Manuel Ponce de Leon

Specific Association of Glycoprotein B with Lipid Rafts during Herpes Simplex Virus Entry

ABSTRACT Herpes simplex virus (HSV) entry requires the interaction of glycoprotein D (gD) with a cellular receptor such as herpesvirus entry mediator (HVEM or HveA) or nectin-1 (HveC). However, the fusion mechanism is still not understood. Since cholesterol-enriched cell membrane lipid rafts are involved in the entry of other enveloped viruses such as human immunodeficiency virus and Ebola virus, we tested whether HSV entry proceeds similarly. Vero cells and cells expressing either HVEM or nectin-1 were treated with cholesterol-sequestering drugs such as methyl-β-cyclodextrin or nystatin and then exposed to virus. In all cases, virus entry was inhibited in a dose-dependent manner, and the inhibitory effect was fully reversible by replenishment of cholesterol. To examine the association of HVEM and nectin-1 with lipid rafts, we analyzed whether they partitioned into nonionic detergent-insoluble glycolipid-enriched membranes (DIG). There was no constitutive association of either receptor with DIG. Binding of soluble gD or virus to cells did not result in association of nectin-1 with the raft-containing fractions. However, during infection, a fraction of gB but not gC, gD, or gH associated with DIG. Similarly, when cells were incubated with truncated soluble glycoproteins, soluble gB but not gC was found associated with DIG. Together, these data favor a model in which HSV uses gB to rapidly mobilize lipid rafts that may serve as a platform for entry and cell signaling. It also suggests that gB may interact with a cellular molecule associated with lipid rafts.

Complement component c3b binds directly to purified glycoprotein c of herpes simplex virus types 1 and 2

Cells infected with herpes simplex virus type 1 (HSV-1), but not HSV-2, express on their surfaces a receptor for the complement component C3b. Receptor activity is markedly enhanced by treatment of the infected cells with neuraminidase. Employing a direct binding assay, consisting of purified HSV glycoproteins immobilized on nitrocellulose and iodinated C3b as a probe, we found that C3b binds directly to gC-1, as well as to gC-2, but not to gB or gD from either serotype. C3b binding was enhanced by treatment of gC-1 or gC-2 with neuraminidase. Endo F or endo H treatment of gC-1 had no effect on C3b binding. However, treatment of gC-2 with these endoglycosidases had a marked negative effect on C3b binding. These results suggest that N-linked oligosaccharides are involved in binding of C3b to gC-2, but not gC-1. Alternatively, removal of N-linked oligosaccharides from gC-2 might adversely affect polypeptide conformation. Glycoprotein C-2 also differs from gC-1 in its effects on the complement cascade. Whereas gC-1 accelerated the decay of the alternative pathway C3 convertase and impaired the efficiency of lysis by the components C5 through C9, gC-2 stabilized the active C3 convertase and had little effect on the late-acting components. The dissimilarity of gC-1 and gC-2 with regard to their effects on the complement cascade may have implications regarding the role of these glycoproteins in confronting the host immune response.

Localization of a Binding Site for Herpes Simplex Virus Glycoprotein D on Herpesvirus Entry Mediator C by Using Antireceptor Monoclonal Antibodies

ABSTRACT The human herpesvirus entry mediator C (HveC), also known as the poliovirus receptor-related protein 1 (PRR1) and as nectin-1, allows the entry of herpes simplex virus type 1 (HSV-1) and HSV-2 into mammalian cells. The interaction of virus envelope glycoprotein D (gD) with such a receptor is an essential step in the process leading to membrane fusion. HveC is a member of the immunoglobulin (Ig) superfamily and contains three Ig-like domains in its extracellular portion. The gD binding site is located within the first Ig-like domain (V domain) of HveC. We generated a panel of monoclonal antibodies (MAbs) against the ectodomain of HveC. Eleven of these, which detect linear or conformational epitopes within the V domain, were used to map a gD binding site. They allowed the detection of HveC by enzyme-linked immunosorbent assay, Western blotting, and biosensor analysis or directly on the surface of HeLa cells and human neuroblastoma cell lines, as well as simian Vero cells. The anti-HveC V-domain MAbs CK6, CK8, and CK41, as well as the previously described MAb R1.302, blocked HSV entry. Their binding to soluble HveC was blocked by the association of gD with the receptor, indicating that their epitopes overlap a gD binding site. Competition assays on an optical biosensor showed that CK6 and CK8 (linear epitopes) inhibited the binding of CK41 and R1.302 (conformational epitopes) to HveC and vice versa. Epitope mapping showed that CK6 and CK8 bound between residues 80 and 104 of HveC, suggesting that part of the gD binding site colocalizes in the same region.

Bimolecular Complementation Defines Functional Regions of Herpes Simplex Virus gB That Are Involved with gH/gL as a Necessary Step Leading to Cell Fusion

ABSTRACT Herpes simplex virus (HSV) entry into cells requires four membrane glycoproteins: gD is the receptor binding protein, and gB and gH/gL constitute the core fusion machinery. Crystal structures of gD and its receptors have provided a basis for understanding the initial triggering steps, but how the core fusion proteins function remains unknown. The gB crystal structure shows that it is a class III fusion protein, yet unlike other class members, gB itself does not cause fusion. Bimolecular complementation (BiMC) studies have shown that gD-receptor binding triggers an interaction between gB and gH/gL and concurrently triggers fusion. Left unanswered was whether BiMC led to fusion or was a by-product of it. We used gB monoclonal antibodies (MAbs) to block different aspects of these events. Non-virus-neutralizing MAbs to gB failed to block BiMC or fusion. In contrast, gB MAbs that neutralize virus blocked fusion. These MAbs map to three functional regions (FR) of gB. MAbs to FR1, which contains the fusion loops, and FR2 blocked both BiMC and fusion. In contrast, MAbs to FR3, a region involved in receptor binding, blocked fusion but not BiMC. Thus, FR3 MAbs separate the BiMC interaction from fusion, suggesting that BiMC occurs prior to fusion. When substituted for wild-type (wt) gB, fusion loop mutants blocked fusion and BiMC, suggesting that loop insertion precedes BiMC. Thus, we postulate that each of the gB FRs are involved in different aspects of the path leading to fusion. Upon triggering by gD, gB fusion loops are inserted into target lipid membranes. gB then interacts with gH/gL, and this interaction is eventually followed by fusion.

Epitope-Mapping Studies Define Two Major Neutralization Sites on the Vaccinia Virus Extracellular Enveloped Virus Glycoprotein B5R

ABSTRACT Vaccinia extracellular enveloped virus (EEV) is critical for cell-to-cell and long-range virus spread both in vitro and in vivo. The B5R gene encodes an EEV-specific type I membrane protein that is essential for efficient EEV formation. The majority of the B5R ectodomain consists of four domains with homology to short consensus repeat domains followed by a stalk. Previous studies have shown that polyclonal antibodies raised against the B5R ectodomain inhibit EEV infection. In this study, our goal was to elucidate the antigenic structure of B5R and relate this to its function. To do this, we produced multimilligram quantities of vaccinia virus B5R as a soluble protein [B5R(275t)] using a baculovirus expression system. We then selected and characterized a panel of 26 monoclonal antibodies (MAbs) that recognize B5R(275t). Five of these MAbs neutralized EEV and inhibited comet formation. Two other MAbs were able only to neutralize EEV, while five others were able only to inhibit comet formation. This suggests that the EEV neutralization and comet inhibition assays measure different viral functions and that at least two different antigenic sites on B5R are important for these activities. We further characterized the MAbs and the antigenic structure of B5R(275t) by peptide mapping and by reciprocal MAb blocking studies using biosensor analysis. The epitopes recognized by neutralizing MAbs were localized to SCR1-SCR2 and/or the stalk of B5R(275t). Furthermore, the peptide and blocking data support the concept that SCR1 and the stalk may be in juxtaposition and may be part of the same functional domain.

Antigenic and Mutational Analyses of Herpes Simplex Virus Glycoprotein B Reveal Four Functional Regions

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

Localization of the gD-binding region of the human herpes simplex virus receptor, HveA.

ABSTRACT During virus entry, herpes simplex virus (HSV) glycoprotein D (gD) binds to one of several human cellular receptors. One of these, herpesvirus entry mediator A (HveA), is a member of the tumor necrosis factor receptor (TNFR) superfamily, and its ectodomain contains four characteristic cysteine-rich pseudorepeat (CRP) elements. We previously showed that gD binds the ectodomain of HveA expressed as a truncated, soluble protein [HveA(200t)]. To localize the gD-binding domain of HveA, we expressed three additional soluble forms of HveA consisting of the first CRP [HveA(76t)], the second CRP [HveA(77–120t)], or the first and second CRPs [HveA(120t)]. Biosensor and enzyme-linked immunosorbent assay studies showed that gD bound to HveA(120t) and HveA(200t) with the same affinity. However, gD did not bind to HveA(76t) or HveA(77–120t). Furthermore, HveA(200t) and HveA(120t), but not HveA(76t) or HveA(77–120t), blocked herpes simplex virus (HSV) entry into CHO cells expressing HveA. We also generated six monoclonal antibodies (MAbs) against HveA(200t). MAbs CW1, -2, and -4 bound linear epitopes within the second CRP, while CW7 and -8 bound linear epitopes within the third or fourth CRPs. None of these MAbs blocked the binding of gD to HveA. In contrast, MAb CW3 recognized a discontinuous epitope within the first CRP of HveA, blocked the binding of gD to HveA, and exhibited a limited ability to block virus entry into cells expressing HveA, suggesting that the first domain of HveA contains at least a portion of the gD binding site. The inability of gD to bind HveA(76t) suggests that additional amino acid residues of the gD binding site may reside within the second CRP.

Vaccinia virus exhibits cell-type-dependent entry characteristics.

Differing and sometimes conflicting data have been reported regarding several aspects of vaccinia virus (VV) entry. To address this, we used a beta-galactosidase reporter virus to monitor virus entry into multiple cell types under varying conditions. Entry into HeLa, B78H1 and L cells was strongly inhibited by heparin whereas entry into Vero and BSC-1 cells was unaffected. Bafilomycin also exhibited variable and cell-type-specific effects on VV entry. Entry into B78H1 and BSC-1 cells was strongly inhibited by bafilomycin whereas entry into Vero and HeLa cells was only partially inhibited suggesting the co-existence of both pH-dependent and pH-independent VV entry pathways in these cell types. Finally, entry into HeLa, B78H1, L and BSC-1 cells exhibited a lag of 6-9 min whereas this delay was undetectable in Vero cells. Our results suggest that VV exploits multiple cell attachment and entry pathways allowing it to infect a broad range of cells.

Dissection of the Antibody Response against Herpes Simplex Virus Glycoproteins in Naturally Infected Humans

ABSTRACT Relatively little is known about the extent of the polyclonal antibody (PAb) repertoire elicited by herpes simplex virus (HSV) glycoproteins during natural infection and how these antibodies affect virus neutralization. Here, we examined IgGs from 10 HSV-seropositive individuals originally classified as high or low virus shedders. All PAbs neutralized virus to various extents. We determined which HSV entry glycoproteins these PAbs were directed against: glycoproteins gB, gD, and gC were recognized by all sera, but fewer sera reacted against gH/gL. We previously characterized multiple mouse monoclonal antibodies (MAbs) and mapped those with high neutralizing activity to the crystal structures of gD, gB, and gH/gL. We used a biosensor competition assay to determine whether there were corresponding human antibodies to those epitopes. All 10 samples had neutralizing IgGs to gD epitopes, but there were variations in which epitopes were seen in individual samples. Surprisingly, only three samples contained neutralizing IgGs to gB epitopes. To further dissect the nature of these IgGs, we developed a method to select out gD- and gB-specific IgGs from four representative sera via affinity chromatography, allowing us to determine the contribution of antibodies against each glycoprotein to the overall neutralization capacity of the serum. In two cases, gD and gB accounted for all of the neutralizing activity against HSV-2, with a modest amount of HSV-1 neutralization directed against gC. In the other two samples, the dominant response was to gD. IMPORTANCE Antibodies targeting functional epitopes on HSV entry glycoproteins mediate HSV neutralization. Virus-neutralizing epitopes have been defined and characterized using murine monoclonal antibodies. However, it is largely unknown whether these same epitopes are targeted by the humoral response to HSV infection in humans. We have shown that during natural infection, virus-neutralizing antibodies are principally directed against gD, gB, and, to a lesser extent, gC. While several key HSV-neutralizing epitopes within gD and gB are commonly targeted by human serum IgG, others fail to induce consistent responses. These data are particularly relevant to the design of future HSV vaccines.

Analysis of the intracellular maturation of the herpes simplex virus type 1 glycoprotein gH in infected and transfected cells

We have expressed the HSV-1 glycoprotein, gH, in transiently transfected COS-1 cells. The expressed protein was retained intracellularly, contained unprocessed carbohydrate, and was unrecognized by the monoclonal antibody, LP11. In addition, the protein was aggregated. These properties suggest that unlike other HSV glycoproteins, gH is misfolded in transfected cells. Pulse-chase studies of HSV-1-infected cells indicate that the kinetics of processing of gH are comparable to those of gB, gC, and gD. Rescue studies suggest that gH may interact with another protein during maturation in infected cells. However, we were unable to detect any stable interaction, although analysis of gH on neutral sucrose gradients shortly after synthesis indicated a possible transient association with a high molecular weight molecule or complex. The processing and cell surface expression of gH were also analyzed in HSV-1 virus mutants lacking gB, gC, or gD. Our results indicate that the maturation and cell surface transport of gH did not require the presence of these HSV-1 glycoproteins. In addition, three truncation mutants were constructed by linker insertion mutagenesis. Each of the three truncated proteins was synthesized, but the proteins were aggregated, contained only endo H-sensitive carbohydrate, and none were secreted.