Structure-based analysis of the herpes simplex virus glycoprotein D binding site present on herpesvirus entry mediator HveA (HVEM)

Binding of herpes simplex virus (HSV) envelope glycoprotein D (gD) to a cell surface receptor is an essential step of virus entry. We recently determined the crystal structure of gD bound to one receptor, HveA. HveA is a member of the tumor necrosis factor receptor family and contains four characteristic cysteine-rich domains (CRDs). The first two CRDs of HveA are necessary and sufficient for gD binding. The structure of the gD-HveA complex reveals that 17 amino acids in HveA CRD1 and 4 amino acids in HveA CRD2 directly contact gD. To determine the contribution of these 21 HveA residues to virus entry, we constructed forms of HveA mutated in each of these contact residues. We determined the ability of the mutant proteins to bind gD, facilitate virus entry, and form HveA oligomers. Our results point to a binding hot spot centered around HveA-Y23, a residue that protrudes into a crevice on the surface of gD. Both the hydroxyl group and phenyl group of HveA-Y23 contribute to HSV entry. Our results also suggest that an intermolecular beta-sheet formed between gD and HveA residues 35 to 37 contributes to binding and that a C37-C19 disulfide bond in CRD1 is a critical component of HveA structure necessary for gD binding. The results argue that CRD2 is required for gD binding mainly to provide structural support for a gD binding site in CRD1. Only one mutant, HveA-R75A, exhibited enhanced gD binding. While some mutations influenced complex formation, the majority did not affect HSV entry, suggesting that most contact residues contribute to HveA receptor function collectively rather than individually. This structure-based dissection of the gD-HveA binding site highlights the contribution of key residues within HveA to gD binding and HSV entry and defines a target region for the design of small-molecule inhibitors.     

Identification of functional regions of herpes simplex virus glycoprotein gD by using linker-insertion mutagenesis.

Glycoprotein gD is a component of the herpes simplex virus (HSV) envelope essential for virus entry into susceptible cells. Previous studies using deletion and point mutations identified a functional domain of HSV-1 gD (gD-1) from residues 231 to 244. However, many of the deletion mutations had global effects on gD-1 structure, thus precluding assessment of the functional role of large portions of the protein. In this study, we constructed a large panel of linker-insertion mutants in the genes for gD-1 and HSV-2 gD (gD-2). The object was to create mutations which would have only localized effects on protein structure but might have profound effects on gD function. The mutant proteins were expressed in transiently transfected L cells. Monoclonal antibodies (MAbs) were used as probes of gD structure. We also examined protein aggregation and appearance of the mutant glycoproteins on the transfected cell surface. A complementation assay measured the ability of the mutant proteins to rescue the infectivity of the gD-null virus, FgD beta, in trans. Most of the mutants were recognized by one or more MAbs to discontinuous epitopes, were transported to the transfected cell surface, and rescued FgD beta virus infectivity. However, some mutants which retained structure were unable to complement FgD beta. These mutants were clustered in four regions of gD. Region III (amino acids 222 to 246) overlaps the region previously defined by gD-1 deletion mutants. The others, from 27 through 43 (region I), from 125 through 161 (region II), and from 277 to 310 (region IV), are newly described. Region IV, immediately upstream of the transmembrane anchor sequence, was previously postulated to be part of a putative stalk structure. However, residues 277 to 300 are directly involved in gD function. The linker-insertion mutants were useful for mapping MAb AP7, a previously ungrouped neutralizing MAb, and provided further information concerning other discontinuous epitopes. The mapping data suggest that regions I through IV are physically near each other in the folded structure of gD and may form a single functional domain.     

Involvement of HVEM receptor in activation of nuclear factor kappaB by herpes simplex virus 1 glycoprotein D

The UV-inactivated herpes simplex virus 1 (HSV-1) and glycoprotein D (gD) of HSV-1 have been shown to activate nuclear factor kappaB (NF-kappaB) in U937 cells, but mechanisms involved in this activation have not been elucidated. Here we report that: (i) UV-inactivated HSV-1 induced an increased NF-kappaB activation in cells expressing human HVEM (for herpesvirus entry mediator) at surface level, naturally or following stable transfection, but not in cells in which this receptor was not detected by flow cytometry analysis, (ii) treatment with soluble gD induced a dose-dependent NF-kappaB activation in THP-1 cells naturally expressing HVEM, and a monoclonal antibody that prevents binding of gD to HVEM significantly reduced NF-kappaB activation by soluble gD in the same cells, (iii) coculture with transfectants expressing wild-type gD on their surface induced an approximately twofold increase in NF-kappaB activation in cells naturally expressing HVEM, while coculture with transfectants expressing a mutated form of gD, lacking its capability to bind HVEM, did not induce a similar effect and (iv) treatment with soluble gD induced a dose-dependent NF-kappaB activation in CHO transfectants expressing HVEM, but not in control CHO transfectants lacking any functional gD receptor. Overall, these results establish that HVEM is involved in NF-kappaB activation by HSV-1 gD.     

Herpes simplex virus glycoprotein D mediates interference with herpes simplex virus infection.

We showed that the expression of a single protein, glycoprotein D (gD-1), specified by herpes simplex virus type 1 (HSV-1) renders cells resistant to infection by HSV but not to infection by other viruses. Mouse (LMtk-) and human (HEp-2) cell lines containing the gene for gD-1 under control of the human metallothionein promoter II expressed various levels of gD-1 constitutively and could be induced to express higher levels with heavy metal ions. Radiolabeled viruses bound equally well to gD-1-expressing and control cell lines. Adsorbed viruses were unable to penetrate cells expressing sufficient levels of gD-1, based on lack of any cytopathic effects of the challenge virus and on failure to detect either the induction of viral protein synthesis or the shutoff of host protein synthesis normally mediated by a virion-associated factor. The resistance to HSV infection conferred by gD-1 expression was not absolute and depended on several variables, including the amount of gD-1 expressed, the dosage of the challenge virus, the serotype of the challenge virus, and the properties of the cells themselves. The interference activity of gD-1 is discussed in relation to the role of gD-1 in virion infectivity and its possible role in permitting escape of progeny HSV from infected cells.     

Potential nectin-1 binding site on herpes simplex virus glycoprotein d

Four glycoproteins (gD, gB, gH, and gL) are essential for herpes simplex virus (HSV) entry into cells. An early step of fusion requires gD to bind one of several receptors, such as nectin-1 or herpesvirus entry mediator (HVEM). We hypothesize that a conformational change in gD occurs upon receptor binding that triggers the other glycoproteins to mediate fusion. Comparison of the crystal structures of gD alone and gD bound to HVEM reveals that upon HVEM binding, the gD N terminus transitions from a flexible stretch of residues to a hairpin loop. To address the contribution of this transition to the ability of gD to trigger fusion, we attempted to "lock" the gD N terminus into a looped conformation by engineering a disulfide bond at its N and C termini. The resulting mutant (gD-A3C/Y38C) failed to trigger fusion in the absence of receptor, suggesting that formation of the loop is not the sole fusion trigger. Unexpectedly, although gD-A3C/Y38C bound HVEM, it failed to bind nectin-1. This was due to the key role played by Y38 in interacting with nectin-1. Since tyrosines are often "hot spot" residues at the center of protein-protein interfaces, we mutated residues that surround Y38 on the same face of gD and tested their binding and functional properties. Our results suggest that this region of gD is important for nectin-1 interaction and is distinct from but partially overlaps the site of HVEM binding. Unique gD mutants with altered receptor usage generated in this study may help dissect the roles played by various HSV receptors during infection.     

Secondary structure of a herpes simplex virus glycoprotein D antigenic domain

The peptide 6-amino caproyl-Pro-Ser-Leu-Lys-Met-Ala-Asp-Pro-Asn-Arg-Phe-Arg-Gly-Lys-Asp-Leu- Pro-6- amino caproate has been synthesized and its secondary structure has been investigated by 1H n.m.r. at 400 MHz. Resonances were assigned from 2D NOESY and COSY spectra, and the secondary structure was determined using NOEs, three-bond coupling constants, and exchange rates of amide protons. The peptide has two tight turns centered on the Pro-Asn and Arg-Gly pairs. The relationship between the secondary structure found here and the antigenic nature of the peptide is discussed.     

Site-directed and linker insertion mutagenesis of herpes simplex virus type 1 glycoprotein H.

The gH-gL complex of herpes simplex virus type 1 (HSV-1) is essential for virion infectivity and virus-induced cell fusion, but functional domains of the gH molecule remain to be defined. We have addressed this question by mutagenesis. A set of linker insertion mutants in HSV-1 gH was generated and tested in transient assays for their ability to complement a gH-negative virus. Insertions at three sites in the C-terminal third of the external domain affected the ability of gH to function in cell-cell fusion and virus entry, while insertions at six sites in the N-terminal half of the external domain induced conformational changes in gH such that it was not recognized by monoclonal antibody LP11, although expression at the cell surface was unchanged. A recombinant virus in which a potential integrin-binding motif, RGD, in gH was changed to the triplet RGE entered cells as efficiently as the wild type, indicating that HSV-1 entry is not mediated by means of the gH-RGD motif binding to cell surface integrins. Furthermore, mutagenesis of the glycosylation site which is positionally conserved in all herpesvirus gH sequences in close proximity to the transmembrane domain generated a recombinant virus that grew in vitro with wild-type single-step kinetics.     

Localization of epitopes of herpes simplex virus type 1 glycoprotein D.

We previously defined eight groups of monoclonal antibodies which react with distinct epitopes of herpes simplex virus glycoprotein D (gD). One of these, group VII antibody, was shown to react with a type-common continuous epitope within residues 11 to 19 of the mature glycoprotein (residues 36 to 44 of the predicted sequence of gD). In the current investigation, we have localized the sites of binding of two additional antibody groups which recognize continuous epitopes of gD. The use of truncated forms of gD as well as computer predictions of secondary structure and hydrophilicity were instrumental in locating these epitopes and choosing synthetic peptides to mimic their reactivity. Group II antibodies, which are type common, react with an epitope within residues 268 to 287 of the mature glycoprotein (residues 293 to 312 of the predicted sequence). Group V antibodies, which are gD-1 specific, react with an epitope within residues 340 to 356 of the mature protein (residues 365 to 381 of the predicted sequence). Four additional groups of monoclonal antibodies appear to react with discontinuous epitopes of gD-1, since the reactivity of these antibodies was lost when the glycoprotein was denatured by reduction and alkylation. Truncated forms of gD were used to localize these four epitopes to the first 260 amino acids of the mature protein. Competition experiments were used to assess the relative positions of binding of various pairs of monoclonal antibodies. In several cases, when one antibody was bound, there was no interference with the binding of an antibody from another group, indicating that the epitopes were distinct. However, in other cases, there was competition, indicating that these epitopes might share some common amino acids.     

Structure-function analysis of soluble forms of herpes simplex virus glycoprotein D.

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry. Truncated forms of gD lacking the transmembrane and cytoplasmic tail regions have been shown to bind to cells and block plaque formation. Using complementation analysis and a panel of gD mutants, we previously identified four regions of gD (regions I to IV) which are important for virus entry. Here, we used baculovirus vectors to overexpress truncated forms of wild-type gD from HSV type 1 (HSV-1) [gD-1(306t)] and HSV-2 [gD-2(306t)] and four mutants, gD-1(inverted delta 34t), gD-1(inverted delta 126t), gD-1(inverted delta 243t), and gD-1(delta 290-299t), each having a mutation in one of the four functional regions. We used an enzyme-linked immunosorbent assay and circular dichroism to analyze the structure of these proteins, and we used functional assays to study the role of gD in binding, penetration, and cell-to-cell spread. gD-1 and gD-2 are similar in antigenic structure and thermal stability but vary in secondary structure. Mutant proteins with insertions in region I or II were most altered in structure and stability, while mutants with insertions in region III or IV were less altered. gD-1(306t) and gD-2(306t) inhibited both plaque formation and cell-to-cell transmission of HSV-1. In spite of obvious structural differences, all of the mutant proteins bound to cells, confirming that binding is not the only function of gD. The region I mutant did not inhibit HSV plaque formation or cell-to-cell spread, suggesting that this region is necessary for the function of gD in these processes. Surprisingly, the other three mutant proteins functioned in all of the in vitro assays, indicating that the ability of gD to bind to cells and inhibit infection does not correlate with its ability to initiate infection as measured by the complementation assay. The region IV mutant, gD-1(delta 290-299t), had an unexpected enhanced inhibitory effect on HSV infection. Taken together, the results argue against a single functional domain in gD. It is likely that different gD structural elements are involved in successive steps of infection.     

Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein D inhibit virus penetration.

Nine monoclonal antibodies specific for glycoprotein D (gD) of herpes simplex virus type 1 were selected for their ability to neutralize virus in the presence of complement. Four of these antibodies exhibited significant neutralization titers in the absence of complement, suggesting that their epitope specificities are localized to site(s) which contribute to the role of gD in virus infectivity. Each of these antibodies was shown to effectively neutralize virus after virion adsorption to cell surfaces, indicating that neutralization did not involve inhibition of virus attachment. Although some of the monoclonal antibodies partially inhibited adsorption of radiolabeled virions, this effect was only observed at concentrations much higher than that required to neutralize virus and did not correlate with complement-independent virus-neutralizing activity. All of the monoclonal antibodies slowed the rate at which virus entered cells, further suggesting that antibody binding of gD inhibits virus penetration. Experiments were carried out to determine the number of different epitopes recognized by the panel of monoclonal antibodies and to identify epitopes involved in complement-independent virus neutralization. Monoclonal antibody-resistant (mar) mutants were selected by escape from neutralization with individual gD-specific monoclonal antibodies. The reactivity patterns of the mutants and antibodies were then used to construct an operational antigenic map for gD. This analysis identified a minimum of six epitopes on gD that could be grouped into four antigenic sites. Antibodies recognizing four distinct epitopes contained in three antigenic sites were found to neutralize virus in a complement-independent fashion. Moreover, mar mutations in these sites did not affect the processing of gD, rate of virus penetration, or the ability of the virus to replicate at high temperature (39 degrees C). Taken together, these results (i) confirm that gD is a major target antigen for neutralizing antibody, (ii) indicate that the mechanism of neutralization can involve inhibition of virus penetration of the cell surface membrane, and (iii) strongly suggest that gD plays a direct role in the virus entry process.     

Antigenic and mutational analyses of herpes simplex virus glycoprotein B reveal four functional regions

Glycoprotein B (gB), along with gD, gH, and gL, is essential for herpes simplex virus (HSV) entry. The crystal structure of the gB ectodomain revealed it to be an elongated multidomain trimer. We generated and characterized a panel of 67 monoclonal antibodies (MAbs). Eleven of the MAbs had virus-neutralizing activity. To organize gB into functional regions within these domains, we localized the epitopes recognized by the entire panel of MAbs and mapped them onto the crystal structure of gB. Most of the MAbs were directed to continuous or discontinuous epitopes, but several recognized discontinuous epitopes that showed some resistance to denaturation, and we refer to them as pseudo-continuous. Each category contained some MAbs with neutralizing activity. To map continuous epitopes, we used overlapping peptides that spanned the gB ectodomain and measured binding by enzyme-linked immunosorbent assay. To identify discontinuous and pseudocontinuous epitopes, a purified form of the ectodomain of gB, gB(730t), was cleaved by alpha-chymotrypsin into two major fragments comprising amino acids 98 to 472 (domains I and II) and amino acids 473 to 730 (major parts of domains III, IV, and V). We also constructed a series of gB truncations to augment the other mapping strategies. Finally, we used biosensor analysis to assign the MAbs to competition groups. Together, our results identified four functional regions: (i) one formed by residues within domain I and amino acids 697 to 725 of domain V; (ii) a second formed by residues 391 to 410, residues 454 to 475, and a less-defined region within domain II; (iii) a region containing residues of domain IV that lie close to domain III; and (iv) the first 12 residues of the N terminus that were not resolved in the crystal structure. Our data suggest that multiple domains are critical for gB function.     

Glycoprotein D of herpes simplex virus encodes a domain which precludes penetration of cells expressing the glycoprotein by superinfecting herpes simplex virus.

Earlier studies have shown that herpes simplex viruses adsorb to but do not penetrate permissive baby hamster kidney clonal cell lines designated the BJ series and constitutively expressing the herpes simplex virus 1 (HSV-1) glycoprotein D (gD). To investigate the mechanism of the restriction, the following steps were done. First, wild-type HSV-1 strain F [HSV-1(F)] virus was passaged blindly serially on clonal line BJ-1 and mutant viruses [HSV-1(F)U] capable of penetration were selected. The DNA fragment capable of transferring the capacity to infect BJ cells by marker transfer contains the gD gene. The mutant gD, designated gDU, differed from wild-type gD only in the substitution of Leu-25 by proline. gDU reacted with monoclonal antibodies which neutralize virus and whose epitopes encompass known functional domains involved in virus entry into cells. It did not react with the monoclonal antibody AP7 previously shown to react with an epitope which includes Leu-25. Second, cell lines expressing gDU constitutively were constructed and cloned. Unlike the clonal cell lines constitutively expressing gD (e.g., the BJ cell line), those expressing gDU were infectable by both HSV-1(F) and HSV-1(F)U. Lastly, exposure of BJ cells to monoclonal antibody AP7 rendered the cells capable of being infected with HSV-1(F). The results indicate that (i) gD expresses a specific function, determined by sequences at or around Leu-25, which blocks entry of virus into cells synthesizing gD, (ii) the gD which blocks penetration by superinfecting virus is located in the plasma membrane, (iii) the target of the restriction to penetration is the identical domain of the gD molecule contained in the envelope of the superinfecting virus, and (iv) the molecular basis of the restriction does not involve competition for a host protein involved in entry, as was previously thought.     

Oligomerization of herpes simplex virus glycoprotein B.

Glycoprotein B (gB) specified by herpes simplex virus can be extracted from virions or infected cells in the form of detergent-stable, heat-dissociable oligomers. The composition of the oligomers and requirements for their formation were investigated. Evidence is presented that the faster-migrating forms of the oligomers are homodimers of gB. Dimerization was shown to occur within minutes of polypeptide synthesis and did not depend on glycosylation, the expression of other viral proteins, or virion morphogenesis. The multiple, electrophoretically distinct forms of gB dimers differ in extent or rate of N-linked oligosaccharide processing and also have other differences that influence electrophoretic mobility.     

Structure of herpes simplex virus glycoprotein D bound to the human receptor nectin-1

Binding of herpes simplex virus (HSV) glycoprotein D (gD) to a cell surface receptor is required to trigger membrane fusion during entry into host cells. Nectin-1 is a cell adhesion molecule and the main HSV receptor in neurons and epithelial cells. We report the structure of gD bound to nectin-1 determined by x-ray crystallography to 4.0 Å resolution. The structure reveals that the nectin-1 binding site on gD differs from the binding site of the HVEM receptor. A surface on the first Ig-domain of nectin-1, which mediates homophilic interactions of Ig-like cell adhesion molecules, buries an area composed by residues from both the gD N- and C-terminal extensions. Phenylalanine 129, at the tip of the loop connecting β-strands F and G of nectin-1, protrudes into a groove on gD, which is otherwise occupied by C-terminal residues in the unliganded gD and by N-terminal residues in the gD/HVEM complex. Notably, mutation of Phe129 to alanine prevents nectin-1 binding to gD and HSV entry. Together these data are consistent with previous studies showing that gD disrupts the normal nectin-1 homophilic interactions. Furthermore, the structure of the complex supports a model in which gD-receptor binding triggers HSV entry through receptor-mediated displacement of the gD C-terminal region.

Authors Summary

Herpes simplex virus (HSV) is a widespread human pathogen. Four viral glycoproteins (gD, gB, gH/gL) are required for HSV entry into host cells. gD binding to a cell surface receptor triggers conformational changes in the other viral glycoproteins leading to membrane fusion and viral entry. Two structurally unrelated cellular protein receptors, nectin-1 and HVEM, can mediate HSV entry upon binding to gD. The structure presented here reveals the molecular basis for the stable interaction between HSV-1 gD and the receptor nectin-1. Comparison with the previously determined structures of the gD/HVEM complex and unliganded gD shows that, despite the fact that the two receptors interact with different binding sites, they both cause a similar conformational change in gD. Therefore, our data point to a conserved mechanism for receptor mediated activation of the HSV entry process. In addition, the gD/Nectin-1 structure reveals that the gD-binding site overlaps with a surface involved in nectin-1 homo-dimerization. This observation explains how gD interferes with the cell adhesion function of nectin-1. Finally, the gD/Nectin-1 complex displays similarities with other viral ligands bound to immunoglobulin-like receptors suggesting a convergent mechanism for receptors selection and usage.     

Fine mapping of antigenic site II of herpes simplex virus glycoprotein D.

Glycoprotein D (gD) is a virion envelope component of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) which plays an important role in viral infection and pathogenesis. Previously, anti-gD monoclonal antibodies (MAbs) were arranged into groups which recognize distinct type-common and type-specific sites on HSV-1 gD (gD-1) and HSV-2 gD (gD-2). Several groups recognize discontinuous epitopes which are dependent on tertiary structure. Three groups, VII, II, and V, recognize continuous epitopes present in both native and denatured gD. Previously, group II consisted of a single MAb, DL6, whose epitope was localized between amino acids 268 and 287. In the study reported here, we extended our analysis of the antigenic structure of gD, concentrating on continuous epitopes. The DL6 epitope was localized with greater precision to residues 272 to 279. Four additional MAbs including BD78 were identified, each of which recognizes an epitope within residues 264 to 275. BD78 and DL6 blocked each other in binding to gD. In addition, a mutant form of gD was constructed in which the proline at 273 was replaced by serine. This change removes a predicted beta turn in gD. Neither antibody reacted with this mutant, indicating that the BD78 and DL6 epitopes overlap and constitute an antigenic site (site II) within residues 264 to 279. A separate antigenic site (site XI) was recognized by MAb BD66 (residues 284 to 301). This site was only six amino acids downstream of site II, but was distinct as demonstrated by blocking studies. Synthetic peptides mimicking these and other regions of gD were screened with polyclonal antisera to native gD-1 or gD-2. The results indicate that sites II, V, VII, and XI, as well as the carboxy terminus, are the major continuous antigenic determinants on gD. In addition, the results show that the region from residues 264 through 369, except the transmembrane anchor, contains a series of continuous epitopes.     

Cellular localization of nectin-1 and glycoprotein D during herpes simplex virus infection

During viral entry, herpes simplex virus (HSV) glycoprotein D (gD) interacts with a specific cellular receptor such as nectin-1 (PRR1/HveC/CD111) or the herpesvirus entry mediator A (HVEM/HveA). Nectin-1 is involved in cell-to-cell adhesion. It is located at adherens junctions, where it bridges cells through homophilic or heterophilic interactions with other nectins. Binding of HSV gD prevents nectin-1-mediated cell aggregation. Since HSV gD affects the natural function of nectin-1, we further investigated the effects of gD expression on nectin-1 during HSV infection or in transfected cells. We also studied the importance of the interaction between nectin-1 and the cytoplasmic protein afadin for HSV entry and spread as well as the effects of infection on this interaction. In these investigations, we used a panel of cells expressing nectin-1 or nectin-1-green fluorescent protein fusions as the only mediators of HSV entry. During HSV infection, nectin-1 localization at adherens junction was dramatically altered in a manner dependent on gD expression. Nectin-1 and gD colocalized at cell contact areas between infected and noninfected cells and at the edges of plaques. This specific accumulation of gD at junctions was driven by expression of nectin-1 in trans on the surface of adjacent cells. Reciprocally, nectin-1 was maintained at junctions by the trans expression of gD in the absence of a cellular natural ligand. Our observations indicate that newly synthesized gD substitutes for nectin-1 of infected cells at junctions with noninfected cells. We propose that gD attracts and maintains the receptor at junctions where it can be used for virus spread.     

Efficacy of recombinant herpes simplex virus 1 glycoprotein D candidate vaccines in mice

To compare the immunogenity of the herpes simplex virus 1 (HSV-1/HHV-1) recombinant glycoprotein D (gD1), as a potential protective vaccine, Balb/c mice were immunized with either gD1/313 (the ectodomain of the gD1 fusion protein consisting of 313 amino acid residues), or the plasmid pcDNA3.1-gD (coding for a full length gD1 protein, FLgD1). A live attenuated HSV-1 (deleted in the gE gene), and a HSV-1 (strain HSZP)-infected cell extract served as positive controls, and three non-structural recombinant HSV-1 fusion proteins (ICP27, UL9/OBP and thymidine kinase--TK) were used as presumed non-protective (negative) controls. Protection tests showed that the LD50 value of the challenging infectious virus increased 90-fold in mice immunized with ICP27, but remained unchanged in other control mice immunized with TK and OBP polypeptides. A significant protection (the LD50 value of challenging virus increased 800-fold) was noted following immunization with gD1/313, while immunization with the gE-del virus and/or the gD1 DNA vaccine resulted in a more than 4,000-fold increase of the challenging virus dose killing 50% of the animals. Using ELISA, elevated antibody titers were detected following immunizations with gD1/313, gE-del virus, and/or HSV-1-infected-cell extract. In addition, all of the three non-structural proteins elicited a good humoral response (with titres ranging from 1:16,000 to 1:128,000). The lowest IgG response (1:8,000) was noted after immunization with the gD1 DNA vaccine. Peripheral blood leukocytes (PBLs) as well as splenocytes from mice immunized with gD1/313, gE-del virus, and gD1-plasmid responded in lymphocyte transformation test (LTT) to the presence of purified gD1/313 antigen. For PBLs, the most significant stimulation of thymidine incorporation was registered at a gD1/313 concentration of 5 microg/100 microl, while the splenocytes from DNA vaccine-immunized mice responded already at a concentration of 1 microg/100 microl.     

Mutations in herpes simplex virus glycoprotein D distinguish entry of free virus from cell-cell spread

Herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) is an essential component of the entry apparatus that is responsible for viral penetration and subsequent cell-cell spread. To test the hypothesis that gD may serve distinguishable functions in entry of free virus and cell-cell spread, mutants were selected for growth on U(S)11cl19.3 cells, which are resistant to both processes due to the lack of a functional gD receptor, and then tested for their ability to enter as free virus and to spread from cell to cell. Unlike their wild-type parent, HSV-1(F), the variants that emerged from this selection, which were named SP mutants, are all capable of forming macroscopic plaques on the resistant cells. This ability is caused by a marked increase in cell-cell spread without a concomitant increase in efficiency of entry of free virus. gD substitutions that arose within these mutants are sufficient to mediate cell-cell spread in U(S)11cl19.3 cells but are insufficient to overcome the restriction to entry of free virions. These results suggest that mutations in gD (i) are sufficient but not necessary to overcome the block to cell-cell spread exhibited by U(S)11cl19.3 cells and (ii) are insufficient to mediate entry of free virus in the same cells.     

Identification of C3b-binding regions on herpes simplex virus type 2 glycoprotein C.

Glycoprotein C from herpes simplex viruses types 1 and 2 (gC-1 and gC-2) acts as a receptor for the C3b fragment of the third component of complement. Our goal is to identify domains on gC involved in C3b receptor activity. Here, we used in-frame linker-insertion mutagenesis of the cloned gene for gC-2 to identify regions of the protein involved in C3b binding. We constructed 41 mutants of gC-2, each having a single, double, or triple insertion of four amino acids at sites spread across the protein. A transient transfection assay was used to characterize the expressed mutant proteins. All of the proteins were expressed on the transfected cell surface, exhibited processing of N-linked oligosaccharides, and bound one or more monoclonal antibodies recognizing distinct antigenic sites on native gC-2. This suggested that each of the mutant proteins was folded into a native structure and that a loss of C3b binding by any of the mutants could be attributed to the disruption of a specific functional domain. When the panel of insertion mutants was assayed for C3b receptor activity, we identified three distinct regions that are important for C3b binding, since an insertion within those regions abolished C3b receptor activity. Region I was located between amino acids 102 and 107, region II was located between residues 222 and 279, and region III was located between residues 307 and 379. In addition, region III has some structural features similar to a conserved motif found in complement receptor 1, the human C3b receptor. Finally, blocking experiments indicated that gC-1 and gC-2 bind to similar locations on the C3b molecule.     

Extensive homology between the herpes simplex virus type 2 glycoprotein F gene and the herpes simplex virus type 1 glycoprotein C gene.

The region of the herpes simplex virus type 2 (HSV-2) genome which maps colinearly with the HSV-1 glycoprotein C (gC) gene has been cloned, and the DNA sequence of a 2.29-kilobase region has been determined. Contained within this sequence is a major open reading frame of 479 amino acids. The carboxyterminal three-fourths of the derived HSV-2 protein sequence showed a high degree of sequence homology to the HSV-1 gC amino acid sequence reported by Frink et al. (J. Virol. 45:634-647, 1983). The amino-terminal region of the HSV-2 sequence, however, showed very little sequence homology to HSV-1 gC. In addition, the HSV-1 gC sequence contained 27 amino acids in the amino-terminal region which were missing from the HSV-2 protein. Computer-assisted analysis of the hydrophilic and hydrophobic properties of the derived HSV-2 sequence demonstrated that the protein contained structures characteristic of membrane-bound glycoproteins, including an amino-terminal signal sequence and carboxy-terminal hydrophobic transmembrane domain and charged cytoplasmic anchor. The HSV-2 protein sequence also contained seven putative N-linked glycosylation sites. These data, in conjunction with mapping studies of Para et al. (J. Virol. 45:1223-1227, 1983) and Zezulak and Spear (J. Virol. 49:741-747, 1984), suggest that the protein sequence derived from the HSV-2 genome corresponds to gF, the HSV-2 homolog of HSV-1 gC.