The prefusion structure of herpes simplex virus glycoprotein B

Cell entry of enveloped viruses requires specialized viral proteins that mediate fusion with the host membrane by substantial structural rearrangements from a metastable pre- to a stable postfusion conformation. This metastability renders the herpes simplex virus 1 (HSV-1) fusion glycoprotein B (gB) highly unstable such that it readily converts into the postfusion form, thereby precluding structural elucidation of the pharmacologically relevant prefusion conformation. By identification of conserved sequence signatures and molecular dynamics simulations, we devised a mutation that stabilized this form. Functionally locking gB allowed the structural determination of its membrane-embedded prefusion conformation at sub-nanometer resolution and enabled the unambiguous fit of all ectodomains. The resulting pseudo-atomic model reveals a notable conservation of conformational domain rearrangements during fusion between HSV-1 gB and the vesicular stomatitis virus glycoprotein G, despite their very distant phylogeny. In combination with our comparative sequence-structure analysis, these findings suggest common fusogenic domain rearrangements in all class III viral fusion proteins.

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

ABSTRACT 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.

Herpes Simplex Virus Glycoprotein C Regulates Low-pH Entry

ABSTRACT Herpes simplex viruses (HSVs) cause significant morbidity and mortality in humans worldwide. Herpesviruses mediate entry by a multicomponent virus-encoded machinery. Herpesviruses enter cells by endosomal low-pH and pH-neutral mechanisms in a cell-specific manner. HSV mediates cell entry via the envelope glycoproteins gB and gD and the heterodimer gH/gL regardless of pH or endocytosis requirements. Specifics concerning HSV envelope proteins that function selectively in a given entry pathway have been elusive. Here, we demonstrate that gC regulates cell entry and infection by a low-pH pathway. Conformational changes in the core herpesviral fusogen gB are critical for membrane fusion. The presence of gC conferred a higher pH threshold for acid-induced antigenic changes in gB. Thus, gC may selectively facilitate low-pH entry by regulating conformational changes in the fusion protein gB. We propose that gC modulates the HSV fusion machinery during entry into pathophysiologically relevant cells, such as human epidermal keratinocytes. IMPORTANCE Herpesviruses are ubiquitous pathogens that cause lifelong latent infections and that are characterized by multiple entry pathways. We propose that herpes simplex virus (HSV) gC plays a selective role in modulating HSV entry, such as entry into epithelial cells, by a low-pH pathway. gC facilitates a conformational change of the main fusogen gB, a class III fusion protein. We propose a model whereby gC functions with gB, gD, and gH/gL to allow low-pH entry. In the absence of gC, HSV entry occurs at a lower pH, coincident with trafficking to a lower pH compartment where gB changes occur at more acidic pHs. This report identifies a new function for gC and provides novel insight into the complex mechanism of HSV entry and fusion.

Herpes Simplex Virus Glycoprotein C Regulates Low pH Entry

Herpes simplex viruses (HSVs) cause significant morbidity and mortality in humans worldwide. Herpesviruses mediate entry by a multi-component, virus-encoded machinery. Herpesviruses enter cells by endosomal low pH and pH-neutral mechanisms in a cell-specific manner. HSV mediates cell entry via envelope glycoproteins gB, gD, and the heterodimer gH/gL regardless of pH or endocytosis requirements. HSV envelope proteins that function selectively in a given entry pathway have been elusive. Here we demonstrate that gC regulates cell entry and infection by a low pH pathway. Conformational changes in the core herpesviral fusogen gB are critical for membrane fusion. The presence of gC conferred a higher pH threshold to acid-induced antigenic changes in gB. Thus, gC may selectively facilitate low pH entry by regulating conformational changes in the fusion protein gB. We propose that gC modulates the HSV fusion machinery during entry into pathophysiologically relevant cells, such as human epidermal keratinocytes.ImportanceHerpesviruses are ubiquitous pathogens that cause lifelong latent infections and are characterized by multiple entry pathways. We propose that herpes simplex virus (HSV) gC plays a selective role in modulating HSV entry by a low pH pathway, such as into epithelial cells. gC facilitates conformational change of the main fusogen gB, a class III fusion protein. We propose a model whereby gC functions with gB, gD, and gH/gL to allow low pH entry. In the absence of gC, HSV entry occurs at a lower pH, coincident with trafficking to a lower pH compartment where gB changes occur at more acidic pHs. This study identifies a new function for gC and provides novel insight into the complex mechanism of HSV entry and fusion.

Identification and Visualization of Functionally Important Domains and Residues in Herpes Simplex Virus Glycoprotein K(gK) Using a Combination of Phylogenetics and Protein Modeling

Alphaherpesviruses are a subfamily of herpesviruses that include the significant human pathogens herpes simplex viruses (HSV) and varicella zoster virus (VZV). Glycoprotein K (gK), conserved in all alphaherpesviruses, is a multi-membrane spanning virion glycoprotein essential for virus entry into neuronal axons, virion assembly, and pathogenesis. Despite these critical functions, little is known about which gK domains and residues are most important for maintaining these functions across all alphaherpesviruses. Herein, we employed phylogenetic and structural analyses including the use of a novel model for evolutionary rate variation across residues to predict conserved gK functional domains. We found marked heterogeneity in the evolutionary rate at the level of both individual residues and domains, presumably as a result of varying selective constraints. To clarify the potential role of conserved sequence features, we predicted the structures of several gK orthologs. Congruent with our phylogenetic analysis, slowly evolving residues were identified at potentially structurally significant positions across domains. We found that using a quantitative measure of amino acid rate variation combined with molecular modeling we were able to identify amino acids predicted to be critical for gK protein structure/function. This analysis yields targets for the design of anti-herpesvirus therapeutic strategies across all alphaherpesvirus species that would be absent from more traditional analyses of conservation.

LIGHT (TNFSF14) Increases Survival and Proliferation of Human Bone Marrow-Derived Mesenchymal Stem Cells Via LTβR

Background: LIGHT (Homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator (HVEM)/tumor necrosis factor (TNF)-related 2, HVEM-L, TNFSF14, or CD258) is a member of TNF superfamily. It is expressed as a homotrimer on activated T cells and also on immune dendritic cells, and has three receptors such as HVEM, LT-¥â receptor (LT-¥âR) and decoy receptor 3 (DcR3). So far, three receptors with distinct cellular expression patterns are described to interact with LIGHT. Follicular DCs and stromal cells bind LIGHT through LT-¥âR. We monitored the effects of LIGHT on human bone marrow-derived mesenchymal stem cells (BM-MSCs). Methods: At first, we checked negative and positive differentiation markers of BM-MSCs. After rhLIGHT treatment, we monitored cell count, viability, proliferation, and cell cycle distribution. Also, PDGF and TGF¥â production by rhLIGHT were examined by ELISA, and biological mechanism were checked by immunoblotting through the rhLIGHT treatment. Results: FACS analysis result showed that LT-¥âR receptor is expressed in human BM-MSCs, but not HVEM indicating that LIGHT binds only LT-¥âR in human BM-MSCs. (Fig. 1A/B). rhLIGHT and LT-¥âR interaction increased cell numbers in BM-MSCs using an inverted microscope for cell number changes. Cell numbers by rhLIGHT enhanced dose-dependently and time-dependently (Fig. 2 A/B). Cell viability and the expression of p-AKT, Bcl-2 and Bcl-xL by rhLIGHT were significantly increased in BM-MSCs and rhLIGHT-induced IkB-¥á degradation activated NF-kB signal (Fig. 3A/B). rhLIGHT increased cell proliferation by increasing S/G2/M phase in BM-MSCs (Fig. 3C and 4D). And cell cycle regulatory proteins were enhanced by rhLIGHT in BM-MSC including cyclin B1, D1, D3, E, and cyclin dependent kinase (CDK) 1 and 2 while CDK inhibitor, p27 was decreased by rhLIGHT treatment (Fig. 3E). Moreover, rhLIGHT-induced PDGF and TGF¥â production by STAT3 and smad3 activation accelerated BM-MSCs proliferation. And we also confirmed differentiation potential of rhLIGHT on BM-MSCs by the staining for adipogenesis (Oil Red O staining), chondrogenesis (Alcian blue staining) and Osteogenesis (Alizarin red Staining). Conclusion: LIGHT and LT-¥âR interaction increases survival and proliferation of human BM-MSCs by activation of survival proteins, anti-apoptotic proteins, CDKs and cyclins. Moreover, LIGHT-induced STAT-3 and smad-3 activation causes PDGF and TGF-¥â production, and they enhance LIGHT signals in human BM-MSCs. We proposed the pathway of LIGHT and LT-¥âR interaction in human BM-MSCs. Therefore, LIGHT may play an important role for therapy of stem cells, and contribute to modification of MSCs. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.