Localization of the Interaction Site of Herpes Simplex Virus Glycoprotein D (gD) on the Membrane Fusion Regulator, gH/gL.

A cascade of protein-protein interactions between four herpes simplex virus (HSV) glycoproteins (gD, gH/gL, and gB) drive fusion between the HSV envelope and host membrane, thereby allowing for virus entry and infection. Specifically, binding of gD to one of its receptors induces a conformational change that allows gD to bind to the regulatory complex gH/gL, which then activates the fusogen gB, resulting in membrane fusion. Using surface plasmon resonance and a panel of anti-gD monoclonal antibodies (MAbs) that sterically blocked the interaction, we previously showed that gH/gL binds directly to gD at sites distinct from the gD receptor binding site. Here, using an analogous strategy, we first evaluated the ability of a panel of uncharacterized anti-gH/gL MAbs to block binding to gD and/or inhibit fusion. We found that the epitopes of four gD-gH/gL-blocking MAbs were located within flexible regions of the gH N terminus and the gL C terminus, while the fifth was placed around gL residue 77. Taken together, our data localized the gD binding region on gH/gL to a group of gH and gL residues at the membrane distal region of the heterodimer. Surprisingly, a second set of MAbs did not block gD-gH/gL binding but instead stabilized the complex by altering the kinetic binding. However, despite this prolonged gD-gH/gL interaction, "stabilizing" MAbs also inhibited cell-cell fusion, suggesting a unique mechanism by which the fusion process is halted. Our findings support targeting the gD-gH/gL interaction to prevent fusion in both therapeutic and vaccine strategies against HSV.IMPORTANCE Key to developing a human HSV vaccine is an understanding of the virion glycoproteins involved in entry. HSV employs multiple glycoproteins for attachment, receptor interaction, and membrane fusion. Determining how these proteins function was resolved, in part, by structural biology coupled with immunological and biologic evidence. After binding, virion gD interacts with a receptor to activate the regulator gH/gL complex, triggering gB to drive fusion. Multiple questions remain, one being the physical location of each glycoprotein interaction site. Using protective antibodies with known epitopes, we documented the long-sought interaction between gD and gH/gL, detailing the region on gD important to create the gD-gH/gL triplex. Now, we have identified the corresponding gD contact sites on gH/gL. Concurrently we discovered a novel mechanism whereby gH/gL antibodies stabilize the complex and inhibit fusion progression. Our model for the gD-gH/gL triplex provides a new framework for studying fusion, which identifies targets for vaccine development.

A single gD glycoprotein can mediate infection by Herpes simplex virus.

Herpes simplex viruses display hundreds of gD glycoproteins, and yet their neutralization requires tens of thousands of antibodies per virion, leading us to ask whether a wild-type virion with just a single free gD is still infective. By quantitative analysis of fluorescently labeled virus particles and virus neutralization assays, we show that entry of a wild-type HSV virion to a cell does indeed require just one or two of the approximately 300 gD glycoproteins to be left unbound by monoclonal antibody. This indicates that HSV entry is an extraordinarily efficient process, functioning at the level of single molecular complexes.

Analysis of a membrane interacting region of herpes simplex virus type 1 glycoprotein H.

Glycoprotein H (gH) of herpes simplex virus type I (HSV-1) is involved in the complex mechanism of membrane fusion of the viral envelope with the host cell. Membrane interacting regions and potential fusion peptides have been identified in HSV-1 gH as well as glycoprotein B (gB). Because of the complex fusion mechanism of HSV-1, which requires four viral glycoproteins, and because there are only structural data for gB and glycoprotein D, many questions regarding the mechanism by which HSV-1 fuses its envelope with the host cell membrane remain unresolved. Previous studies have shown that peptides derived from certain regions of gH have the potential to interact with membranes, and based on these findings we have generated a set of peptides containing mutations in one of these domains, gH-(626-644), to investigate further the functional role of this region. Using a combination of biochemical, spectroscopic, and nuclear magnetic resonance techniques, we showed that the alpha-helical nature of this stretch of amino acids in gH is important for membrane interaction and that the aromatic residues, tryptophan and tyrosine, are critical for induction of fusion.

Peptides containing membrane-interacting motifs inhibit herpes simplex virus type 1 infectivity.

Herpes simplex virus (HSV) membrane fusion represents an attractive target for anti-HSV therapy. To investigate the structural basis of HSV membrane fusion and identify new targets for inhibition, we have investigated the different membranotropic domains of HSV-1 gH envelope glycoprotein. We observed that fusion peptides when added exogenously are able to inhibit viral fusion likely by intercalating with viral fusion peptides upon adopting functional structure in membranes. Interestingly, peptides analogous to the predicted HSV-1 gH loop region inhibited viral plaque formation more significantly. Their inhibitory effect appears to be a consequence of their ability to partition into membranes and aggregate within them. Circular dichroism spectra showed that peptides self-associate in aqueous and lipidic solutions, therefore the inhibition of viral entry may occur via peptides association with their counterpart on wild-type gH. The antiviral activity of HSV-1 peptides tested provides an attractive basis for the development of new fusion peptide inhibitors corresponding to regions outside the fusion protein heptad repeat regions.

The identification and characterization of fusogenic domains in herpes virus glycoprotein B molecules.

The molecular mechanism of entry of herpes viruses requires a multicomponent fusion system. Virus entry and cell-cell fusion of Herpes simplex virus (HSV) requires four glycoproteins: gD, gB and gH/gL. The role of gB remained elusive until recently, when the crystal structure of HSV-1 gB became available. Glycoprotein B homologues represent the most highly conserved group of herpes virus glycoproteins; however, despite the high degree of sequence and structural conservation, differences in post-translational processing are observed for different members of this virus family. Whereas gB of HSV is not proteolytically processed after oligomerization, most other gB homologues are cleaved by a cellular protease into subunits that remain linked through disulfide bonds. Proteolytic cleavage is common for activation of many other viral fusion proteins, so it remains difficult to envisage a common role for different herpes virus gB structures in the fusion mechanism. We selected bovine herpes virus type 1 (BoHV-1) and herpes simplex virus type 1 (HSV-1) as representative viruses expressing cleaved and uncleaved gBs, and have screened their amino acid sequences for regions of highly interfacial hydrophobicity. Synthetic peptides corresponding to such regions were tested for their ability to induce the fusion of large unilamellar vesicles and to inhibit herpes virus infection. These results underline that several regions of the gB protein are involved in the mechanism of membrane interaction.

A herpes simplex virus gD-YFP fusion glycoprotein is transported separately from viral capsids in neuronal axons.

Two models describing how alphaherpesviruses exit neurons differ with respect to whether nucleocapsids and envelope glycoproteins travel toward axon termini separately or as assembled enveloped virions. Recently, a pseudorabies virus glycoprotein D (gD)-green fluorescent protein fusion was found to colocalize with viral capsids, supporting anterograde transport of enveloped virions. Previous antibody staining experiments demonstrated that herpes simplex virus (HSV) glycoproteins and capsids are separately transported in axons. Here, we generated an HSV expressing a gD-yellow fluorescent protein (YFP) fusion and found that gD-YFP and capsids were transported separately in neuronal axons. Anti-gD antibodies colocalized with gD-YFP, indicating that gD-YFP behaves like wild-type HSV gD.

Two-color fluorescence analysis of individual virions determines the distribution of the copy number of proteins in herpes simplex virus particles.

We present a single virion method to determine absolute distributions of copy number in the protein composition of viruses and apply it to herpes simplex virus type 1. Using two-color coincidence fluorescence spectroscopy, we determine the virion-to-virion variability in copy numbers of fluorescently labeled tegument and envelope proteins relative to a capsid protein by analyzing fluorescence intensity ratios for ensembles of individual dual-labeled virions and fitting the resulting histogram of ratios. Using EYFP-tagged capsid protein VP26 as a reference for fluorescence intensity, we are able to calculate the mean and also, for the first time to our knowledge, the variation in numbers of gD, VP16, and VP22 tegument. The measurement of the number of glycoprotein D molecules was in good agreement with independent measurements of average numbers of these glycoproteins in bulk virus preparations, validating the method. The accuracy, straightforward data processing, and high throughput of this technique make it widely applicable to the analysis of the molecular composition of large complexes in general, and it is particularly suited to providing insights into virus structure, assembly, and infectivity.

Evidence for a role of the membrane-proximal region of herpes simplex virus type 1 glycoprotein H in membrane fusion and virus inhibition.

We have identified a putative membrane-interacting domain preceding the transmembrane domain of the Herpes simplex virus type 1 (HSV-1) glycoprotein H (gH). Peptides derived from this region interact strongly with membranes and show a high tendency to partition at the interface. This region is predicted to bind at the membrane interface by adopting an alpha helical structure. Peptides representing either the HSV-1 gH pretransmembrane region or a scrambled control with a different hydrophobic profile at the point of interface have been studied. The peptides derived from this domain of gH induce the fusion of liposomal membranes, adopt helical conformations in membrane mimetic environments and are able to inhibit HSV-1 infectivity. The pretransmembrane region appears to be a common feature in viral fusion proteins of several virus families, and such a feature might be related to their fusogenic function. The identification of membrane-interacting regions capable of modifying the biophysical properties of phospholipid membranes lends weight to the view that such domains might function directly in the fusion process and could facilitate the future development of HSV-1 entry inhibitors.

Analysis of synthetic peptides from heptad-repeat domains of herpes simplex virus type 1 glycoproteins H and B.

Human herpesviruses enter cells by fusion of their own membrane with a cellular membrane through the concerted action of multiple viral proteins and cellular receptors. Two conserved viral glycoproteins, gB and gH, are required for herpes simplex virus type 1 (HSV-1)-mediated membrane fusion, but little is known of how these proteins cooperate during entry. Both glycoproteins were shown to contain heptad repeat (HR) sequences predicted to form alpha-helical coiled coils, and the inhibitory activity against infection of four sets of synthetic peptides corresponding to HR1 and HR2 of gB and gH was tested. The interactions between these HR peptides were also investigated by circular dichroism, native polyacrylamide-gel electrophoresis and size exclusion high-performance liquid chromatography. gH coiled-coil peptides were more effective than gB coiled-coils peptides in inhibiting virus infectivity. The peptides did not impair fusion when added to cells immediately after infection. In contrast, inhibition of infection was observed, albeit to various extents, when peptides were added to virus before or during inoculation. The results of biophysical analyses were indicative of the existence of an interaction between HR1 and HR2 of gH and suggest that the HRs of gB and gH do not interact with each other.

Evidence for resonance optical trapping of individual fluorophore-labeled antibodies using single molecule fluorescence spectroscopy.

We report single molecule fluorescence studies of the diffusion of individual multiple fluorophore-labeled antibodies in solution, which show that a trapping potential of about 3.6 k(B)T can be obtained at laser powers below 1 mW with resonant excitation. Individual antibodies can be trapped for up to 140 ms, and bound antibodies can also be used to trap a single virion for up to 1 s. Selective resonance trapping to sort and manipulate fluorophore-labeled biomolecules and complexes may be possible.

Herpes simplex virus tegument protein VP16 is a component of primary enveloped virions.

Immunogold electron microscopy was used to determine whether the tegument proteins VP13/14, VP22, and VP16 of herpes simplex virus type 1 (HSV1) are components of primary enveloped virions. Whereas VP13/14 and VP22 were not detected in virus particles in the perinuclear space and were present in only mature extracellular virions, VP16 was acquired prior to primary envelopment of the virus at the inner nuclear membrane. This finding highlights potential similarities and differences between HSV1 and the related alphaherpesvirus, pseudorabies virus, in which the homologues of all three of these tegument proteins are not incorporated into the virion until secondary envelopment.

Characterization of a highly glycosylated form of the human cytomegalovirus HLA class I homologue gpUL18.

Human cytomegalovirus (HCMV) gpUL18 is a HLA class I (HLA-I) homologue with high affinity for the inhibitory receptor LIR-1/ILT2. The previously described 67 kDa form of gpUL18 is shown here to be sensitive to endoglycosidase-H (EndoH). A novel form of gpUL18 with a molecular mass of approximately 160 kDa and resistance to EndoH was identified in cells infected with HCMV strain AD169 or the low passage HCMV isolates Merlin and Toledo. The 67 kDa EndoH-sensitive gpUL18 glycoform was detected earlier in a productive infection (from 24 h post-infection) than the slower-migrating EndoH-resistant glycoform (from 72 h post-infection). Deletion of the US2-US11 region from the HCMV genome was associated with a substantial up-regulation of endogenous HLA-I in infected cells, but had no obvious effect on the gpUL18 expression pattern. Vaccinia virus and adenovirus vectors were used to further analyse gpUL18 expression. Depending on the delivery vector system, differences in the electrophoretic motility of the EndoH-resistant >105 kDa form of gpUL18, but not the EndoH-sensitive 67 kDa form, were observed; post-translational modification of the higher molecular mass glycoform appears to be influenced by active virus infection and vector delivery. The EndoH-sensitive 67 kDa gpUL18 had a rapid turnover, while the maturation to the EndoH-resistant >105 kDa form was relatively slow and inefficient. However, synthesis of the EndoH-resistant >105 kDa form was enhanced with elevated levels of beta2-microglobulin. When expressed by using an adenovirus vector, both the EndoH-sensitive 67 kDa and the EndoH-resistant >105 kDa gpUL18 forms could be detected on the cell surface.

Fusogenic domains in herpes simplex virus type 1 glycoprotein H.

Infection of eukaryotic cells by enveloped viruses requires fusion between the viral envelope and the cellular plasma or endosomal membrane. The actual merging of the two membranes is mediated by viral envelope glycoproteins, which generally contain a highly hydrophobic region termed the fusion peptide. The entry of herpesviruses is mediated by three conserved proteins: glycoproteins B, H (gH), and L. However, how fusion is executed remains unknown. Herpes simplex virus type 1 gH exhibits features typical of viral fusion glycoproteins, and its ectodomain seems to contain a putative internal fusion peptide. Here, we have identified additional internal segments able to interact with membranes and to induce membrane fusion of large unilamellar vesicles. We have applied the hydrophobicity-at-interface scale proposed by Wimley and White (Wimley, W. C., and White, S. H. (1996) Nat. Struct. Biol. 3, 842-848) to identify six hydrophobic stretches within gH with a tendency to partition into the membrane interface, and four of them were able to induce membrane fusion. Experiments in which equimolar mixtures of gH peptides were used indicated that different fusogenic regions may act in a synergistic way. The functional and structural characterization of these segments suggests that herpes simplex virus type 1 gH possesses several fusogenic internal peptides that could participate in the actual fusion event.

Herpes simplex virus type 1 glycoprotein H binds to alphavbeta3 integrins.

Glycoprotein H (gH) homologues are found in all members of the herpes virus family, and gH is one of the virion envelope glycoproteins that is essential for virus entry. In this study, a recombinant soluble form of Herpes simplex virus type 1 (HSV-1) gH, in which the ectodomain is fused to the Fc-binding region of IgG, has been generated. This was expressed in mammalian cells together with gL and the resulting gHFc-gL heterodimer was purified using Protein A Sepharose. Low-affinity cell binding assays showed that gHFc-gL bound specifically to Vero cells and mutation of a potential integrin-binding motif, Arg-Gly-Asp (RGD), in gH abolished binding. CHO cells failed to bind in this assay. However, CHO cells expressing the human alphavbeta3 integrin bound efficiently to gHFc-gL, suggesting that HSV-1 gH can bind to cells using alphavbeta3 integrins and that this binding is mediated by the RGD motif in the gH ectodomain.

Alphaherpesvirus glycoprotein M causes the relocalization of plasma membrane proteins.

Herpesvirus glycoprotein M (gM) is a multiple-spanning integral membrane protein found within the envelope of mature herpesviruses and is conserved throughout the Herpesviridae. gM is defined as a non-essential glycoprotein in alphaherpesviruses and has been proposed as playing a role in controlling final envelopment in a late secretory-pathway compartment such as the trans-Golgi network (TGN). Additionally, gM proteins have been shown to inhibit cell-cell fusion in transfection-based assays by an as yet unclear mechanism. Here, the effect of pseudorabies virus (PRV) gM and the herpes simplex virus type 1 (HSV-1) gM/UL49A complex on the fusion events caused by the HSV-1 glycoproteins gB, gD, gH and gL was investigated. Fusion of cells expressing HSV-1 gB, gD, gH and gL was efficiently inhibited by both PRV gM and HSV-1 gM/UL49A. Furthermore, expression of PRV gM or HSV-1 gM/UL49A, which are themselves localized to the TGN, caused both gD and gH/L to be relocalized from the plasma membrane to a juxtanuclear compartment, suggesting that fusion inhibition is caused by the removal of 'fusion' proteins from the cell surface. The ability of gM to cause the relocalization of plasma membrane proteins was not restricted to HSV-1 glycoproteins, as other viral and non-viral proteins were also affected. These data suggest that herpesvirus gM (gM/N) can alter the membrane trafficking itineraries of a broad range of proteins and this may have multiple functions.

Molecule by molecule direct and quantitative counting of antibody-protein complexes in solution.

We have used two-color fluorescence coincidence detection to directly count individual protein-antibody complexes of protein G or herpes simplex virus labeled with one or more red- and blue-excited antibodies. This allowed quantitative measurement of the concentration of the protein-antibody complexes over 3 orders of magnitude down to the femtomolar level. Single molecule measurements in diluted serum are also possible. The sample preparation is simple, takes place in solution, and requires no separation. Both the antibody affinity and complex dissociation rate are important in determining the sensitivity of the method. At present, the sensitivity limit of 50 fM is determined by the encounter rate of the labeled analyte with the probe volume. This method can be used to detect and quantitate proteins and to measure the stoichiometry, equilibrium constant, and dissociation rate of protein-protein complexes at low concentrations.

Analysis of the requirement for glycoprotein m in herpes simplex virus type 1 morphogenesis.

A mutant of herpes simplex virus type 1 lacking both glycoprotein M and glycoprotein E was marginally compromised in terms of its in vitro growth characteristics. This finding is in marked contrast to a similar mutant of the related alphaherpesvirus, pseudorabies virus (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999), and suggests that the glycoprotein requirements for virion assembly may vary among different members of this family of viruses.

Analysis of the role of the membrane-spanning and cytoplasmic tail domains of herpes simplex virus type 1 glycoprotein D in membrane fusion.

Glycoprotein D (gD) of herpes simplex virus type 1 is a type 1 membrane protein in the virus envelope that binds to receptor molecules on the cell surface and which induces cell-cell fusion when co-expressed with gB, gH and gL. A chimeric gD molecule in which the membrane anchor and cytoplasmic tail domains were replaced with analogous regions from the human CD8 molecule was as competent as wild-type gD at mediating membrane fusion and virus entry. However, when gD was tethered to the membrane by means of a glycosylphosphatidylinositol (gpi)-anchor sequence, which binds only to the outer leaflet of the lipid bilayer, it was unable to function in cell-cell fusion assays. This chimera was incorporated into virions as efficiently as wild-type gD and yet virus particles containing gpi-linked gD entered cells more slowly than virions containing wild-type gD in their envelopes, suggesting that gD must be anchored in both leaflets of a lipid bilayer for it to function in both cell fusion and virus entry.

Expression of the UL16 glycoprotein of Human Cytomegalovirus protects the virus-infected cell from attack by natural killer cells.

BACKGROUND: Human Cytomegalovirus (HCMV) has acquired through evolution a number of genes to try to evade immune recognition of the virus-infected cell. Many of these mechanisms act to inhibit the MHC class I antigen presentation pathway, but any virus-infected cell which has down-regulated cell surface expression of MHC class I proteins, to avoid CTL attack, would be expected to become susceptible to lysis by Natural Killer cells. Surprisingly, however, HCMV infected fibroblasts were found to be resistant to NK cell mediated cytotoxicity. Expression of the UL16 glycoprotein could represent one mechanism to help the virus to escape from NK cell attack, as it has been shown to bind, in vitro, some of the ligands for NKG2D, the NK cell activating receptor. Here, we explored the role of UL16, in the context of a viral infection, by comparing the susceptibility to NK lysis of cells infected with HCMV and cells infected with a UL16 deletion mutant of this virus. RESULTS: Cells infected with the UL16 knockout virus were killed at substantially higher levels than cells infected with the wild-type virus. This increased killing could be correlated with a UL16-dependent reduction in surface expression of ligands for the NK cell activating receptor NKG2D. CONCLUSIONS: Expression of the UL16 glycoprotein was associated with protection of HCMV-infected cells from NK cell attack. This observation could be correlated with the downregulation of cell surface expression of NKG2D ligands. These data represent a first step towards understanding the mechanism(s) of action of the UL16 protein.

The human cytomegalovirus protein UL16 mediates increased resistance to natural killer cell cytotoxicity through resistance to cytolytic proteins.

Several reports have shown that human cytomegalovirus (HCMV)-infected cells are resistant to NK lysis. These studies have focused on receptor-ligand interactions, and different HCMV proteins have been indicated to mediate inhibitory NK signals. Here, we report that the HCMV protein UL16 is of major importance for the ability of HCMV-infected cells to resist NK cell-mediated cytotoxicity. Fibroblasts infected with the UL16 deletion mutant HCMV strain exhibited a 70% increased sensitivity to NK killing at 7 days postinfection compared to AD169-infected cells. Interestingly, HCMV-infected cells did not appear to engage inhibitory molecules on NK cells, since the levels of granzyme B were not reduced in supernatants obtained from NK cell cocultures with infected target cells compared to uninfected target cells. Furthermore, HCMV-infected cells, but not cells infected with the UL16 deletion mutant HCMV strain, exhibited a significantly increased resistance to the action of cytolytic proteins, including perforin, granzyme B, streptolysin O, and porcine NK lysin. In addition, fluorescence-activated cell sorting for UL16-positive transfected cells resulted in protection levels of 90% compared to control cells carrying the green fluorescent protein vector. Thus, the UL16 protein mediates an increased protection against the action of cytolytic proteins released by activated NK cells, possibly by a membrane-stabilizing mechanisms, rather than by delivering negative signals to NK cells.