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.     

Antibody against extracellular vaccinia virus (EV) protects mice through complement and Fc receptors

Protein-based subunit smallpox vaccines have shown their potential as effective alternatives to live virus vaccines in animal model challenge studies. We vaccinated mice with combinations of three different vaccinia virus (VACV) proteins (A33, B5, L1) and examined how the combined antibody responses to these proteins cooperate to effectively neutralize the extracellular virus (EV) infectious form of VACV. Antibodies against these targets were generated in the presence or absence of CpG adjuvant so that Th1-biased antibody responses could be compared to Th2-biased responses to the proteins with aluminum hydroxide alone, specifically with interest in looking at the ability of anti-B5 and anti-A33 polyclonal antibodies (pAb) to utilize complement-mediated neutralization in vitro. We found that neutralization of EV by anti-A33 or anti-B5 pAb can be enhanced in the presence of complement if Th1-biased antibody (IgG2a) is generated. Mechanistic differences found for complement-mediated neutralization showed that anti-A33 antibodies likely result in virolysis, while anti-B5 antibodies with complement can neutralize by opsonization (coating). In vivo studies found that mice lacking the C3 protein of complement were less protected than wild-type mice after passive transfer of anti-B5 pAb or vaccination with B5. Passive transfer of anti-B5 pAb or monoclonal antibody into mice lacking Fc receptors (FcRs) found that FcRs were also important in mediating protection. These results demonstrate that both complement and FcRs are important effector mechanisms for antibody-mediated protection from VACV challenge in mice.     

Possible role of brain salt-inducible kinase 1 in responses to central sodium in Dahl rats

In Dahl salt-sensitive (S) rats, Na(+) entry into the cerebrospinal fluid (CSF) and sympathoexcitatory and pressor responses to CSF Na(+) are enhanced. Salt-inducible kinase 1 (SIK1) increases Na(+)/K(+)-ATPase activity in kidney cells. We tested the possible role of SIK1 in regulation of CSF [Na(+)] and responses to Na(+) in the brain. SIK1 protein and activity were lower in hypothalamic tissue of Dahl S (SS/Mcw) compared with salt-resistant SS.BN13 rats. Intracerebroventricular infusion of the protein kinase inhibitor staurosporine at 25 ng/day, to inhibit SIK1 further increased mean arterial pressure (MAP) and HR but did not affect the increase in CSF [Na(+)] or hypothalamic aldosterone in Dahl S on a high-salt diet. Intracerebroventricular infusion of Na(+)-rich artificial CSF caused significantly larger increases in renal sympathetic nerve activity, MAP, and HR in Dahl S vs. SS.BN13 or Wistar rats on a normal-salt diet. Intracerebroventricular injection of 5 ng staurosporine enhanced these responses, but the enhancement in Dahl S rats was only one-third that in SS.BN13 and Wistar rats. Staurosporine had no effect on MAP and HR responses to intracerebroventricular ANG II or carbachol, whereas the specific protein kinase C inhibitor GF109203X inhibited pressor responses to intracerebroventricular Na(+)-rich artificial CSF or ANG II. These results suggest that the SIK1-Na(+)/K(+)-ATPase network in neurons acts to attenuate sympathoexcitatory and pressor responses to increases in brain [Na(+)]. The lower hypothalamic SIK1 activity and smaller effect of staurosporine in Dahl S rats suggest that impaired activation of neuronal SIK1 by Na(+) may contribute to their enhanced central responses to sodium.     

Antibody-induced conformational changes in herpes simplex virus glycoprotein gD reveal new targets for virus neutralization

As the receptor-binding protein of herpes simplex virus (HSV), gD plays an essential role in virus entry. In its native state, the last 56 amino acids of the ectodomain C terminus (C-term) occlude binding to its receptors, herpesvirus entry mediator (HVEM) and nectin-1. Although it is clear that movement of the C-term must occur to permit receptor binding, we believe that this conformational change is also a key event for triggering later steps leading to fusion. Specifically, gD mutants containing disulfide bonds that constrain the C-term are deficient in their ability to trigger fusion following receptor binding. In this report, we show that two newly made monoclonal antibodies (MAbs), MC2 and MC5, have virus-neutralizing activity but do not block binding of gD to either receptor. In contrast, all previously characterized neutralizing anti-gD MAbs block binding of gD to a receptor(s). Interestingly, instead of blocking receptor binding, MC2 significantly enhances the affinity of gD for both receptors. Several nonneutralizing MAbs (MC4, MC10, and MC14) also enhanced gD-receptor binding. While MC2 and MC5 recognized different epitopes on the core of gD, these nonneutralizing MAbs recognized the gD C-term. Both the neutralizing capacity and rate of neutralization of virus by MC2 are uniquely enhanced when MC2 is combined with MAb MC4, MC10, or MC14. We suggest that MC2 and MC5 prevent gD from performing a function that triggers later steps leading to fusion and that the epitope for MC2 is normally occluded by the C-term of the gD ectodomain.     

Herpes Simplex Virus Glycoprotein B Associates with Target Membranes via Its Fusion Loops

Herpes simplex virus (HSV) glycoproteins gB, gD, and gH/gL are necessary and sufficient for virus entry into cells. Structural features of gB are similar to those of vesicular stomatitis virus G and baculovirus gp64, and together they define the new class III group of fusion proteins. Previously, we used mutagenesis to show that three hydrophobic residues (W174, Y179, and A261) within the putative gB fusion loops are integral to gB function. Here we expanded our analysis, using site-directed mutagenesis of each residue in both gB fusion loops. Mutation of most of the nonpolar or hydrophobic amino acids (W174, F175, G176, Y179, and A261) had severe effects on gB function in cell-cell fusion and null virus complementation assays. Of the six charged amino acids, mutation of H263 or R264 also negatively affected gB function. To further analyze the mutants, we cloned the ectodomains of the W174R, Y179S, H263A, and R264A mutants into a baculovirus expression system and compared them with the wild-type (WT) form, gB730t. As shown previously, gB730t blocks virus entry into cells, suggesting that gB730t competes with virion gB for a cell receptor. All four mutant proteins retained this function, implying that fusion loop activity is separate from gB-receptor binding. However, unlike WT gB730t, the mutant proteins displayed reduced binding to cells and were either impaired or unable to bind naked, cholesterol-enriched liposomes, suggesting that it may be gB-lipid binding that is disrupted by the mutations. Furthermore, monoclonal antibodies with epitopes proximal to the fusion loops abrogated gB-liposome binding. Taken together, our data suggest that gB associates with lipid membranes via a fusion domain of key hydrophobic and hydrophilic residues and that this domain associates with lipid membranes during fusion.Herpes simplex virus (HSV) entry into cells requires four viral envelope glycoproteins (gB, gD, and the heterodimer gH/gL) as well as a cell surface gD receptor (reviewed in references 31, 42, 43, and 49). When gD binds its receptor, it undergoes conformational changes that are essential to activate the fusion machinery, gB and gH/gL. In addition to being essential for virus entry, both gH/gL and gB play important roles in primary fusion events that occur during egress of the capsid from the nuclei of infected cells (22). gB and gH/gL constitute the core fusion machinery of all members of the Herpesviridae.The mechanisms by which gB and gH/gL function individually and in concert during fusion are topics of intense investigations. Peptides based on predicted heptad repeats in gH block virus entry and have the ability to bind and disrupt model membranes (24, 26, 27). In addition, gH/gL can achieve hemifusion of adjacent cells in the absence of other herpesvirus proteins (50). These studies imply that gH/gL has fusogenic properties. Previously, we showed that both virion gB and soluble wild-type (WT) gB (gB730t), but not gD or gH/gL, bind to cells and associate with lipid rafts (10). Like gH/gL, several synthetic gB peptides induced the fusion of large unilamellar vesicles and inhibited herpesvirus infection (23, 24). Thus, it appears that both gB and gH/gL may be fusion proteins, a theory strengthened by data showing that either gB or gH/gL is sufficient for membrane fusion during nuclear egress (22). Additionally, gB730t blocks virus entry into cells deficient in heparan sulfate proteoglycans (HSPGs), suggesting that it competes with virion gB for an obligate cell surface receptor (9). A recent study suggested that paired immunoglobulin-like type 2 receptor alpha (PILRα) may serve this role for at least some cell types (47).The crystal structure of gB is now known for both HSV type 1 (HSV-1) (32) and Epstein-Barr virus (EBV) (6). Interestingly, gB is structurally related to two other viral fusion proteins, the vesicular stomatitis virus (VSV) G protein (45) and the baculovirus gp64 protein (34). VSV G, gB, and most recently, baculovirus gp64 were placed into a newly formed group of fusion proteins, the class III proteins. Class III fusion proteins have similar individual domain structures and contain a central three-stranded coiled coil reminiscent of the class I proteins. Whereas class I proteins have an N-terminal fusion peptide, class III proteins have internal bipartite fusion loops within domain I (shown in Fig. ​Fig.1A1A for gB) which are similar to the single fusion loop of class II fusion proteins. However, the class II fusion loop is composed entirely of hydrophobic amino acids, whereas the fusion loops of gB have both hydrophobic and charged residues (32, 34, 45). Unlike G or gp64, which are the sole fusion proteins for their respective viruses, gB requires gH/gL to function in fusion and entry.Open in a separate windowFIG. 1.HSV gB hydrophobic ridge is surrounded by charged residues on the surface of the molecule. A ribbon diagram of the HSV protomer (A) and molecular surface representation of the trimer (B) are shown. In each, one protomer is colored by secondary structure succession, using blue (domain I), green (domain II), yellow (domain III), orange (domain IV), and red (domain V). The box in panel A shows the primary amino acid sequences of the fusion loops. The box in panel B shows the base of the gB trimer, rotated 90°. For the boxes in both panels A and B, highlighted hydrophobic residues are colored in blue and charged residues are shown in red. All structural figures were generated, in part, using PyMOL Molecular Graphics System software.In our previous study, we used site-directed mutagenesis to show that three hydrophobic amino acids within the gB loops (W174, Y179, and A261) are essential for gB function (29). Similar studies of VSV G, gp64, and EBV gB support the notion that hydrophobic amino acids of both fusion loops are critical for fusion (34, 44, 51) and together constitute a fusion domain. Recently, bimolecular complementation was used to show that gB and gH/gL interact with each other concomitantly with fusion and that this interaction is triggered by binding of gD to its cellular receptor (3, 4). Thus, gB may function cooperatively with gH/gL, yet each may have some fusogenic potential on its own.The goal of the experiments reported here was twofold. First, we wanted to complete our mutagenic analysis of all of the residues in the two putative fusion loops of HSV gB. Our data show that the two fusion loops constitute a structural “subdomain” wherein key hydrophobic amino acids form a ridge that is supported on both sides by charged residues. We hypothesize that two charged residues on one side of the ridge enhance the ability of the hydrophobic residues to interact with target membranes and to function in fusion.Our second goal was to assess the effects of mutations in the fusion loops on the function of gB in cell binding, blocking of entry, and insertion into lipid membranes. Therefore, we constructed recombinant baculoviruses, with each carrying the gene for a truncated version (residues 31 to 730) of one of four mutant forms of gB (W174R, Y179S, H263A, and R264A). We found that the mutant proteins were able to efficiently block virus entry, suggesting that the fusion loops do not participate in protein-receptor binding. However, all four mutant proteins were impaired in cell binding compared to WT gB730t. Whereas WT gB730t associated with liposomes in a flotation assay, soluble truncated forms of HSV gD and gH/gL did not, consistent with our previous finding that gB730t associates with lipid rafts on cell surfaces (8). In contrast to WT gB730t, the gB mutant proteins were either impaired or unable to bind liposomes. Our data suggest that gB has an intrinsic ability to associate with a target membrane via its fusion domain.     

Stable association of herpes simplex virus with target membranes is triggered by low pH in the presence of the gD receptor, HVEM

Using a liposome-binding assay, we investigated the requirements for activation of herpes simplex virus (HSV) into a state capable of membrane interaction. Virions were mixed with liposomes along with the ectodomain of one of three gD receptors (HVEMt, nectin-1t, or nectin-2t) and incubated under different pH and temperature conditions. Virions failed to associate with liposomes in the presence of nectin-1 or nectin-2 at any temperature or pH tested. In contrast, HVEMt triggered association of HSV with liposomes at pH 5.3 or 5.0 when incubated at 37 degrees C, suggesting that HVEM binding and mildly acidic pH at a physiological temperature provide coactivation signals, allowing virus association with membranes. Virions incubated with HVEMt at 37 degrees C without liposomes rapidly lost infectivity upon exposure to pH 5.0, suggesting that these conditions lead to irreversible virus inactivation in the absence of target membranes. Consistent with the idea that soluble receptor molecules provide a trigger for HSV entry, HVEMt promoted virus entry into receptor-deficient CHO K1 cells. However, in B78H1 cells, HVEMt promoted virus entry with markedly lower efficiency. Interestingly, HSV entry into receptor-bearing CHO K1 cells has been shown to proceed via a pH-dependent manner, whereas HSV entry into receptor-bearing B78H1 cells is pH independent. Based on these observations, we propose that the changes triggered by HVEM and mildly acidic pH that allow liposome association are similar or identical to changes that occur during pH-dependent HSV entry.     

Replication regions of Sinorhizobium meliloti plasmids

The replication (rep) regions of small plasmids from three Sinorhizobium meliloti strains were cloned by marker rescue. Two unique replication regions were identified, one of which was common to two different strains. Plasmid pBB83 carried a 7.2 kbp rep region from a 42 kbp plasmid, and pBB84 carried a 4.5 kbp rep region from a 36 kbp plasmid. The cloned rep regions were of different compatibility types, and were capable of displacing their parent plasmids from S. meliloti. Neither could function in a PolA- strain of Escherichia coli. The cloned replication regions were less stable in S. meliloti than their parent plasmids. The rep genes for each plasmid were localized to less than 2.5 kbp segments. Sequencing data revealed that the pBB83 Rep protein is uncommon, with partial identity to a protein encoded by a plasmid from S. meliloti GR4 [Mercado-Blanco, J., Olivares, J., 1994. The large nonsymbiotic plasmid pRmeGR4a of Rhizobium meliloti GR4 encodes a protein involved in replication that has homology with the RepC protein of Agrobacterium plasmids. Plasmid 32, 75-79]. However, the cloned DNA fragment also contains a truncated segment of the common repABC genes, suggesting that the parent plasmid contained two sets of replication genes. Other genes and an IS-element within the insert are most closely related to sequences derived from the Rhizobiaceae family, suggesting that the plasmid has a limited host range. In contrast, the pBB84 rep region contained genes similar to those associated with several broad host-range plasmids, and its Rep protein is related to that of a Pseudomonas aeruginosa broad host-range plasmid, pVS1 [Heeb, S., Itoh, Y., Nishijyo, T., Schnider, U., Keel, C., Wade, J., Walsh, U., O'Gara, F., Haas, D., 2000. Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria. Mol. Plant-Microbe Interact. 13, 232-237]. The pBB84 rep region also includes a probable origin of replication, consisting of DNA boxes flanking a series of direct repeats and an AT-rich sequence.     

The importance of sediment phosphorus release in the restoration of very shallow lakes (The Norfolk Broads,England) and implications for biomanipulation

Phosphorus release from the sediments of very shallow lakes, the Norfolk Broads, can be as high as 278 mgP m^-2 d^-1. These high rates are associated with high total sediment Fe:P ratios and occur when sulphide from sulphate reduction removes Fe(II) from the pore water. There is also evidence that bioturbation from benthic chironomids can enhance phosphorus release rates, particularly in sediments low in total iron. The release of phosphorus from the sediments of these lakes is delaying restoration following the control of phosphorus from sewage discharges. Biomanipulation is being used in these lakes to create clear water and re-establish aquatic macrophytes. This removal of fish has allowed larger populations of benthic chironomid larvae to develop which may result in an increase in the rate of phosphorus release and changes to the pore profiles of dissolved phosphorus, soluble iron and free sulphide.     

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.     

Effect of modulating cardiac A1 adenosine receptor expression on protection with ischemic preconditioning

Activation of A(1) adenosine receptors (A(1)ARs) may be a crucial step in protection against myocardial ischemia-reperfusion (I/R) injury; however, the use of pharmacological A(1)AR antagonists to inhibit myocardial protection has yielded inconclusive results. In the current study, we have used mice with genetically modified A(1)AR expression to define the role of A(1)AR in intrinsic protection and ischemic preconditioning (IPC) against I/R injury. Normal wild-type (WT) mice, knockout mice with deleted (A(1)KO(-/-)) or single-copy (A(1)KO(+/-)) A(1)AR, and transgenic mice (A(1)TG) with increased cardiac A(1)AR expression underwent 45 min of left anterior descending coronary artery occlusion, followed by 60 min of reperfusion. Subsets of each group were preconditioned with short durations of ischemia (3 cycles of 5 min of occlusion and 5 min of reperfusion) before index ischemia. Infarct size (IF) in WT, A(1)KO(+/-), and A(1)KO(-/-) mice was (in % of risk region) 58 +/- 3, 60 +/- 4, and 61 +/- 2, respectively, and was less in A(1)TG mice (39 +/- 4, P < 0.05). A strong correlation was observed between A(1)AR expression level and response to IPC. IF was significantly reduced by IPC in WT mice (35 +/- 3, P < 0.05 vs. WT), A(1)KO(+/-) + IPC (48 +/- 4, P < 0.05 vs. A(1)KO(+/-)), and A(1)TG + IPC mice (24 +/- 2, P < 0.05 vs. A(1)TG). However, IPC did not decrease IF in A(1)KO(-/-) + IPC mice (63 +/- 2). In addition, A(1)KO(-/-) hearts subjected to global I/R injury demonstrated diminished recovery of developed pressure and diastolic function compared with WT controls. These findings demonstrate that A(1)ARs are critical for protection from myocardial I/R injury and that cardioprotection with IPC is relative to the level of A(1)AR gene expression.