Characterization of a recombinant herpes simplex virus which expresses a glycoprotein D lacking asparagine-linked oligosaccharides.

Glycoprotein D (gD) is an envelope component of herpes simplex virus essential for virus penetration. gD contains three sites for addition of asparagine-linked carbohydrates (N-CHO), all of which are utilized. Previously, we characterized mutant forms of herpes simplex virus type 1 gD (gD-1) lacking one or all three N-CHO addition sites. All of the mutants complemented the infectivity of a gD-minus virus, F-gD beta, to the same extent as wild-type gD. Here, we show that recombinant viruses containing mutations in the gD-1 gene which eliminate the three N-CHO signals are viable. Two such viruses, called F-gD(QAA)-1 and F-gD(QAA)-2, were independently isolated, and the three mutations in the gD gene in one of these viruses were verified by DNA sequencing. We also verified that the gD produced in cells infected by these viruses is devoid of N-CHO. Plaques formed by both mutants developed more slowly than those of the wild-type control virus, F-gD(WT), and were approximately one-half the size of the wild-type. One mutant, F-gD(QAA)-2, was selected for further study. The QAA mutant and wild-type gD proteins extracted from infected cells differed in structure, as determined by the binding of monoclonal antibodies to discontinuous epitopes. However, flow cytometry analysis showed that the amount and structure of gD found on infected cell surfaces was unaffected by the presence or absence of N-CHO. Other properties of F-gD(QAA)-2 were quite similar to those of F-gD(WT). These included (i) the kinetics of virus production as well as the intracellular and extracellular virus titers; (ii) the rate of virus entry into uninfected cells; (iii) the levels of gB, gC, gE, gH, and gI expressed by infected cells; and (iv) the turnover time of gD. Thus, the absence of N-CHO from gD-1 has some effect on its structure but very little effect on its function in virus infection in cell culture.     

Disulfide bond structure of glycoprotein D of herpes simplex virus types 1 and 2.

Glycoprotein D (gD) is a structural component of the herpes simplex virus envelope which is essential for virus penetration. The function of this protein is highly dependent on its structure, and its structure is dependent on maintenance of three intact disulfide bonds. gD contains six cysteines in its ectodomain whose spacing is conserved among all its homologs in other alphaherpesviruses as well as Marek's disease virus. For other proteins, conservation of cysteine spacing correlates with conservation of disulfide bond structure. We have now solved the disulfide bond structure of gD-1 and gD-2 of herpes simplex virus types 1 and 2, respectively. Two approaches were used. First, we constructed 15 double-Cys mutants of gD-1, representing all possible disulfide pairs. In each case, codons for cysteines were changed to serine. We reasoned that if two cysteines normally form a disulfide bond, double mutations which eliminate one proper bond should be less harmful to gD structure than double mutations which eliminate two disulfide bonds. The mutated genes were cloned into a eucaryotic expression vector, and the proteins were expressed in transiently transfected cells. Three double mutations, Cys-1,5, Cys-2,6, and Cys-3,4 permitted gD-1 folding, processing, transport to the cell surface, and function in virus infection, whereas 12 other double mutations each produced a malfolded and nonfunctional protein. Thus, the three functional double-Cys mutants may represent the actual partners in disulfide bond linkages. The second approach was to define the actual disulfide bond structure of gD by biochemical means. Purified native gD-2 was cleaved by CNBr and proteases, and the peptides were separated by high-performance liquid chromatography. Disulfide-linked peptides were subjected to N-terminal amino acid sequencing. The results show that cysteine 1 (amino acid [aa] 66) is bonded to cysteine 5 (aa 189), cysteine 2 (aa 106) is bonded to cysteine 6 (aa 202), and cysteine 3 (aa 118) is bonded to cysteine 4 (aa 127). Thus, the biochemical analysis of gD-2 agrees with the genetic analysis of gD-1. A similar disulfide bond arrangement is postulated to exist in other gD homologs.     

A herpes simplex virus mutant in which glycoprotein D sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells.

Herpes simplex virus (HSV) glycoprotein gD is a major component of the virion envelope and is thought to play an important role in the initial stages of viral infection and stimulates the production of high titers of neutralizing antibodies. We assumed that gD plays an essential role in virus replication, and so to complement viruses with mutations in the gD gene we constructed a cell line, denoted VD60, which is capable of expressing high levels of gD after infection with HSV. A recombinant virus, designated F-gD beta, in which sequences encoding gD and a nonessential glycoprotein, gI, were replaced by Escherichia coli beta-galactosidase sequences, was selected on the basis that it produced blue plaques on VD60 cell monolayers under agarose overlays containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). F-gD beta was able to replicate normally on complementing VD60 cells. However, F-gD beta was unable to form plaques on noncomplementing Vero cells. Virions lacking gD were produced in normal amounts by Vero cells infected with F-gD beta, and the virus particles were distributed throughout the cytoplasm and on the cell surface, suggesting that gD is not essential for HSV envelopment and egress. Virions lacking gD were able to bind to cells, but were unable to initiate synthesis of viral early polypeptides. Plaque production of F-gD beta particles lacking gD was enhanced by polyethylene glycol treatment, suggesting that gD is essential for penetration of HSV into cells. Other HSV glycoproteins have been implicated in the entry of virus into cells, and thus this process appears to involve multiple interactions at the cell surface.     

Deletions in herpes simplex virus glycoprotein D define nonessential and essential domains.

Herpes simplex virus glycoprotein D (gD) is a major component of the virion envelope and infected cell membranes and is essential for virus entry into cells. We have recently shown that gD interacts with a limited number of cell surface receptors which are required for virus penetration into cells. To define domains of gD which are required for aspects of virus replication including receptor binding, deletion mutations of 5 to 14 amino acids were constructed by using oligonucleotide-directed mutagenesis. Plasmids containing mutant genes for gD were assayed for the ability to rescue a recombinant virus, F-gD beta, in which beta-galactosidase sequences replace gD-coding sequences. Effects on global folding and posttranslational processing of the molecules were assessed by using a panel of monoclonal antibodies which recognize both continuous and discontinuous epitopes. A region near the amino terminus (residues 7 to 21) of gD which is recognized by monoclonal antibodies able to neutralize herpes simplex virus in the absence of complement was not essential for function. In addition, virtually all of the cytoplasmic domain of gD and an extracellular domain close to the membrane were dispensable. In contrast, deletion mutations in the central region of the molecule, save for one exception, led to alterations in global folding of the molecule and maturation of the protein was inhibited.     

Absence of asparagine-linked oligosaccharides from glycoprotein D of herpes simplex virus type 1 results in a structurally altered but biologically active protein.

Glycoprotein D (gD) of herpes simplex virus contains three utilized sites (Asn-X-Ser/Thr) for addition of asparagine-linked carbohydrates (N-CHO). Previously, we used oligonucleotide-directed mutagenesis to alter serine or threonine residues to alanine at each N-CHO addition site. Studies with monoclonal antibodies showed that a mutant protein lacking all three sites (now designated AAA) was structurally altered because of the amino acid change at residue 96 as well as the absence of the N-CHO. In this study, we constructed additional single mutations at site 1 (residues 94 and 96) and found that in most cases, the amino acid change itself adversely affected the conformation of gD. However, changing asparagine 94 to glutamine (Q) at site 1 had the least effect on gD. We constructed a second triple mutant, QAA, which lacked all three N-CHO signals. The antigenic conformation of QAA was similar to that of gD produced in the presence of tunicamycin (TM-gD). However, binding of MAbs to the AAA protein or to single mutants altered at site 1 was reduced compared with TM-gD. Wild-type gD and QAA proteins were equally susceptible to digestion by trypsin or Staphylococcus aureus V8 protease. In contrast, the AAA protein was more sensitive to trypsin but less sensitive to V8, again suggesting conformational alterations of the AAA protein. Despite what appeared to be large changes in structure, each mutant complemented the infectivity of a virus lacking gD (F-gD beta). We conclude that the N-CHO and amino acids at N-CHO site 1 play an important role in forming and/or maintaining gD structure, but none of the N-CHO are required for gD to function in the complementation assay.     

The contribution of cysteine residues to antigenicity and extent of processing of herpes simplex virus type 1 glycoprotein D.

Glycoprotein D (gD) is an envelope component of herpes simplex virus types 1 (gD-1) and 2 (gD-2). The gD-1 polypeptide contains seven cysteine residues among its 369 amino acids; six are located on the N-terminal or luminal portion of the glycoprotein, and a seventh is located in the transmembrane region. Previous studies used a panel of monoclonal antibodies (MAbs) to define gD epitopes as continuous or discontinuous. Purified gD, denatured by reduction and alkylation, loses discontinuous epitopes, whereas continuous epitopes are retained. The contribution of disulfide bonds to maintenance of discontinuous epitopes is, therefore, significant. In the present study, our objective was to determine the contribution of individual cysteine residues to folding of gD-1 into its native conformation. Site-directed oligonucleotide mutagenesis was used to create seven mutants, each with a serine residue replacing a cysteine. The mutated genes were cloned into a eucaryotic expression vector and transfected into COS-1 cells, and the proteins were separated by nondenaturing polyacrylamide gel electrophoresis, followed by immunoblotting. Replacement of cysteine 7 (residue 333) had only a minimal effect on the antigenic properties of gD-1. In contrast, replacement of any one of the other six cysteine residues resulted in either a major reduction or a complete loss of binding of those MAbs that recognize discontinuous epitopes, with no effect on the binding of MAbs which recognize continuous epitopes. These mutations also had profound effects on the extent of oligosaccharide processing of gD-1. This was determined by digestion of the expressed proteins with various endoglycosidases, followed by electrophoresis and Western blotting (immunoblotting) to observe any mobility changes. Three mutant gD proteins which did not express discontinuous epitopes contained only high-mannose-type oligosaccharides, suggesting that processing had not proceeded beyond the precursor stage. Two mutant forms of gD exhibited reduced binding of MAbs to discontinuous epitopes. A small proportion of the molecules which accumulated at 48 h posttransfection contained complex oligosaccharides. One mutant exhibited reduced binding of MAbs to discontinuous epitopes, but was present at 48 h posttransfection only in the precursor form. The cysteine 7 mutant was processed to the same extent as wild-type gD. We conclude that the first six cysteine residues are critical to the correct folding, antigenic structure, and processing of gD-1, and we speculate that they form three disulfide-bonded pairs.     

Mutant analysis of herpes simplex virus-induced cell surface antigens: resistance to complement-mediated immune cytolysis.

BHK-21 cells infected with temperature-sensitive mutants of herpes simplex virus type 1 strain KOS representing 16 complementation groups were tested for susceptibility to complement-mediated immune cytolysis at permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures. Only cells infected by mutants in complementation group E were resistant to immune cytolysis in a temperature-sensitive manner compared with wild-type infections. The expression of group E mutant cell surface antigens during infections at 34 and 39 degrees C was characterized by a combination of cell surface radioiodination, specific immunoprecipitation, and gel electrophoretic analysis of immunoprecipitates. Resistance to immune lysis at 39 degrees C correlated with the absence of viral antigens exposed at the cell surface. Intrinsic radiolabeling of group E mutant infections with [14C]glucosamine revealed that normal glycoproteins were produced at 34 degrees C but none were synthesized at 39 degrees C. The effect of 2-deoxy-D-glucose on glycosylation of group E mutants at 39 degrees C suggested that the viral glycoprotein precursors were not synthesized. The complementation group E mutants failed to complement herpes simplex virus type 1 mutants isolated by other workers. These included the group B mutants of strain KOS, the temperature-sensitive group D mutants of strain 17, and the LB2 mutant of strain HFEM. These mutants should be considered members of herpes simplex virus type 1 complementation group 1.2, in keeping with the new herpes simplex virus type 1 nomenclature.     

Immunogenicity of herpes simplex virus glycoproteins gC and gB and their role in protective immunity.

The relative antigenicity of the individual herpes simplex virus type 1 (KOS) glycoproteins gC and gB was analyzed in BALB/c mice by using KOS mutants altered in their ability to present these antigens on cell surface membranes during infection. The mutants employed were as follows: syn LD70 , a non-temperature-sensitive mutant defective in the synthesis of cell surface membrane gC; tsF13 , a temperature-sensitive mutant defective in the processing of the precursor form of gB to the mature cell surface form at 39 degrees C; and ts606 , an immediate early temperature-sensitive mutant defective in the production of all early and late proteins including the glycoproteins. By comparing the relative susceptibility to immunolysis of mouse 3T3 cells infected at 39 degrees C with wild-type virus, presenting the full complement of the glycoprotein antigens, gC, gB, and gD, with target cells infected with mutants presenting only subsets of these antigens, we determined that a major portion of cytolytic antibody contained in hyperimmune anti-herpes simplex virus type 1 (KOS) mouse antiserum was directed against glycoproteins gC and gB. The relative immunogenicity of wild-type and mutant virus-infected cells also was compared in BALB/c mice. Immunogen lacking the mature form of gB induced a cytolytic antibody titer comparable to that of the wild-type virus, whereas that lacking the mature form of gC showed a 70% reduction in titer. The absence of the mature cell surface forms of gB and gC in immunogen preparations resulted in a 4- to 15-fold reduction in in virus neutralizing titer. Animals immunized with ts606 -infected cells (39 degrees C) induced relatively little virus-specific cytolytic and neutralizing antibody. Analysis of the glycoprotein specificities of these antisera by radioimmunoprecipitation showed that the antigens immunoprecipitated reflected the viral plasma membrane glycoprotein profiles of the immunogens. The absence of the mature forms of gC or gB in the immunizing preparation did not appreciably affect the immunoprecipitating antibody response to other antigens. Mice immunized with wild-type and mutant virus-infected cells were tested for their resistance to intracranial and intraperitoneal challenge with the highly virulent WAL strain of herpes simplex virus type 1. Despite the observed alterations in serum virus-specific antibody induced with the individual immunogens, all animals survived an intraperitoneal challenge of 10 50% lethal doses. However, differences in the survival of animals were obtained upon intracranial challenge.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

The phenotype in vitro and in infected cells of herpes simplex virus 1 alpha trans-inducing factor (VP16) carrying temperature-sensitive mutations introduced by substitution of cysteines.

alpha trans-inducing factor (alpha TIF, VP16, Vmw65) is an essential structural protein of herpes simplex virus, being required for virion assembly. The protein also forms complexes with host proteins and a response element and transactivates the alpha genes which carry this element. The protein contains an acidic carboxyl terminus required for transactivation and a much larger amino-terminal domain required for promoter recognition. We report the first set of temperature-sensitive (ts) mutations deliberately introduced into the protein by substitution of the cysteine codons with those specifying glycine at positions 78, 102, and 176, either singly or in combinations. We report the following results. (i) All mutated proteins synthesized in vitro formed complexes with the DNA response element at room temperature. However, the mutant with the triple substitution and two mutants with substitutions in two of the three cysteines exhibited a ts phenotype at 33 and 37 degrees C, and one exhibited a ts phenotype only at 37 degrees C. (ii) Replacement of wild-type alpha TIF with genes carrying substitutions in any two cysteines conferred a ts phenotype for replication at 39.5 degrees C. Shift-down experiments indicated that the 10(4)- to 10(5)-fold reduction in virus yield at the nonpermissive temperature was due to the disfunction of alpha TIF late in infection, presumably in virion maturation. (iii) The alpha TIF expressed in cells infected with mutant viruses exhibited the same ts phenotype in protein-DNA complex formation as those expressed in vitro from mutated plasmids. Although the virus carrying the alpha TIF substitutions at Cys-102 and Cys-176 failed to induce a reporter gene linked to the alpha 4 promoter at 39.5 degrees C, it replicated as well as the parent virus in cells maintained for the first 10 h of infection at 39.5 degrees C. We conclude the following. (i) Formation of DNA-protein complexes containing alpha TIF is a poor prognosticator of alpha TIF function. (ii) The data presented here and in the literature strongly support the hypothesis that the secondary structure of the alpha TIF is very sensitive to deletions or insertions which probably affect the interaction of alpha TIF with both viral proteins in the virion and cellular proteins during infection. As a consequence, deletion-insertion mutagenesis may not shed useful information on the role of transactivating function of alpha TIF in infection. (iii) Since cysteines may play a role in stabilizing the secondary structure of proteins, substitutions of cysteines may be a powerful technique for site-specific construction of ts mutants in essential viral proteins.     

Truncated forms of glycoprotein D of herpes simplex virus 1 capable of blocking apoptosis and of low-efficiency entry into cells form a heterodimer dependent on the presence of a cysteine located in the shared transmembrane domains

Earlier studies have shown that herpes simplex virus 1 (HSV-1) virions of mutant lacking glycoprotein D (gD) and made in either complementing (gD(-/+) stocks) or noncomplementing cells (gD(-/-) stocks) induce apoptosis. Subsequent studies have shown that apoptosis induced by gD(-/-) mutant virus stocks can be blocked by in trans delivery of viral genes that encode either intact gD or a mixture of two genes encoding the glycoprotein ectodomain plus transmembrane domain (gD-B) and transmembrane domain plus the cytoplasmic carboxyl terminus of the protein (gD-D), respectively. Since the presence of the transmembrane domains was critical for precluding apoptosis in the bipartite system, the question arose whether the two components, gD-B and gD-D, form a heterodimer mediated by an unpaired cysteine located in the transmembrane domain. We report the following. (i) The substitution of the unpaired cysteine with serine in either gD-B or gD-D truncated forms of gD disabled the ability of gD-D and gD-B to block apoptosis. (ii) Immunoprecipitation of gD-D coprecipitated gD-B only from lysates of cells transduced with gD-D and gD-B containing the cysteine in the transmembrane domains. Replacement of cysteine with serine ablated coprecipitation of the components. (ii) The mixture of gD-D and gD-B complemented at a low level gD(-/+) virions. We conclude that the gD-B and gD-D can form a heterodimer dependent on the presence of cysteines in the transmembrane domain and the heterodimer can substitute for intact gD but at a much reduced efficiency.     

A post-alpha gene function turns off the capacity of a host protein to bind DNA in cells infected with herpes simplex virus 1.

HEp-2 cell proteins electrophoretically separated in denaturing polyacrylamide gels and electrically transferred to nitrocellulose sheets contain a polypeptide which efficiently binds linear native DNA end labeled with 32P but not denatured DNA. The polypeptide has an apparent molecular weight of ca. 130,000. The activity of the protein was stable, and no appreciable turnover was observed after exposure of uninfected cells to inhibitory concentrations of cycloheximide for intervals of up to 24 h. However, the activity was absent from lysates of cells harvested 6 h or later postinfection with wild-type viruses. To identify the viral function involved in the loss of DNA-binding activity, we tested the lysates of cells infected with several mutants. Thus, the DNA-binding activity was unaffected in cells infected with a temperature-sensitive mutant (herpes simplex virus 1 tsLB2) in the alpha 4 gene and was maintained at a nonpermissive temperature (39 degrees C). Experiments involving (i) temperature shift-down of cells infected with tsLB2 in the presence of cycloheximide, (ii) withdrawal of cycloheximide in the presence and absence of actinomycin D from cells infected with wild-type virus, (iii) infection of cells at 33 and 39 degrees C with herpes simplex virus 1 tsHA1 carrying a temperature-sensitive lesion in the beta 8 gene, and (iv) infection of cells in the presence of inhibitory concentrations of phosphonoacetate led to the conclusion that the viral functions responsible for the loss of DNA-binding capacity were specified by either beta or gamma genes not dependent on viral DNA synthesis for their expression.     

Amino-terminal sequence of glycoprotein D of herpes simplex virus types 1 and 2.

Glycoprotein D (gD) of herpes simplex virus is a structural component of the virion envelope which stimulates production of high titers of herpes simplex virus type-common neutralizing antibody. We carried out automated N-terminal amino acid sequencing studies on radiolabeled preparations of gD-1 (gD of herpes simplex virus type 1) and gD-2 (gD of herpes simplex virus type 2). Although some differences were noted, particularly in the methionine and alanine profiles for gD-1 and gD-2, the amino acid sequence of a number of the first 30 residues of the amino terminus of gD-1 and gD-2 appears to be quite similar. For both proteins, the first residue is a lysine. When we compared our sequence data for gD-1 with those predicted by nucleic acid sequencing, the two sequences could be aligned (with one exception) starting at residue 26 (lysine) of the predicted sequence. Thus, the first 25 amino acids of the predicted sequence are absent from the polypeptides isolated from infected cells.     

Identification of nonessential disulfide bonds and altered conformations in the v-sis protein, a homolog of the B chain of platelet-derived growth factor.

The protein encoded by v-sis, the oncogene of simian sarcoma virus, is homologous to the B chain of platelet-derived growth factor (PDGF). There are eight conserved Cys residues between PDGF-B and the v-sis protein. Both native PDGF and the v-sis protein occur as disulfide-bonded dimers, probably containing both intramolecular and intermolecular disulfide bonds. Oligonucleotide-directed mutagenesis was used to change the Cys codons to Ser codons in the v-sis gene. Four single mutants lacked detectable biological activity, indicating that Cys-127, Cys-160, Cys-171, and Cys-208 are required for formation of a biologically active v-sis protein. The other four single mutants retained biological activity as determined in transformation assays, indicating that Cys-154, Cys-163, Cys-164, and Cys-210 are dispensable for biological activity. Double and triple mutants containing three of these altered sites were constructed, some of which were transforming as well. The v-sis proteins encoded by biologically active mutants displayed significantly reduced levels of dimeric protein compared with the wild-type v-sis protein, which dimerized very efficiently. Furthermore, a mutant with a termination codon at residue 209 exhibited partial transforming activity. This study thus suggests that the minimal region required for transformation consists of residues 127 to 208. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the v-sis proteins encoded by some of the biologically active mutants exhibited an altered conformation when compared with the wild-type v-sis protein, and suggested that Cys-154 and Cys-163 participate in a nonessential disulfide bond.     

Fine structure analysis of type-specific and type-common antigenic sites of herpes simplex virus glycoprotein D

The fine structure of the antigenic determinants of herpes simplex virus type 1 and 2 glycoprotein D (gD) was analyzed to determine whether structural differences underlie the differential immunogenicity of these glycoproteins. A region common to herpes simplex virus type 1 and 2 gD (amino acid residues 11 to 19) and two sites specific for herpes simplex virus type 2 gD (one determined by proline at position 7, the other determined by asparagine at position 21) were localized within the N-terminal 23 amino acids of gD by synthesis of peptides and comparison of their cross-reactivity with antisera raised to herpes simplex virus type 1 and 2 gD. The secondary structure of these peptides, as predicted by computer analysis, is discussed in relation to their immunogenicity.     

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.     

Characterization of a heparan sulfate octasaccharide that binds to herpes simplex virus type 1 glycoprotein D

Herpes simplex virus type 1 utilizes cell surface heparan sulfate as receptors to infect target cells. The unique heparan sulfate saccharide sequence offers the binding site for viral envelope proteins and plays critical roles in assisting viral infections. A specific 3-O-sulfated heparan sulfate is known to facilitate the entry of herpes simplex virus 1 into cells. The 3-O-sulfated heparan sulfate is generated by the heparan sulfate d-glucosaminyl-3-O-sulfotransferase isoform 3 (3-OST-3), and it provides binding sites for viral glycoprotein D (gD). Here, we report the purification and structural characterization of an oligosaccharide that binds to gD. The isolated gD-binding site is an octasaccharide, and has a binding affinity to gD around 18 microm, as determined by affinity coelectrophoresis. The octasaccharide was prepared and purified from a heparan sulfate oligosaccharide library that was modified by purified 3-OST-3 enzyme. The molecular mass of the isolated octasaccharide was determined using both nanoelectrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The results from the sequence analysis suggest that the structure of the octasaccharide is a heptasulfated octasaccharide. The proposed structure of the octasaccharide is DeltaUA-GlcNS-IdoUA2S-GlcNAc-UA2S-GlcNS-IdoUA2S-GlcNH(2)3S6S. Given that the binding of 3-O-sulfated heparan sulfate to gD can mediate viral entry, our results provide structural information about heparan sulfate-assisted viral entry.     

Protection by herpes simplex virus glycoprotein D against Fas-mediated apoptosis: role of nuclear factor kappaB

Signals involved in protection against apoptosis by herpes simplex virus 1 (HSV-1) were investigated. Using U937 monocytoid cells as an experimental model, we have demonstrated that HSV-1 rendered these cells resistant to Fas-induced apoptosis promptly after infection. UV-inactivated virus as well as the envelope glycoprotein D (gD) of HSV-1, by itself, exerted a protective effect on Fas-induced apoptosis. NF-kappaB was activated by gD, and protection against Fas-mediated apoptosis by gD was abolished in cells stably transfected with a dominant negative mutant I-kappaBalpha, indicating that NF-kappaB activation plays a role in the antiapoptotic activity of gD in our experimental model. Moreover, NF-kappaB-dependent protection against Fas-mediated apoptosis was associated with decreased levels of caspase-8 activity and with the up-regulation of intracellular antiapoptotic proteins.     

Complexes between herpes simplex virus glycoproteins gD, gB, and gH detected in cells by complementation of split enhanced green fluorescent protein

The interactions between herpes simplex virus gD and its nectin1 receptor or between gD, gB, and gH were analyzed by complementation of the N and C portions of split enhanced green fluorescent protein (EGFP) fused to the glycoproteins. The gD(N)-Nect(C) complex was readily detected; the gD(N)-gC(C) complex was undetectable, highlighting the specificity of the assay. Split EGFP complementation was detected between proteins designated gD(N)+gH(C), gD(N)+gB(C), and gH(N)+gB(C)+wtgD (gB was deleted of endocytosis motifs), both in cells transfected with two-tree glycoproteins and in syncytia. The in situ assay provides evidence that gD interacts with gH and gB independently of each other and supports a model whereby gH and gB in complex exert their activities to gD.     

In vitro characterization of a thermolabile herpes simplex virus DNA-binding protein.

The major herpes simplex virus DNA-binding protein, ICP8, was purified from cells infected with the herpes simplex virus type 1 temperature-sensitive strain tsHA1. tsHA1 ICP8 bound single-stranded DNA in filter binding assays carried out at room temperature and exhibited nonrandom binding to single-stranded bacteriophage fd DNA circles as determined by electron microscopy. The filter binding assay results and the apparent nucleotide spacing of the DNA complexed with protein were identical, within experimental error, to those observed with wild-type ICP8. Thermal inactivation assays, however, showed that the DNA-binding activity of tsHA1 ICP8 was 50% inactivated at approximately 39 degrees C as compared with 45 degrees C for the wild-type protein. Both wild-type and tsHA1 ICP8 were capable of stimulating viral DNA polymerase activity at permissive temperatures. The stimulatory effect of both proteins was lost at 39 degrees C.