Biomedical subjects

G H Cohen

Publications and source records attributed to G H Cohen.

At least 37 records · Page 2Linked to original sources

PubMed1998

A cell surface protein with herpesvirus entry activity (HveB) confers susceptibility to infection by mutants of herpes simplex virus type 1, herpes simplex virus type 2, and pseudorabies virus.

Certain mutant strains of herpes simplex virus type 1 (HSV-1) are unable to infect cells in which entry is dependent on HVEM, the previously described herpesvirus entry mediator designated here as herpesvirus entry protein A (HveA). These mutant viruses can infect other cells where entry is apparently dependent on other co-receptors. The mutant virus HSV-1(KOS)Rid1 was used to screen a human cDNA expression library for ability of transfected plasmids to convert resistant Chinese hamster ovary cells to susceptibility to virus entry. A plasmid expressing the previously described poliovirus receptor-related protein 2 (Prr2) was isolated on the basis of this activity. This protein, designated here as HveB, was shown to mediate the entry of three mutant HSV-1 strains that cannot use HVEM as co-receptor, but not wild-type HSV-1 strains. HveB also mediated the entry of HSV-2 and pseudorabies virus but not bovine herpesvirus type 1. HveB was expressed in some human neuronal cell lines, fibroblastic cells, keratinocytes, and primary activated T lymphocytes. Antibodies specific for HveB blocked infection of HveB-expressing CHO cells and a human fibroblastic cell strain HEL299. Differences in ability of HSV-1 and HSV-2 strains to use HveB for entry should influence the types of cells that can be infected and thereby account in part for serotype and strain differences in tissue tropism and pathogenicity.

Alphaherpesvirinae↗

PubMed1998

Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor.

A human member of the immunoglobulin superfamily was shown to mediate entry of several alphaherpesviruses, including herpes simplex viruses (HSV) 1 and 2, porcine pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1). This membrane glycoprotein is poliovirus receptor-related protein 1 (Prr1), designated here as HveC. Incubation of HSV-1 with a secreted form of HveC inhibited subsequent infection of a variety of cell lines, suggesting that HveC interacts directly with the virus. Poliovirus receptor (Pvr) itself mediated entry of PRV and BHV-1 but not of the HSV strains tested. HveC was expressed in human cells of epithelial and neuronal origin; it is the prime candidate for the coreceptor that allows both HSV-1 and HSV-2 to infect epithelial cells on mucosal surfaces and spread to cells of the nervous system.

Alphaherpesvirinae↗

PubMed1998

LIGHT, a new member of the TNF superfamily, and lymphotoxin alpha are ligands for herpesvirus entry mediator.

Herpes simplex virus (HSV) 1 and 2 infect activated T lymphocytes by attachment of the HSV envelope glycoprotein D (gD) to the cellular herpesvirus entry mediator (HVEM), an orphan member of the tumor necrosis factor receptor superfamily. Here, we demonstrate that HVEM binds two cellular ligands, secreted lymphotoxin alpha (LTalpha) and LIGHT, a new member of the TNF superfamily. LIGHT is a 29 kDa type II transmembrane protein produced by activated T cells that also engages the receptor for the LTalphabeta heterotrimer but does not form complexes with either LTalpha or LTbeta. HSV1 gD inhibits the interaction of HVEM with LIGHT, and LIGHT and gD interfere with HVEM-dependent cell entry by HSV1. This characterizes herpesvirus gD as a membrane-bound viokine and establishes LIGHT-HVEM as integral components of the lymphotoxin cytokine-receptor system.

Amino Acid Sequence↗

PubMed1998

The gH-gL complex of herpes simplex virus (HSV) stimulates neutralizing antibody and protects mice against HSV type 1 challenge.

The herpes simplex virus type 1 (HSV-1) gH-gL complex which is found in the virion envelope is essential for virus infectivity and is a major antigen for the host immune system. However, little is known about the precise role of gH-gL in virus entry, and attempts to demonstrate the immunologic or vaccine efficacy of gH and gL separately or as the gH-gL complex have not succeeded. We constructed a recombinant mammalian cell line (HL-7) which secretes a soluble gH-gL complex, consisting of gH truncated at amino acid 792 (gHt) and full-length gL. Purified gHt-gL reacted with gH- and gL-specific monoclonal antibodies, including LP11, which indicates that it retains its proper antigenic structure. Soluble forms of gD (gDt) block HSV infection by interacting with specific cellular receptors. Unlike soluble gD, gHt-gL did not block HSV-1 entry into cells, nor did it enhance the blocking capacity of gD. However, polyclonal antibodies to the complex did block entry even when added after virus attachment. In addition, these antibodies exhibited high titers of complement-independent neutralizing activity against HSV-1. These sera also cross-neutralized HSV-2, albeit at low titers, and cross-reacted with gH-2 present in extracts of HSV-2-infected cells. To test the potential for gHt-gL to function as a vaccine, BALB/c mice were immunized with the complex. As controls, other mice were immunized with gD purified from HSV-infected cells or were sham immunized. Sera from the gD- or gHt-gL-immunized mice exhibited high titers of virus neutralizing activity. Using a zosteriform model of infection, we challenged mice with HSV-1. All animals showed some evidence of infection at the site of virus challenge. Mice immunized with either gD or gHt-gL showed reduced primary lesions and exhibited no secondary zosteriform lesions. The sham-immunized control animals exhibited extensive secondary lesions. Furthermore, mice immunized with either gD or gHt-gL survived virus challenge, while many control animals died. These results suggest that gHt-gL is biologically active and may be a candidate for use as a subunit vaccine.

Animals↗

PubMed1998

Herpes simplex virus type 1 glycoprotein gC mediates immune evasion in vivo.

Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact and C3-deficient animals. Using a vaginal infection model in complement-intact guinea pigs, we showed that gC-null virus grows to lower titers and produces less severe vaginitis than wild-type or gC rescued virus, indicating a role for gC in virulence. To determine the importance of complement, studies were performed with C3-deficient guinea pigs; the results demonstrated significant increases in vaginal titers of gC-null virus, while wild-type and gC rescued viruses showed nonsignificant changes in titers. Similar findings were observed for mice where gC null virus produced significantly less disease than gC rescued virus at the skin inoculation site. Proof that C3 is important was provided by studies of C3 knockout mice, where disease scores of gC-null virus were significantly higher than in complement-intact mice. The results indicate that gC-null virus is approximately 100-fold (2 log10) less virulent that wild-type virus in animals and that gC-C3 interactions are involved in pathogenesis.

Animals↗

PubMed1998

Monoclonal antibodies to distinct sites on herpes simplex virus (HSV) glycoprotein D block HSV binding to HVEM.

HVEM (for herpesvirus entry mediator) is a member of the tumor necrosis factor receptor superfamily and mediates entry of many strains of herpes simplex virus (HSV) into normally nonpermissive Chinese hamster ovary (CHO) cells. We used sucrose density centrifugation to demonstrate that purified HSV-1 KOS virions bind directly to a soluble, truncated form of HVEM (HVEMt) in the absence of any other cell-associated components. Therefore, HVEM mediates HSV entry by serving as a receptor for the virus. We previously showed that soluble, truncated forms of HSV glycoprotein D (gDt) bind to HVEMt in vitro. Here we show that antibodies specific for gD, but not the other entry glycoproteins gB, gC, or the gH/gL complex, completely block HSV binding to HVEM. Thus, virion gD is the principal mediator of HSV binding to HVEM. To map sites on virion gD which are necessary for its interaction with HVEM, we preincubated virions with gD-specific monoclonal antibodies (MAbs). MAbs that recognize antigenic sites Ib and VII of gD were the only MAbs which blocked the HSV-HVEM interaction. MAbs from these two groups failed to coprecipitate HVEMt in the presence of soluble gDt, whereas the other anti-gD MAbs coprecipitated HVEMt and gDt. Previous mapping data indicated that site VII includes amino acids 11 to 19 and site Ib includes 222 to 252. The current experiments indicate that these sites contain residues important for HSV binding to HVEM. Group Ib and VII MAbs also blocked HSV entry into HVEM-expressing CHO cells. These results suggest that the mechanism of neutralization by these MAbs is via interference with the interaction between gD in the virus and HVEM on the cell. Group Ia and II MAbs failed to block HSV binding to HVEM yet still neutralized HVEM-mediated entry, suggesting that these MAbs block entry at a step other than HVEM binding.

Animals↗

PubMed1998

HveA (herpesvirus entry mediator A), a coreceptor for herpes simplex virus entry, also participates in virus-induced cell fusion.

The purpose of this study was to determine whether a cell surface protein that can serve as coreceptor for herpes simplex virus type 1 (HSV-1) entry, herpesvirus entry mediator (previously designated HVEM but renamed HveA), also mediates HSV-1-induced cell-cell fusion. We found that transfection of DNA from KOS-804, a previously described HSV-1 syncytial (Syn) strain whose Syn mutation was mapped to an amino acid substitution in gK, induced numerous large syncytia on HveA-expressing Chinese hamster ovary cells (CHO-HVEM12) but not on control cells (CHO-C8). Antibodies specific for gD as well as for HveA were effective inhibitors of KOS-804-induced fusion, consistent with previously described direct interactions between gD and HveA. Since mutations in gD determine the ability of HSV-1 to utilize HveA for entry, we examined whether the form of virally expressed gD also influenced the ability of HveA to mediate fusion. We produced a recombinant virus carrying the KOS-804 Syn mutation and the KOS-Rid1 gD mutation, which significantly reduces viral entry via HveA, and designated it KOS-SR1. KOS-SR1 DNA had a markedly reduced ability to induce syncytia on CHO-HVEM12 cells and a somewhat enhanced ability to induce syncytia on CHO-C8 cells. These results support previous findings concerning the relative abilities of KOS and KOS-Rid1 to infect CHO-HVEM12 and CHO-C8 cells. Thus, HveA mediates cell-cell fusion as well as viral entry and both activities of HveA are contingent upon the form of gD expressed by the virus.

Animals↗

PubMed1998

Examination of the kinetics of herpes simplex virus glycoprotein D binding to the herpesvirus entry mediator, using surface plasmon resonance.

Previously, we showed that truncated soluble forms of herpes simplex virus (HSV) glycoprotein D (gDt) bound directly to a truncated soluble form of the herpesvirus entry mediator (HveAt, formerly HVEMt), a cellular receptor for HSV. The purpose of the present study was to determine the affinity of gDt for HveAt by surface plasmon resonance and to compare and contrast the kinetics of an expanded panel of gDt variants in binding to HveAt in an effort to better understand the mechanism of receptor binding and virus entry. Both HveAt and gDt are dimers in solution and interact with a 2:1 stoichiometry. With HveAt, gD1(306t) (from the KOS strain of HSV-1) had a dissociation constant (KD) of 3.2 x 10(-6) M and gD2(306t) had a KD of 1.5 x 10(-6) M. The interaction between gDt and HveAt fits a 1:1 Langmuir binding model, i.e., two dimers of HveAt may act as one binding unit to interact with one dimer of gDt as the second binding unit. A gD variant lacking all signals for N-linked oligosaccharides had an affinity for HveAt similar to that of gD1(306t). A variant lacking the bond from cysteine 1 to cysteine 5 had an affinity for HveAt that did not differ from that of the wild type. However, variants with double cysteine mutations that eliminated either of the other two disulfide bonds showed decreased affinity for HveAt. This result suggests that two of the three disulfide bonds of gD are important for receptor binding. Four nonfunctional gDt variants, each representing one functional domain of gD, were also studied. Mutations in functional regions I and II drastically decreased the affinity of gDt for HveAt. Surprisingly, a variant with an insertion in functional region III had a wild-type level of affinity for HveAt, suggesting that this domain may function in virus entry at a step other than receptor binding. A variant with a deletion in functional region IV [gD1(Delta290-299t)] exhibited a 100-fold enhancement in affinity for HveAt (KD = 3.3 x 10(-8) M) due mainly to a 40-fold increase in its kinetic on rate. This agrees with the results of other studies showing the enhanced ability of gD1(Delta290-299t) to block infection. Interestingly, all the variants with decreased affinities for HveAt exhibited decreased kinetic on rates but only minor changes in their kinetic off rates. The results suggest that once the complex between gDt and HveAt forms, its stability is unaffected by a variety of changes in gD.

Animals↗

PubMed1998

Structural and antigenic analysis of a truncated form of the herpes simplex virus glycoprotein gH-gL complex.

The herpes simplex virus (HSV) gH-gL complex is essential for virus infectivity and is a major antigen for the host immune system. The association of gH with gL is required for correct folding, cell surface trafficking, and membrane presentation of the complex. Previously, a mammalian cell line was constructed which produces a secreted form of gHt-gL complex lacking the transmembrane and cytoplasmic tail regions of gH. gHt-gL retains a conformation similar to that of its full-length counterpart in HSV-infected cells. Here, we examined the structural and antigenic properties of gHt-gL. We first determined its stoichiometry and carbohydrate composition. We found that the complex consists of one molecule each of gH and gL. The N-linked carbohydrate (N-CHO) site on gL and most of the N-CHO sites on gH are utilized, and both proteins also contain O-linked carbohydrate and sialic acid. These results suggest that the complex is processed to the mature form via the Golgi network prior to secretion. To determine the antigenically active sites of gH and gL, we mapped the epitopes of a panel of gH and gL monoclonal antibodies (MAbs), using a series of gH and gL C-terminal truncation variant proteins produced in transiently transfected mammalian cells. Sixteen gH MAbs (including H6 and 37S) reacted with the N-terminal portion of gH between amino acids 19 and 276. One of the gH MAbs, H12, reacted with the middle portion of gH (residues 476 to 678). Nine gL MAbs (including 8H4 and VIII 62) reacted with continuous epitopes within the C-terminal portion of gL, and this region was further mapped within amino acids 168 to 178 with overlapping synthetic peptides. Finally, plasmids expressing the gH and gL truncations were employed in cotransfection assays to define the minimal regions of both gH and gL required for complex formation and secretion. The first 323 amino acids of gH and the first 161 amino acids of gL can form a stable secreted hetero-oligomer with gL and gH792, respectively, while gH323-gL168 is the smallest secreted hetero-oligomer. The first 648 amino acids of gH are required for reactivity with MAbs LP11 and 53S, indicating that a complex of gH648-gL oligomerizes into the correct conformation. The data suggest that both antigenic activity and oligomeric structure require the amino-terminal portions of gH and gL.

Amino Acid Sequence↗

PubMed1998

Herpes simplex virus glycoprotein D can bind to poliovirus receptor-related protein 1 or herpesvirus entry mediator, two structurally unrelated mediators of virus entry.

Several cell membrane proteins have been identified as herpes simplex virus (HSV) entry mediators (Hve). HveA (formerly HVEM) is a member of the tumor necrosis factor receptor family, whereas the poliovirus receptor-related proteins 1 and 2 (PRR1 and PRR2, renamed HveC and HveB) belong to the immunoglobulin superfamily. Here we show that a truncated form of HveC directly binds to HSV glycoprotein D (gD) in solution and at the surface of virions. This interaction is dependent on the native conformation of gD but independent of its N-linked glycosylation. Complex formation between soluble gD and HveC appears to involve one or two gD molecules for one HveC protein. Since HveA also mediates HSV entry by interacting with gD, we compared both structurally unrelated receptors for their binding to gD. Analyses of several gD variants indicated that structure and accessibility of the N-terminal domain of gD, essential for HveA binding, was not necessary for HveC interaction. Mutations in functional regions II, III, and IV of gD had similar effects on binding to either HveC or HveA. Competition assays with neutralizing anti-gD monoclonal antibodies (MAbs) showed that MAbs from group Ib prevented HveC and HveA binding to virions. However, group Ia MAbs blocked HveC but not HveA binding, and conversely, group VII MAbs blocked HveA but not HveC binding. Thus, we propose that HSV entry can be mediated by two structurally unrelated gD receptors through related but not identical binding with gD.

Amino Acid Sequence↗

PubMed1998

Functional region IV of glycoprotein D from herpes simplex virus modulates glycoprotein binding to the herpesvirus entry mediator.

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry and has four functional regions (I to IV) important for this process. We previously showed that a truncated form of a functional region IV variant, gD1(Delta290-299t), had an enhanced ability to block virus entry and to bind to the herpesvirus entry mediator (HveAt; formerly HVEMt), a cellular receptor for HSV. To explore this phenotype further, we examined other forms of gD, especially ones with mutations in region IV. Variant proteins with deletions of amino acids between 277 and 300 (region IV), as well as truncated forms lacking C-terminal residues up to amino acid 275 of gD, were able to block HSV entry into Vero cells 1 to 2 logs better than wild-type gD1(306t). In contrast, gD truncated at residue 234 did not block virus entry into Vero cells. Using optical biosensor technology, we recently showed that gD1(Delta290-299t) had a 100-fold-higher affinity for HveAt than gD1(306t) (3.3 x 10(-8) M versus 3.2 x 10(-6) M). Here we found that the affinities of other region IV variants for HveAt were similar to that of gD1(Delta290-299t). Thus, the affinity data follow the same hierarchy as the blocking data. In each case, the higher affinity was due primarily to a faster kon rather than to a slower koff. Therefore, once the gDt-HveAt complex formed, its stability was unaffected by mutations in or near region IV. gD truncated at residue 234 bound to HveAt with a lower affinity (2.0 x 10(-5) M) than did gD1(306t) due to a more rapid koff. These data suggest that residues between 234 and 275 are important for maintaining stability of the gDt-HveAt complex and that functional region IV is important for modulating the binding of gD to HveA. The binding properties of any gD1(234t)-receptor complex could account for the inability of this form of gDt to block HSV infection.

Animals↗

PubMed1997

Mechanism of complement inactivation by glycoprotein C of herpes simplex virus.

Glycoprotein C (gC) of both herpes simplex virus type 1 (HSV-1) and HSV-2 interacts with complement C3b and protects the virus from complement-mediated neutralization. To study the mechanism by which gC modulates complement activation, we expressed both gC-1 and gC-2 in a baculovirus expression system. Baculovirus recombinants containing gC genes spanning the entire gC-1 sequence (gC-1-TMR) or only the extracellular domain(s) of gC-1, gC-2, or a deletion mutant of gC-1 lacking residues 33 through 123 were expressed in sf9 insect cells. Binding of the expressed proteins to human C3 and C3 fragments was assessed by direct and competition ELISA. All four expressed proteins bound to C3, C3b, and C3c but not to C3d, suggesting 1) that the binding sites for these proteins are located in the C3c region of C3; and 2) that gC, in contrast to other C3-binding proteins, interacts with native C3. We have also examined the interaction of native C3 with gC-1 expressed on the HSV-1-infected cells. Analogous to recombinant proteins, gC-1 expressed on the infected cells also bound to native C3. The ability of baculovirus-expressed gCs to inhibit the interaction of C3b with its ligands was also analyzed. We found that gC-1, but not gC-2, inhibited the binding of C5 and properdin to C3b and also inhibited the alternative pathway-mediated lysis of rabbit erythrocytes. Inhibition of alternative pathway-mediated lysis and properdin binding to C3b, but not of C5 binding to C3b, required the transmembrane segment of the gC-1. The specificity of gC interactions was examined by studying the interaction of gC with C3 from various species. In contrast to properdin, both gCs bound to cobra C3; this finding suggests that gC-1 and properdin bind to different sites on C3b. Further analyses suggested that gC-1 sterically hindered access of C5 and properdin to C3b.

Binding, Competitive↗

PubMed1997

Antigenic structure of soluble herpes simplex virus (HSV) glycoprotein D correlates with inhibition of HSV infection.

Soluble forms of herpes simplex virus (HSV) glycoprotein D (gD) block viral penetration. Likewise, most HSV strains are sensitive to gD-mediated interference by cells expressing gD. The mechanism of both forms of gD-mediated inhibition is thought to be at the receptor level. We analyzed the ability of different forms of soluble, truncated gD (gDt) to inhibit infection by different strains of HSV-1 and HSV-2. Strains that were resistant to gD-mediated interference were also resistant to inhibition by gDt, thereby suggesting a link between these two phenomena. Virion gD was the major viral determinant for resistance to inhibition by gDt. An insertion-deletion mutant, gD-1(delta 290-299t), had an enhanced inhibitory activity against most strains tested. The structure and function of gDt proteins derived from the inhibition-resistant viruses rid1 and ANG were analyzed. gD-1(ridlt) and gD-1(ANGt) had a potent inhibitory effect on plaque formation by wild-type strains of HSV but, surprisingly, little or no effect on their parental strains. As measured by quantitative enzyme-linked immunosorbent assay with a diverse panel of monoclonal antibodies, the antigenic structures of gD-1(rid1t) and gD-1(ANGt) were divergent from that of the wild type yet were similar to each other and to that of gD-1 (delta 290-299t). Thus, three different forms of gD have common antigenic changes that correlate with enhanced inhibitory activity against HSV. We conclude that inhibition of HSV infectivity by soluble gD is influenced by the antigenic conformation of the blocking gDt as well as the form of gD in the target virus.

Animals↗

PubMed1997

Glycoprotein D of herpes simplex virus (HSV) binds directly to HVEM, a member of the tumor necrosis factor receptor superfamily and a mediator of HSV entry.

Glycoprotein D (gD) is a structural component of the herpes simplex virus (HSV) envelope which is essential for virus entry into host cells. Chinese hamster ovary (CHO-K1) cells are one of the few cell types which are nonpermissive for the entry of many HSV strains. However, when these cells are transformed with the gene for the herpesvirus entry mediator (HVEM), the resulting cells, CHO-HVEM12, are permissive for many HSV strains, such as HSV-1(KOS). By virtue of its four cysteine-rich pseudorepeats, HVEM is a member of the tumor necrosis factor receptor superfamily of proteins. Recombinant forms of gD and HVEM, gD-1(306t) and HVEM(200t), respectively, were used to demonstrate a specific physical interaction between these two proteins. This interaction was dependent on native gD conformation but independent of its N-linked oligosaccharides, as expected from previous structure-function studies. Recombinant forms of gD derived from HSV-1(KOS)rid1 and HSV-1(ANG) did not bind to HVEM(200t), explaining the inability of these viruses to infect CHO-HVEM12 cells. A variant gD protein, gD-1(delta290-299t), showed enhanced binding to HVEM(200t) relative to the binding of gD-1(306t). Competition studies showed that gD-1(delta290-299t) and gD-1(306t) bound to the same region of HVEM(200t), suggesting that the differences in binding to HVEM are due to differences in affinity. These differences were also reflected in the ability of gD-1(delta290-299t) but not gD-1(306t) to block HSV type 1 infection of CHO-HVEM12 cells. By gel filtration chromatography, the complex between gD-1(delta290-299t) and HVEM(200t) had a molecular mass of 113 kDa and a molar ratio of 1:2. We conclude that HVEM interacts directly with gD, suggesting that HVEM is a receptor for virion gD and that the interaction between these proteins is a step in HSV entry into HVEM-expressing cells.

Animals↗

PubMed1996

Crystallization and preliminary X-ray analysis of herpes simplex virus neutralizing antibody Fab fragment DL11.

An Fab fragment of a virus-neutralizing monoclonal antibody (DL11) that binds to herpes simplex virus glycoprotein D (HSV gD) has been purified, sequenced and crystallized. The biological activity of the purified Fab was verified by enzyme-linked immunosorbant assay, flow cytometry and by neutralization of HSV infectivity. The crystals have the space group P1 with cell dimensions a = 40.2, b = 49.2, c = 63.9 A, alpha = 76.1, beta = 77.4, gamma = 71.6 degrees. The unit-cell volume is consistent with it containing a single Fab molecule. The crystals grow to a maximum size of 0.7 x 0.3 x 0.3 mm and diffract X-rays to greater than 2.2 A resolution. The amino-acid sequences of the variable regions of the heavy and light chains of DL11 have been determined. These have been compared to those for other known Fab structures in the Protein Data Bank for selection of a starting model for crystallographic refinement by the molecular-replacement method.

Journal Article↗

PubMed1996

Refined structure of the monoclonal antibody HyHEL-5 with its antigen hen egg-white lysozyme.

The crystal structure of the complex of the antibody Fab, HyHEL-5, with its antigen, hen egg-white lysozyme, has been refined at 2.65 A resolution to an R value of 0.196. The resulting model has significantly better stereochemistry than the previously reported model of the complex, PDB reference 2HFL, and sufficiently improved phases, permitting the reliable location of a number of water molecules. No major conformational differences are observed between this structure and that previously reported, although small differences occur throughout the complex. 82 water molecules have been assigned, of which three are in the antibody-antigen interface involved in a hydrogen-bonding network. Three other waters are trapped within the interface between V(H) and V(L) and a fourth water molecule is observed near the interface but buried below the lysozyme surface as observed in crystal structures of lysozyme alone.

Journal Article↗

PubMed1996

Interactions of protein antigens with antibodies.

There are now several crystal structures of antibody Fab fragments complexed to their protein antigens. These include Fab complexes with lysozyme, two Fab complexes with influenza virus neuraminidase, and three Fab complexes with their anti-idiotype Fabs. The pattern of binding that emerges is similar to that found with other protein-protein interactions, with good shape complementarity between the interacting surfaces and reasonable juxtapositions of polar residues so as to permit hydrogen-bond formation. Water molecules have been observed in cavities within the interface and on the periphery, where they often form bridging hydrogen bonds between antibody and antigen. For the most part the antigen is bound in the middle of the antibody combining site with most of the six complementarity-determining residues involved in binding. For the most studied antigen, lysozyme, the epitopes for four antibodies occupy approximately 45% of the accessible surface area. Some conformational changes have been observed to accompany binding in both the antibody and the antigen, although most of the information on conformational change in the latter comes from studies of complexes with small antigens.

Animals↗

PubMed1996

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.

Animals↗