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. 2004 Aug 24;101(34):12414-21.
doi: 10.1073/pnas.0404211101. Epub 2004 Jul 23.

Mutations in herpes simplex virus glycoprotein D that prevent cell entry via nectins and alter cell tropism

Sharmila Manoj  1 , Cheryl R Jogger, Dawn Myscofski, Miri Yoon, Patricia G Spear
Affiliations

Mutations in herpes simplex virus glycoprotein D that prevent cell entry via nectins and alter cell tropism

Sharmila Manoj et al. Proc Natl Acad Sci U S A. .

Abstract

Glycoprotein D (gD) determines which cells can be infected by herpes simplex virus (HSV) by binding to one of the several cell surface receptors that can mediate HSV entry or cell fusion. These receptors include the herpesvirus entry mediator (HVEM), nectin-1, nectin-2, and sites in heparan sulfate generated by specific 3-O-sulfotransferases. The objective of the present study was to identify residues in gD that are critical for physical and functional interactions with nectin-1 and nectin-2. We found that double or triple amino acid substitutions at positions 215, 222, and 223 in gD caused marked reduction in gD binding to nectin-1 and a corresponding inability to function in cell fusion or entry of HSV via nectin-1 or nectin-2. These substitutions either enhanced or did not significantly inhibit functional interactions with HVEM and modified heparan sulfate. These and other results demonstrate that different domains of gD, with some overlap, are critical for functional interactions with each class of entry receptor. Viral entry assays, using gD mutants described here and previously, revealed that nectins are the principal entry receptors for selected human cell lines of neuronal and epithelial origin, whereas HVEM or nectins could be used to mediate entry into a T lymphocyte line. Because T cells and fibroblasts can be infected via HVEM, HSV strains carrying gD mutations that prevent entry via nectins may establish transient infections in humans, but perhaps not latent infections of neurons, and are therefore candidates for development of safe live virus vaccines and vaccine vectors.

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Figures

Fig. 1.
Fig. 1.
Binding of HSV-1 and HSV-2 wild-type and mutant gD:Fcs to CHO-K1 cells expressing human forms of HVEM and nectin-1. Serial dilutions of concentrated culture supernatants containing known concentrations of the wild-type or mutant forms of HSV-1 gD:Fc (A and C) or HSV-2 gD:Fc (B and D) were incubated with confluent monolayers of CHO-HVEM cells (A and B) or CHO-nectin-1 cells (C and D) in 96-well format. After incubation and washing, the cells were fixed, and then cell-bound gD:Fc was quantified by an Fc detection system. The values presented (HRP reaction product detected at OD380) are means and standard deviations of triplicate determinations and are representative of three independent experiments with similar results.
Fig. 2.
Fig. 2.
Binding of HSV-1 wild-type and mutant gD:Fcs to CHO cells expressing 3-OST-3A and cell fusion activity of wild-type and mutant HSV-1 gD with target CHO cells expressing 3-OST-3A. (Upper) CHO-K1 cells transfected with a plasmid expressing 3-OST-3A were plated in 96-well dishes and incubated with culture supernatants containing each of the forms of gD:Fc indicated (or no gD:Fc) at 1 μg/ml. Binding of the gD:Fcs to the cells was quantified and the results are presented as described in the legend to Fig. 1. (Lower) CHO-K1 cells were cotransfected with plasmids expressing gB, gD (wild type or mutant or empty vector for the –gD control), gH, gL, and T7 polymerase and were mixed 1:1 with CHO-K1 cells transfected with a plasmid expressing 3-OST-3A and a plasmid carrying the luceriferase gene under control of the T7 promoter or with CHO-K1 cells transfected with just the luciferase plasmid (control CHO cells). The cell mixtures were replated in 96-well dishes at 6 h after transfection. After 18 h of incubation, luciferase activity was quantified as a measure of cell fusion. The bars for the 3-OST-3A-expressing CHO-K1 cells are stacked over the bars for control CHO-K1 cells to show the slightly enhanced fusion of the control cells observed with some mutant forms of HSV-1 gD. The values presented (luciferase activity in arbitrary units) are means and standard deviations of triplicate determinations and are representative of three independent experiments with similar results.
Fig. 3.
Fig. 3.
Cell fusion activities of HSV-1 and HSV-2 gD mutants with target BHK-95-19 cells expressing HVEM, nectin-1, or nectin-2. BHK-95-19 cells were transfected with plasmids expressing the HSV-1 or HSV-2 glycoproteins (gB, gD, gH, and gL) and T7 polymerase (effector cells) or with plasmids expressing one of the entry receptors indicated and the luciferase reporter plasmid (target cells). Controls included effector cells that received empty vector instead of a gD-expressing plasmid and target cells that received empty vector instead of a receptor-expressing plasmid. The cell fusion assay was performed as described in the legend to Fig. 2. The results for each mutant gD (or for the control with no gD) are normalized to the cell fusion activity observed for wild-type gD, set at 100%. Percent of wild-type cell fusion activity = [(luciferase activity for mutant gD in the presence of receptor–luciferase activity for mutant gD in the absence of receptor)/(luciferase activity for wild-type gD in the presence of receptor–luciferase activity for wild-type gD in the absence of receptor)] × 100. The results shown are the means and standard deviations for at least three independent experiments, each done in triplicate.
Fig. 4.
Fig. 4.
Viral entry activities of the HSV-1 and HSV-2 gD mutants in CHO-HVEM and CHO-nectin-1 cells. HSV-1 and HSV-2 gD-negative mutant viruses were passed through cells expressing each of the wild-type and mutant forms of gD so that the viral envelopes would incorporate the various forms of gD (complementation). Mutants Q27P and Δ7–32 have been described (22). The complemented viruses, which can express β-gal from inserts in the viral genome, were used to inoculate CHO-HVEM and CHO-nectin-1 cells in 96-well dishes. After 24 h, the cells were lysed, and β-gal activity (OD405 for detection of the reaction product) was quantified as a measure of viral entry. The results are normalized to the viral entry activity observed with wild-type gD, set at 100%. Percent of wild-type gD activity = [(absorbance for mutant gD–absorbance in the absence of gD)/(absorbance for wild-type gD–absorbance in the absence of gD)] × 100. The results shown are the means and standard deviations for at least three independent experiments, each done in triplicate.
Fig. 5.
Fig. 5.
Viral entry activities of the HSV-1 and HSV-2 gD mutants in human neuroblastoma cell lines, IMR-5 and SH-SY5Y. IMR-5 cells and SH-SY5Y were plated in 96-well dishes and inoculated with complemented viruses prepared as described in the legend to Fig. 4. Viral entry was quantified and the results were normalized to viral entry activities obtained with wild-type gD, as described for Fig. 4, for two independent experiments.
Fig. 6.
Fig. 6.
Viral entry activities of the HSV-2 gD mutants in two human epithelial cell lines, A431 and C33A, and in the Jurkat T cell line. The experiments were done as described in the legends to Figs. 4 and 5. Viral entry was quantified and the results were normalized to viral entry activities obtained with wild-type gD, as described for Fig. 4, for two independent experiments.
Fig. 7.
Fig. 7.
Locations of the critical amino acid substitutions (D215, R222, and F223, brown) in gD crystallized alone (Upper) and gD crystallized with HVEM (Lower) (HVEM is not shown). β-Strands are yellow, and α-helices are red except for the contact sites with HVEM (amino acids 7–15 and 24–32), which are dark green. The entire ectodomain of gD is ≈316 aa. A truncated form of gD used for crystallization (amino acids 1–285) yielded structures in which only the first 255 (minus 1–13) or 259 aa were visible. The structures shown are based on the coordinates deposited in the Protein Data Bank (37) for entries 1JMA and 1L2G (19). Molecular graphics images were produced by using the ucsf chimera package (38) from the Computer Graphics Laboratory, University of California, San Francisco (supported by National Institutes of Health Grant P41 RR-01081).
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