Multiple cell surface molecules (herpesvirus entry mediator [HVEM], nectin-1, nectin-2, and 3-O-sulfated heparan sulfate) can serve as entry receptors for herpes simplex virus type 1 (HSV-1) or HSV-2 and also as receptors for virus-induced cell fusion. Viral glycoprotein D (gD) is the ligand for these receptors. A previous study showed that HVEM makes contact with HSV-1 gD at regions within amino acids 7 to 15 and 24 to 32 at the N terminus of gD. In the present study, amino acid substitutions and deletions were introduced into the N termini of HSV-1 and HSV-2 gDs to determine the effects on interactions with all of the known human and mouse entry/fusion receptors, including mouse HVEM, for which data on HSV entry or cell fusion were not previously reported. A cell fusion assay was used to assess functional activity of the gD mutants with each entry/fusion receptor. Soluble gD:Fc hybrids carrying each mutation were tested for the ability to bind to cells expressing the entry/fusion receptors. We found that deletions overlapping either or both of the HVEM contact regions, in either HSV-1 or HSV-2 gD, severely reduced cell fusion and binding activity with all of the human and mouse receptors except nectin-1. Amino acid substitutions described previously for HSV-1 (L25P, Q27P, and Q27R) were individually introduced into HSV-2 gD and, for both serotypes, were found to be without effect on cell fusion and the binding activity for nectin- Human herpes simplex virus type 1 (HSV-1) and HSV-2, porcine pseudorabies virus (PRV), and bovine herpesvirus type 1 (BHV-1) are members of the alphaherpesvirus subfamily and have similar viral and cellular requirements for entry into cells (29). Initial attachment is usually mediated by interaction of virion envelope glycoprotein C (gC) and/or gB with cell surface heparan sulfate. The subsequent interaction of virion gD with one of its receptors triggers the penetration of virus, which occurs by fusion of the viral envelope with a cell membrane, and requires four viral glycoproteins, gB, gD, gH, and gL. HSV-induced cell fusion requires the same four viral glycoproteins-gB, gD, gH, and gL-as well as a gD receptor (28,32). Transfection of cells with plasmids expressing gB, gD, gH, and gL is sufficient, in the absence of virus infection, to induce fusion with target cells expressing HSV entry receptors (2,6,21,23).

Either herpesvirus entry mediator (HVEM, TNFRSF14) or nectin-1 (PVRL1) is sufficient for herpes simplex virus (HSV) infection of cultured cells. The contribution of individual receptors to infection in vivo and to disease is less clear. To assess this, Tnfrsf14(-/-) and/or Pvrl1(-/-) mice were challenged intravaginally with HSV-2. Infection of the vaginal epithelium occurred in the absence of either HVEM or nectin-1 but was virtually undetectable when both receptors were absent, indicating that either HVEM or nectin-1 was necessary. Absence of nectin-1 (but not HVEM) reduced efficiency of infection of the vaginal epithelium and viral spread to the nervous system, attenuating neurological disease and preventing external lesion development. While nectin-1 proved not to be essential for infection of the nervous system, it is required for the full manifestations of disease. This study illustrates the value of mutant mice for understanding receptor contributions to disease caused by a human virus.

One of the herpes simplex virus envelope glycoproteins, designated gD, is the principal determinant of cell recognition for viral entry. Other viral glycoproteins, gB, gH and gL, cooperate with gD to mediate the membrane fusion that is required for viral entry and cell fusion. Membrane fusion is triggered by the binding of gD to one of its receptors. These receptors belong to three different classes of cell surface molecules. This review summarizes recent findings on the structure and function of gD. The results presented indicate that gD may assume more than one conformation, one in the absence of receptor, another when gD is bound to the herpesvirus entry mediator, a member of the TNF receptor family, and a third when gD is bound to nectin-1, a cell adhesion molecule in the immunoglobulin superfamily. Finally, information and ideas are presented about a membrane-proximal region of gD that is required for membrane fusion, but not for receptor binding, and that may have a role in activating the fusogenic activity of gB, gH and gL.

Herpes simplex virus (HSV) glycoprotein B (gB) is one of the four viral glycoproteins required for viral entry and cell fusion and is highly conserved among herpesviruses. Mutants of HSV type 2 gB were generated by substituting conserved residues in the cytoplasmic tail with alanine or by deleting 41 amino acids from the C-terminus. Some of the mutations abolished cell fusion activity and also prevented transport of gB to the cell surface, identifying residues in the gB cytoplasmic tail that are critical for intracellular transport of this glycoprotein. These mutations also prevented production of infectious virus, possibly because the mutant forms of gB were not transported to the site of envelopment. Other mutations, particularly the deletion, significantly enhanced cell fusion activity. These mutations, as well as others described previously, identify regions of the gB cytoplasmic domain that modulate cell fusion activity.

Membrane fusion induced by herpes simplex virus (HSV) requires the action of four viral membrane glycoproteins (gB, gD, gH, and gL) and the binding of gD to one of its receptors, such as the herpesvirus entry mediator or nectin-1. The related animal herpesvirus, pseudorabies virus (PRV), encodes a homologous set of glycoproteins and its gD can also use nectin-1 as an entry receptor. We show here that PRV gD, when coexpressed with HSV gB, gH, and gL, cannot substitute for HSV gD in inducing fusion with target cells expressing nectin-1. Chimeric gD molecules composed of HSV and PRV sequences can substitute, provided the first 285 aa are from HSV gD. Because the first 261 aa were sufficient for receptor binding, this suggested that amino acids 262-285 contain a region required for cell fusion but not for receptor binding. Deletions from amino acids 250 -299 failed to identify a specific subregion critical for cell fusion, except possibly for amino acids 250 -255, which also influenced receptor binding. Instead, presence of a flexible stalk between the membrane and receptor-binding domain appears to be required, perhaps to enable conformational changes in gD on receptor binding and subsequent interactions of undefined regions of gD with the other glycoproteins required for membrane fusion. E nveloped viruses of humans and animals invade cells by inducing fusion between the viral envelope and a cell membrane. Viral envelope glycoproteins initiate and mediate this fusion. In some cases, a single viral glycoprotein can mediate binding of virus to the cell surface and fusion with a cell membrane. In other cases, two viral glycoproteins or subunits of a single translation product are required for binding and fusion (reviewed in ref. 1). In the case of herpes simplex virus (HSV), four distinct glycoproteins (gB, gD, gH, and gL) are required for membrane fusion, whereas the initial attachment of virus to cell can be mediated by gB or gC binding to cell surface heparan sulfate (reviewed in refs. 2 and 3). The initiation of membrane fusion requires the interaction of gD with one of its receptors. These include the herpesvirus entry mediator (HVEM); nectin-1 and nectin-2, cell adhesion molecules in the Ig superfamily; and specific sites in heparan sulfate generated by particular 3-O-sulfotransferases (reviewed in ref. 4).It remains unclear why HSV, and herpesviruses in general, require multiple envelope glycoproteins to induce membrane fusion. It seems unlikely that gD is an actual fusogen. All known viral fusogens must be anchored to the viral envelope as a transmembrane protein, whereas a glycosylphosphatidylinositol-linked gD ectodomain is functional for cell fusion (5) and soluble forms of the gD ectodomain can complement the entry defect of a gD-negative HSV (6). It has been proposed that interactions of gD with one of its receptors causes conformational changes in gD that enable it to activate the fusogenic activity of gB, a homooligomer, and͞or gH-gL, a heterodimer (6-8).HSV-1 gD is a 369-residue type 1 membrane glycop...