Structural and antigenic analysis of a truncated form of the herpes simplex virus glycoprotein gH-gL complex

Abstract

The herpes simplex virus (HSV) gH-gL complex is essential for virus infectivity and is a major antigen for the host immune system. The association of gH with gL is required for correct folding, cell surface trafficking, and membrane presentation of the complex. Previously, a mammalian cell line was constructed which produces a secreted form of gHt-gL complex lacking the transmembrane and cytoplasmic tail regions of gH. gHt-gL retains a conformation similar to that of its full-length counterpart in HSV-infected cells. Here, we examined the structural and antigenic properties of gHt-gL. We first determined its stoichiometry and carbohydrate composition. We found that the complex consists of one molecule each of gH and gL. The N-linked carbohydrate (N-CHO) site on gL and most of the N-CHO sites on gH are utilized, and both proteins also contain O-linked carbohydrate and sialic acid. These results suggest that the complex is processed to the mature form via the Golgi network prior to secretion. To determine the antigenically active sites of gH and gL, we mapped the epitopes of a panel of gH and gL monoclonal antibodies (MAbs), using a series of gH and gL C-terminal truncation variant proteins produced in transiently transfected mammalian cells. Sixteen gH MAbs (including H6 and 37S) reacted with the N-terminal portion of gH between amino acids 19 and 276. One of the gH MAbs, H12, reacted with the middle portion of gH (residues 476 to 678). Nine gL MAbs (including 8H4 and VIII 62) reacted with continuous epitopes within the C-terminal portion of gL, and this region was further mapped within amino acids 168 to 178 with overlapping synthetic peptides. Finally, plasmids expressing the gH and gL truncations were employed in cotransfection assays to define the minimal regions of both gH and gL required for complex formation and secretion. The first 323 amino acids of gH and the first 161 amino acids of gL can form a stable secreted hetero-oligomer with gL and gH792, respectively, while gH323-gL168 is the smallest secreted hetero-oligomer. The first 648 amino acids of gH are required for reactivity with MAbs LP11 and 53S, indicating that a complex of gH648-gL oligomerizes into the correct conformation. The data suggest that both antigenic activity and oligomeric structure require the amino-terminal portions of gH and gL.

FIG. 1

The stoichiometry of gHt and gL in the complex. (A) HL-7 cells were grown in medium containing [³⁵S]cysteine. gHt-gL was purified and applied to SDS-PAGE gel. The gel was dried, and the disintegrations per minute (dpm) in each band were determined by phosphorimaging. (B) Fifty micrograms of purified gHt-gL was applied to a Superose 12 gel filtration column. One-milliliter fractions were collected and analyzed for gHt and gL by ELISA, with 37S MAb for gHt and 8H4 MAb for gL. The peaks are labeled ①, ②, and ③. Abs 405nm, absorbance at 405 nm. (C) Purified gHt-gL was mixed with sample buffer containing 0.1% SDS in the absence of reducing reagent. Samples were resolved on a 4 to 12% gradient SDS-PAGE gel and analyzed by Western blotting with R83 to detect gHt and 8H4 to detect gL. ▪ indicates the band detected by both R83 and 8H4.

FIG. 2

Analysis of carbohydrates on gH and gL. Purified gHt-gL was incubated with no enzyme (untreated control) or with glycosidases and neuraminidase in the indicated combinations. The digests were resolved on a 10% denaturing polyacrylamide gel. Following transfer to a nitrocellulose membrane, one blot was probed with R83 anti-gH antibody (A). The bound antibody was detected with goat anti-rabbit IgG-peroxidase and chemiluminescent substrate. A second blot was probed with anti-gH MAb 8H4 (B) and then with goat anti-mouse IgG-peroxidase and chemiluminescent substrate. The molecular weight after each treatment of gH and gL was calculated according to molecular size markers on the gel (data not shown). The contributions of N-CHO, O-CHO, and sialic acid to the molecular weights of gH (C) and gL (D) were estimated from the difference between the untreated controls (A and B, lanes 1) and the Endo-F-treated (A and B, lanes 4), O-glycanase-treated (A and B, lanes 6), and neuraminidase-treated (A and B, lanes 7) samples.

FIG. 3

Schematic stick figures of full-length HSV-1 gH and gL and the C-terminal truncation mutants. Plasmids were constructed to express truncated forms of gH, a deletion mutant of gH pCMV3del(276–323), full-length gL (pCMV3gL), and truncated forms of gL. The signal peptides (signal), TMR, positions of the cysteine residues (C) and predicted N-CHO sites (open balloons) and predicted O-CHO sites (open hexagons), and the names of the plasmids are indicated.

FIG. 4

Epitope mapping of anti-gH antibodies. CHO cells were transfected with gH truncation or deletion mutants. (A) Cell extracts were immunoprecipitated with R137, electrophoresed on a 12% denaturing polyacrylamide gel, transferred to nitrocellulose, and probed with MAb H6. Cell extracts from cells mock transfected (lane 1) or transfected with pCMV3gHdel(276–323) (lane 2), pSR123 (lane 3), or pCMV3gH_trunc(323) (lane 4) are shown. (B) Cell extracts were electrophoresed on a 12% denaturing polyacrylamide gel, transferred to nitrocellulose, and probed with MAb H12. (C) Cell extracts were electrophoresed on a 12% denaturing polyacrylamide gel, transferred to nitrocellulose, and probed with R137. Secondary antibodies were then added, and the blots were visualized by ECL. Lanes for panels B and C are cell extracts from pSR124- (lane 1), pSR123- (lane 2), and mock- (lane 3) transfected cells.

FIG. 5

Reactivity of anti-gL antibodies with gL168 or with full-length gL. CHO-K1 cells were transfected with pMN115 (gL168) (lanes 1, 3, 5, 7, and 9) or pCMV3gL (gL) (lanes 2, 4, 6, 8, and 10). Cell extracts were prepared and resolved on a 12% polyacrylamide denaturing gel. After Western blotting, separate strips of the membrane were probed with antibodies L1, L2, L3, 8H4, or αUL1-1, as indicated below the gel. Secondary antibodies were then added, and ECL was used to visualize the bands.

FIG. 6

Mapping of anti-gL antibody epitopes with synthetic peptides. (A) Diagram depicting the sequences of the set of overlapping synthetic peptides mimicking the gL sequence. The location of each peptide within the gL sequence is indicated. (B) Dot blot analysis of anti-gL antibodies with the peptides. Two microliters of each peptide (4 μg/dot) was spotted onto nitrocellulose membrane strips. After blocking, antibodies were added to each strip, and the reactivity was detected by ECL with goat anti-mouse peroxidase or goat anti-rabbit peroxidase.

FIG. 7

Positions of the epitopes for MAbs to gH and gL mapped in this study. Schematic figures depict the linear amino acid sequences of gH and gL. The hatched bars depict the locations of the epitopes of anti-gH and anti-gL antibodies. The position of MAb 52S is according to the amino acid change (residue 536) of two MAR mutants selected by 52S (13).

FIG. 8

Expression of gH truncations by transfected CHO-K1 cells. (A) Culture supernatants were immunoprecipitated (IP) with gH-specific MAb H6, electrophoresed on a 12% denaturing polyacrylamide gel, transferred to nitrocellulose, and probed with R137. (B) Cell extracts were immunoprecipitated (IP) with gH-specific MAb H6, electrophoresed on a 12% denaturing polyacrylamide gel, transferred to nitrocellulose, and probed with R137. The bands corresponding to the various gH truncations are indicated with arrows. In both A and B, cells were transfected with pSR162 (lanes 1); pSR124 (lanes 2); pSR123 (lanes 3); pCMV3gH323 (lanes 4); and pSR125 (lanes 5).

FIG. 9

Determination of the shortest C-terminal truncation of gH that forms a complex with full-length gL and is secreted. CHO-K1 cells were cotransfected with pCMV3gL, encoding full-length gL, and a plasmid encoding one of the five gH truncations. Culture supernatants were immunoprecipitated (IP) with MAbs H6 (anti-gH) and 8H4 (anti-gL) (A) or with LP11 (anti-gH-gL) (B). Proteins were resolved on a 12% denaturing polyacrylamide gel, transferred to nitrocellulose, and probed with R137. Lanes contain culture supernatants of cells cotransfected with pSR162 and pCMV3gL (lanes 1), pSR124 and pCMV3gL (lanes 2), pSR123 and pCMV3gL (lanes 3), pCMV3gH323 and pCMV3gL (lanes 4), or pCMV3gL (lanes 5) or of mock-transfected cells.

FIG. 10

Determination of the minimal size of gL that forms a complex with gH792 and is secreted. CHO-K1 cells were cotransfected with pSR162, encoding gH792, and plasmids encoding one of the seven gL truncations. (A and B) Culture supernatants were immunoprecipitated (IP) with anti-gH MAb H6 and anti-HA MAb 12CA5 (directed at the HA epitope present in each of the gL truncations). The precipitated proteins were resolved on a 16% polyacrylamide denaturing gel and transferred to nitrocellulose. The blot was cut in half, and the top half was probed with R137 to detect gH (A); the bottom half was probed with αUL1-1 to detect gL (B). (C and D) Cell extracts were immunoprecipitated with H6 and 12CA5. The precipitated proteins were resolved on a 16% polyacrylamide denaturing gel and transferred to nitrocellulose. The blot was cut in half, the top half was probed with R137 to detect gH (C), and the bottom half was probed with αUL1-1 to detect gL (D). Lanes contain cells transfected with pSR 162 and pMN115 (lane 1); pMN110 (lane 2); pMN109 (lane 3); pMN108 (lane 4); pMN107 (lane 5); pMN112 (lane 6); and pMN114 (lane 7).

FIG. 11

The smallest complexes formed and secreted by cells cotransfected with plasmids expressing truncated gH and truncated gL. CHO-K1 cells were cotransfected with gH truncation mutants and pMN115 (A) or pMN110 (B and C). (A) Culture supernatants were immunoprecipitated (IP) with anti-gH MAb H6, and the precipitates were resolved on 12% denaturing polyacrylamide gels, transferred to nitrocellulose, and probed with R137. Lane 1, pSR162 plus pMN115; lane 2, pSR124 plus pMN115; lane 3, pSR123 plus pMN115; lane 4, pCMV3gH323 plus pMN115; lane 5, pMN115. (B) Culture supernatants were immunoprecipitated (IP) with anti-gH MAb H6, and the precipitates were resolved on 12% denaturing polyacrylamide gels, transferred to nitrocellulose, and probed with R137. (C) Cell extracts were immunoprecipitated (IP) with anti-gH MAb H6 and anti-HA MAb 12CA5. Protein was resolved on 12% polyacrylamide denaturing gel, transferred to nitrocellulose, and probed with R137 and aUL1-1. Lanes in panels B and C are cells transfected with pSR162 plus pMN110 (lane 1); pSR124 plus pMN110 (lane 2); pSR123 plus pMN110 (lane 3); pCMV3gH323 plus pMN110 (lane 4); and pMN110 (lane 5). The positions of bands representing truncated gHs are indicated by arrows. The positions of IgG heavy chain (HC) and light chain (LC) are also indicated.

FIG. 12

A proposed model for gH-gL structure. We speculate that the gH-gL complex contains one gH and one gL. The C termini of gH and gL are labeled coo⁻. The complex is anchored to the membrane through the TMR of gH. The gray area shows the antigenic active sites of gH and gL. Mutations affecting membrane fusion are located in the dot-shaded area of gH (10, 48). The positions of gH residue 323 and gL residue 161 are indicated as gH323 and gL161, respectively. These are the smallest regions required for complex formation and secretion. The positions of cysteines on both gH and gL are shown, and the possible disulfide bonds on gH are indicated by broken lines. The locations of N-linked and O-linked CHO sites are also shown.