Function of herpes simplex virus type 1 gD mutants with different receptor-binding affinities in virus entry and fusion

Abstract

We have studied the receptor-specific function of four linker-insertion mutants of herpes simplex virus type 1 glycoprotein D (gD) representing each of the functional regions of gD. We used biosensor analysis to measure binding of the gD mutants to the receptors HVEM (HveA) and nectin-1 (HveC). One of the mutants, gD(inverted Delta 34t), failed to bind HVEMt but showed essentially wild-type (WT) affinity for nectin-1t. The receptor-binding kinetics and affinities of the other three gD mutants varied over a 1,000-fold range, but each mutant had the same affinity for both receptors. All of the mutants were functionally impaired in virus entry and cell fusion, and the levels of activity were strikingly similar in these two assays. gD(inverted Delta 34)-containing virus was defective on HVEM-expressing cells but did enter nectin-1-expressing cells to about 60% of WT levels. This showed that the defect of this form of gD on HVEM-expressing cells was primarily one of binding and that this was separable from its later function in virus entry. gD(inverted Delta 243t) showed WT binding affinity for both receptors, but virus containing this form of gD had a markedly reduced rate of entry, suggesting that gD(inverted Delta 243) is impaired in a postbinding step in the entry process. There was no correlation between gD mutant activity in fusion or virus entry and receptor-binding affinity. We conclude that gD functions in virus entry and cell fusion regardless of its receptor-binding kinetics and that as long as binding to a functional receptor occurs, entry will progress.

FIG. 1.

Structure of gD in complex with HVEM, showing the locations of FR and of linker-insertions. The structure shown (3) contains HVEM residues 4 to 105 (shown in blue) and gD residues 1 to 259. Residues 1 and 259 of gD are numbered and prefixed with an asterisk. FRI (residues 27 to 43), FRII (residues 125 to 161), and FRIII (residues 222 to 246) are shown in red, yellow, and green, respectively. FRIV (residues 277 to 310) was not resolved in the structure. The Ig-like core of gD, with the exception of the C", D, and E β-strands that are part of FRII, is shown in grey, as is the short stretch of amino acids downstream of FRIII. The curved grey arrow adjacent to gD residue 259 indicates that the C-terminal portion of gD (including FRIV) is thought to fold back on the Ig-like core. Arrows point to the sites of linker insertions for the FRI, FRII, and FRIII mutants gD(∇34), gD(∇126), and gD(∇243), respectively. The pink arrow shows the approximate viewpoint used in Fig. 8. (This figure was prepared using the SwissPdb Viewer [18] from PDB file 1JMA.)

FIG. 2.

Complementation analysis of FR mutants on VD60 cells. Titers of FR mutant gD-complemented virus are shown as percentages of the titer obtained with WT gD-complemented virus. Results are the mean of four experiments, except for the results for gD(∇34), which are the mean of six. Error bars represent ±1 standard deviation about the mean.

FIG. 3.

Expression of HVEM (A) and nectin-1 (B) on receptor-defined complementing cell lines and parental lines. Cells were incubated with dilutions of antibodies at 4°C for 2 h and then fixed and processed as a CELISA. OD 405nm, optical density at 405 nm.

FIG. 4.

Complementation analysis of FR mutants on B78A-gD (HVEM) (A) and B78C10-gD (nectin-1) (B) cells. Titers of FR mutant gD-complemented virus are shown as percentages of the titer obtained with WT gD-complemented virus. Data are from a representative experiment.

FIG. 5.

Activity of gD mutants in cell fusion. Effector CHOK1 cells expressing WT gB, gH, gL, and WT or mutant gD were mixed with receptor-expressing target cells. (A) Equal expression of each form of gD was checked by CELISA on effector cells with gD polyclonal serum R7. (B and C) RLU obtained with effector cells expressing each FR mutant form of gD fusing with HVEM target cells (B) or nectin-1 target cells (C) are plotted as percentages of RLU obtained with effector cells expressing WT glycoproteins. Data in panel A are from a representative experiment. Data in panels B and C are the mean of three experiments, each carried out in quadruplicate. Error bars represent ±1 standard deviation about the mean. OD 405nm, optical density at 405 nm.

FIG. 6.

Virus entry (A) and cell fusion (B) mediated by human and Vero nectin-1. (A) HSV-1 KOStk12 was titrated on cells transiently expressing human or Vero nectin-1 or control cells transfected with vector. Entry activity is shown as the mean slope of β-galactosidase activity over 50 min. (B) Target CHOK1 cells transiently expressing human or Vero nectin-1 were used in a fusion assay. Data shown are from a representative experiment, carried out in quadruplicate. Error bars in panel B represent ±1 standard deviation about the mean RLU.

FIG. 7.

Rate of entry of FR mutant-complemented virus into VD60 cells. Equal numbers of PFU of each virus preparation were added to cells on ice. After a 90-min adsorption, the temperature was shifted to 37°C to allow entry to progress. Remaining extracellular virus was inactivated by acid treatment at the times indicated. The plaque number at each time point was expressed as a percentage of the plaque number obtained with no acid. Curves were generated using the curve-fitting option of the Delta Graph program. Data shown are the mean of three experiments. Error bars represent ±1 standard deviation about the mean.

FIG. 8.

Detail of the gD/HVEM interface. Residues 4 to 105 (boxed numbers) of HVEM and residues 1 to 43 of gD are shown. The bulk of gD has been removed to allow this view; the viewer is looking through the N-terminal hairpin loop of gD onto the apposed surface of HVEM. The approximate viewing angle is indicated by the pink arrow in Fig. 1. HVEM is shown in blue, gD FRI is in red, and the rest of gD is grey. HVEM residue 23 (shown in space-filling format in green) is visible through the binding pocket formed by gD residues 11, 14, and 25 (each shown in yellow). A β-strand of HVEM (residues 35 to 37 [shown in pink]) forms an intermolecular antiparallel β-sheet with gD residues 27 to 29, which are at the start of FRI.