These include immune system evasion via the ability to bind to the Fc domains of human immunoglobulin G (IgG), enhancement of viral cell-to-cell spread, and virulence (reviewed in references 8, 13, 16, and 17). Much remains to be understood about which regions of the gE-gI complex are important for each function and the overall molecular basis for each function. To allow for molecular characterization of gE-gI functions, we previously expressed soluble forms of herpes simplex virus type 1 (HSV-1) gE and gI in CHO cells and showed that the glycoproteins assembled into stable complexes (5). We determined that the stoichiometry of the gE-gI complex was 1:1. We also demonstrated that soluble gE-gI complexes bound human immunoglobulins with a 1:1 stoichiometry and with K_d values in the range of 200 to 400 nM. In the present studies, we undertook investigations of the domain structure of gE-gI complexes, with the goals of obtaining further insights into protein domains important for the formation of the gE-gI complex and for the function of the gE-gI complex in viral spread and Fc binding. Other studies have identified segments of gE and gI that are important for the gE-gI interaction and the gE-gI-IgG interaction (1, 2, 11). While the studies map gE-gI and gE-gI-IgG interactions to the linear sequence of gE or gI, little insight is available about gE-gI interactions in the context of the three-dimensional structure of the protein complex. Limited-proteolysis experiments have been valuable for providing structure-function correlations and information about domain organization in other systems (4, 14, 15). Here we used limited proteolytic analysis to obtain insights into the domain structure of soluble gE-gI. We showed that the extracellular domain of gE contains a C-terminal and an N-terminal domain, with the domain boundary in the vicinity of residue 188 of mature gE. Subsequently, we analyzed the ability of each gE domain to form complexes with gI, as well as to interact with the Fc domains of immunoglobulins. We interpret the results of these studies using sequence alignments of gE from several alphaherpesviruses.

Proteolytic digestion of gE-gI complexes yields information about a domain boundary. Soluble gE-gI was purified from transfected CHO cells using human IgG-based affinity chromatography as previously described (5), and was subjected to limited proteolytic analysis at 4°C with three different proteases. Five to 20 µg of the gE-gI protein was digested with either 0.12 to 0.5 µg of trypsin (in a buffer containing 100 mM Tris-HCl [pH 8.5]), 0.25 to 1 µg of chymotrypsin (in 100 mM Tris-HCl-10 mM CaCl₂ [pH 7.8]), or 1 to 4 µg of endoproteinase Glu-C (in 50 mM phosphate buffer [pH 7.8]). All proteolytic enzymes were obtained from Roche Molecular Biochemicals. Reactions were quenched by addition of the protease inhibitor N-tosyl-L-lysine chloromethyl ketone (TLCK) (at 50 µg/ml, for trypsin) or aprotinin (at 1 µg/ml, for chymotrypsin and endoproteinase Glu-C). Samples were boiled in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer and analyzed using an SDS-12% PAGE gel. Digestion of soluble gE-gI with all three enzymes resulted in the degradation of gE into smaller fragments in the molecular size range of 20 to 30 kDa, as well as some fragments smaller than 20 kDa (Fig. 1). Remarkably, gI was stable to digestion by all three enzymes.

View larger version (71K): [in this window] [in a new window]   FIG. 1.   Digestions of soluble gE-gI with three different proteases show that gI is stable while gE is degraded into 20- to 25-kDa fragments. Soluble gE-gI complexes were subjected to limited proteolytic digestion with trypsin, endoproteinase Glu-C (endo Glu-C), or chymotrypsin at 4°C for 30, 30, or 120 min, respectively. Products of tryptic digestion were analyzed by SDS-PAGE. Lane 1, intact gE-gI complexes (20 µg); lane 2, trypsin-digested gE-gI (20 µg); lane 3, endoproteinase Glu-C-digested gE-gI (5 µg); lane 4, chymotrypsin digested gE-gI (5 µg).

View larger version (58K): [in this window] [in a new window]   FIG. 2.   Analysis of tryptic digestion products yields information about a domain boundary in gE. (A) Soluble gE-gI complexes were digested with trypsin (substrate/enzyme ratio, 80:1 by weight) at 4°C, and digested proteins were separated on a Mono Q column. (B) Chromatographic peaks 1 and 2 from panel A were analyzed by SDS-PAGE followed by N-terminal sequencing. Lanes 1 and 2, proteins contained in peak 1, including gI (with the N-terminal sequence LVVRG) and gE fragments with SWPSA as the N-terminal sequence (initiating at residue 189 of mature gE). Lanes 3 and 4, proteins contained in peak 2, including gI (with the N-terminal sequence LVVRG) and gE fragments with the N-terminal sequence GTPKT (initiating at the N terminus of mature gE) or GPTQK (initiating at residue 23 of mature gE).

2E9 is a monoclonal antibody specific for gI and for gE-gI complexes. To facilitate analyses of complex formation between gE and gI, we raised monoclonal antibodies against gE-gI complexes by immunizing mice with 50 µg of purified soluble gE-gI complexes. The bleeds and hybridoma lines were screened by enzyme-linked immunosorbent assays (ELISA) using ELISA plates coated with 10 µg of purified soluble gE-gI/ml. Goat anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad) at a 1/1,000 dilution was used as the secondary antibody. Mouse immunoglobulins do not bind to gE-gI via the Fc domains, and goat IgG binds only weakly to gE-gI (9); thus, the screen was designed to identify antibodies with Fab (rather than Fc) specificity for a component of the gE-gI complex. Hybridoma lines that were positive by ELISA relative to control lines were further screened by immunoprecipitation with metabolically labeled CHO cells expressing either soluble gE, gI, or both (5). A wash protocol more rigorous than that previously described (5) was used (50 mM Tris-HCl, 150 mM NaCl, 0.02% NaN₃, 1 mM EDTA, 0.1% NP-40, and 0.25% gelatin, pH 7.5) in the immunoprecipitation assays in order to minimize nonspecific binding. All the hybridoma supernatants, including those from control cell lines and media alone, immunoprecipitated soluble gE-gI complexes from CHO cells expressing gE and gI, whereas none of the hybridoma lines immunoprecipitated gE from CHO cells expressing soluble gE (data not shown). Since soluble gE-gI, but not soluble gE alone, binds to the Fc domains of IgG with high affinity (5), these observations indicated that the Fc regions of bovine immunoglobulins present in the cell culture medium interacted with gE-gI complexes and interfered with the assay to identify the hybridoma lines with Fab reactivity toward a component of the gE-gI complex. One hybridoma line, 2E9, immunoprecipitated gI from CHO cells expressing gI. Ascites fluid was generated using the 2E9 hybridoma line. In metabolic-labeling experiments with this ascites fluid, 2E9 immunoprecipitated soluble gE-gI complexes from CHO cells expressing those proteins whereas ascites fluid generated using a second hybridoma line (3D3) did not (Fig. 3, fifth and eighth lanes). Thus, Fc-mediated immunoprecipitation of gE-gI complexes is not observed using antibodies contained in ascites fluids from mice, as expected from previous observations that the Fc domains of murine immunoglobulins do not interact with gE-gI complexes (9). 2E9 ascites fluid could also immunoprecipitate soluble gI from a CHO cell line expressing soluble gI alone, but not soluble gE from a cell line expressing soluble gE alone (Fig. 3, sixth and seventh lanes). 2E9 ascites fluid was used for further analyses of gI complexes with truncated gE fragments.

View larger version (83K): [in this window] [in a new window]   FIG. 3.   The 2E9 hybridoma line recognizes gI and gE-gI complexes. CHO cells expressing both soluble gE and gI, or either soluble gE or soluble gI alone, or untransfected CHO-K1 cells as negative controls were metabolically labeled with [35S]methionine/cysteine (ICN Biomedicals) for 5 h. Cell supernatants were immunoprecipitated with 10 µg of rabbit anti-HSV IgG to visualize the expressed HSV proteins, or with 0.05 ml of 2E9 ascites fluid, or with 0.05 ml of a control ascites fluid (generated using the 3D3 hybridoma line). The 30-kDa band observed in the second lane is a spontaneously derived proteolytic fragment of gE, often visualized in cells that express gE.

NgE but not CgE forms a stable complex with gI. To establish whether either the N-terminal or the C-terminal gE domain could associate with gI, we coexpressed gI along with truncation mutants of gE encoding either residues 1 to 188 appended to a hexahistidine tag (NgE) or residues 189 to 399 (CgE) (numbering corresponds to mature gE). The DNA sequence encoding HSV-1 gE in the PCRII vector (5) was modified by PCR to generate sequences encoding NgE and CgE. For NgE, the gE DNA sequence was truncated at the position corresponding to residue 188 of mature gE and a sequence encoding a hexahistidine epitope tag was appended. For generating CgE, bridge PCR was used to fuse the sequence encoding the gE leader peptide with that encoding CgE. PCR products encoding NgE and CgE were cloned individually into the unique XhoI and NotI sites of the pBJ5-GS expression vector (5). Each of these constructs was transfected into CHO cells along with the previously described pBJ5-GS-gI (5). Cells resistant to the drug methionine sulfoximine were selected as previously described (5). Cells secreting NgE and gI (NgE+gI) or CgE and gI (CgE+gI) were identified by immunoprecipitation of supernatants of metabolically labeled cells using the anti HSV-1 polyclonal antibody (ScyTek Laboratories), and clonal lines were obtained (Fig. 4, anti-HSV immunoprecipitations). CgE migrates faster than NgE on SDS-PAGE gels (Fig. 4), even though CgE is larger by 17 amino acids. We believe that this might arise due to charge differences between the two proteins that result from the introduction of a hexahistidine tag on NgE but not CgE.

View larger version (56K): [in this window] [in a new window]   FIG. 4.   NgE but not CgE forms stable complexes with gI, as shown by coimmunoprecipitation analyses with antibodies specific for gE and gI. CHO cells expressing soluble gE and gI, or cells expressing NgE and gI, or CgE and gI, were metabolically labeled with [35S]methionine/cysteine (ICN Biomedicals) for 5 h. Cell supernatants were immunoprecipitated with the indicated antibodies, and proteins were visualized by SDS-PAGE and phosphorimaging analyses. Antibodies used were anti-gH (an irrelevant antibody), to assess nonspecific binding; anti-HSV, to visualize expressed HSV proteins; anti-His, which recognizes the hexahistidine epitope tag present on NgE; 1108, which recognizes an unknown epitope present in gE and CgE; and 2E9, which is directed against gI.

Soluble gE-gI complexes, but not NgE-gI or the CgE+gI combination, function as an Fc receptor for IgG. Early studies have suggested that cells transfected with genes encoding both gE and gI have enhanced IgG binding activity compared to cells transfected with gE alone (3, 6, 7, 10). Using linker insertion mutagenesis, it has also been shown that mutations in the C-terminal region of the extracellular domain of gE, at positions 215, 244, 265, 304, 313, 319, 335, 351, 360, and 369 of the mature gE sequence, could disrupt the gE-gI-IgG interaction (1). All of these residues fall within the CgE domain. Using an immunofluorescence-based monomeric IgG binding assay, Basu et al. have also reported that gE residues 5 to 397, 91 to 397, and 163 to 397, when fused between residues 244 and 246 of gD, could bind to monomeric IgG in the absence of gI (1). Thus, we compared the relative abilities of soluble gE, soluble gI, gE-gI complexes, and the CgE+gI and NgE+gI combinations to bind Fc using immunoprecipitation-based experiments with metabolically labeled proteins. The use of intact human IgG in these experiments is complicated by our observations that most commercial human IgG preparations contain low levels of anti-HSV antibodies that react with gE and gI by binding via the Fab ends (data not shown). Thus, we used human Fc (Jackson ImmunoResearch) or IgG purified from normal rabbit serum (Jackson ImmunoResearch) in our assays. Neither the CgE+gI combination nor the NgE+gI combination is able to bind human Fc or rabbit IgG under conditions where soluble gE-gI binds (Fig. 5). Additionally, neither gE alone nor gI alone shows specific binding to human Fc or rabbit IgG. Based on these observations, it appears that if soluble gE or CgE is able to bind IgG, these interactions must be significantly reduced in affinity compared to the interaction of soluble gE-gI with IgG. Thus, as previously suggested (3, 6, 7), gI plays a critical role in the binding of monomeric IgG by gE-gI complexes, either by directly participating in Fc binding or, alternatively, through an indirect effect on gE domain structure and conformation.

View larger version (47K): [in this window] [in a new window]   FIG. 5.   Soluble gE-gI complexes, but not the NgE-gI complexes or CgE+gI combination, function as an Fc receptor for IgG. gE-gI complexes have previously been shown to interact with the Fc domains of human and rabbit immunoglobulins but not mouse immunoglobulins. CHO cells expressing soluble gE and gI, or cells expressing either NgE plus gI, CgE plus gI, gE alone, or gI alone, were metabolically labeled with [35S]methionine/cysteine (ICN Biomedicals) for 5 h. Cell supernatants were immunoprecipitated with either anti-HSV-1, human Fc [hIgG(Fc)], rabbit IgG (rIgG) (purified from normal rabbit serum), or mouse IgG (mIgG). Immunoprecipitated proteins were visualized by SDS-PAGE followed by phosphorimaging analyses. The 30-kDa band observed in the second panel (with the gE-expressing cell line) is a spontaneously derived proteolytic fragment of gE, often visualized in cells that express gE.