Sharon H. Willis

Herpes simplex virus glycoprotein D bound to the human receptor HveA.

Herpes simplex virus (HSV) infection requires binding of the viral envelope glycoprotein D (gD) to cell surface receptors. We report the X-ray structures of a soluble, truncated ectodomain of gD both alone and in complex with the ectodomain of its cellular receptor HveA. Two bound anions suggest possible binding sites for another gD receptor, a 3-O-sulfonated heparan sulfate. Unexpectedly, the structures reveal a V-like immunoglobulin (Ig) fold at the core of gD that is closely related to cellular adhesion molecules and flanked by large N- and C-terminal extensions. The receptor binding segment of gD, an N-terminal hairpin, appears conformationally flexible, suggesting that a conformational change accompanying binding might be part of the viral entry mechanism.

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.

Function of Herpes Simplex Virus Type 1 gD Mutants with Different Receptor-Binding Affinities in Virus Entry and Fusion

ABSTRACT We have studied the receptor-specific function of four linker-insertion mutants of herpes simplex virus type 1 glycoprotein D (gD) representing each of the functional regions of gD. We used biosensor analysis to measure binding of the gD mutants to the receptors HVEM (HveA) and nectin-1 (HveC). One of the mutants, gD(∇34t), failed to bind HVEMt but showed essentially wild-type (WT) affinity for nectin-1t. The receptor-binding kinetics and affinities of the other three gD mutants varied over a 1,000-fold range, but each mutant had the same affinity for both receptors. All of the mutants were functionally impaired in virus entry and cell fusion, and the levels of activity were strikingly similar in these two assays. gD(∇34)-containing virus was defective on HVEM-expressing cells but did enter nectin-1-expressing cells to about 60% of WT levels. This showed that the defect of this form of gD on HVEM-expressing cells was primarily one of binding and that this was separable from its later function in virus entry. gD(∇243t) showed WT binding affinity for both receptors, but virus containing this form of gD had a markedly reduced rate of entry, suggesting that gD(∇243) is impaired in a postbinding step in the entry process. There was no correlation between gD mutant activity in fusion or virus entry and receptor-binding affinity. We conclude that gD functions in virus entry and cell fusion regardless of its receptor-binding kinetics and that as long as binding to a functional receptor occurs, entry will progress.

Antibody-Induced Conformational Changes in Herpes Simplex Virus Glycoprotein gD Reveal New Targets for Virus Neutralization

ABSTRACT 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.

Crystallization and preliminary diffraction studies of the ectodomain of the envelope glycoprotein D from herpes simplex virus 1 alone and in complex with the ectodomain of the human receptor HveA

Gycoprotein D (gD) is a glycoprotein expressed on the surface of several human and animal alpha herpes viruses. Binding of gD to cell-surface receptors has been shown to be necessary for herpes simplex virus 1 and 2 (HSV-1 and HSV-2) cell entry. The gD ectodomain consists of 316 residues and has no sequence homology to any other proteins of known structure. Two fragments of the HSV-1 gD ectodomain (gD(22-260): residues 22-260 and gD(285): residues 1-285) have been crystallized in two crystal forms. The complex between gD(285) and the ectodomain of HveA, a gD cellular receptor member of the tumor necrosis factor (TNFR) superfamily, has also been crystallized. Moreover, insect-cell-expressed selenomethionine-substituted gD(285) has been purified and crystallized alone and in complex with HveA.

Expression and Purification of Secreted Forms of HSV Glycoproteins from Baculovirus-Infected Insect Cells.

Herpes simplex virus (HSV) remains a major human pathogen worldwide (25 causing cold sores, eye and genital infections, blindness, encephalitis, and neonatal infections. Most adults have antibodies against the oral form of the virus HSV-1 (9), and a significant number are infected with the genital form, HSV-2. Both serotypes establish lifelong latent infections and reactivate periodically to produce recurrent disease (25). After infection, virus-encoded glycoproteins are expressed on all cellular membranes and are major targets of the hosts immune response. The virion envelope contains 10 glycoproteins that are important for infection and pathogenesis of HSV-1 and HSV-2. Because HSV contains so many glycoproteins, sorting out their functions in virus entry remains a difficult task. Our approach has focused on establishing structure-function relationships of the individual glycoproteins with particular emphasis on gC and gD. After many years of studying the properties of these proteins in HSV-infected and plasmid-transfected mammalian cells, we have now begun to overexpress the proteins using a baculovirus expression system.

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

ABSTRACT The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.

Maturation of the Gag core decreases the stability of retroviral lipid membranes

To better understand how detergents disrupt enveloped viruses, we monitored the biophysical stability of murine leukemia virus (MLV) virus-like particles (VLPs) against a panel of commonly used detergents using real-time biosensor measurements. Although exposure to many detergents, such as Triton X-100 and Empigen, results in lysis of VLP membranes, VLPs appeared resistant to complete membrane lysis by a significant number of detergents, including Tween 20, Tween 80, Lubrol, and Saponin. VLPs maintained their structural integrity after exposure to Tween 20 at concentrations up to 500-fold above its CMC. Remarkably, VLPs containing immature cores composed of unprocessed (uncleaved) Gag polyprotein were significantly more resistant to detergent lysis than VLPs with mature cores. Although the maturity of retroviral Gag is known to influence the stability of the protein core structure itself, our studies suggest that the maturity of the Gag core also influences the stability of the lipid bilayer surrounding the core.

Isolation of state-dependent monoclonal antibodies against the 12-transmembrane domain glucose transporter 4 using virus-like particles

Significance Generating mAbs against the native extracellular epitopes of multispanning membrane proteins is challenging, and as a result, few nonpeptidic mAbs against transporters have ever been isolated. Our approach here using virus-like particles and divergent host species for immunizations provides a means to overcome these challenges. The specific mAbs isolated here recognize native GLUT4 on the cell surface and can distinguish its different conformational states, thus representing some of the only state-specific mAbs ever isolated against any transporter. Epitope mapping of these mAbs revealed their binding sites as well as the mechanisms by which amino acids control the inward-open and outward-open states of GLUT4. Our studies demonstrate a valuable platform to isolate functional mAbs against important multispanning membrane proteins. The insulin-responsive 12-transmembrane transporter GLUT4 changes conformation between an inward-open state and an outward-open state to actively facilitate cellular glucose uptake. Because of the difficulties of generating conformational mAbs against complex and highly conserved membrane proteins, no reliable tools exist to measure GLUT4 at the cell surface, follow its trafficking, or detect the conformational state of the protein. Here we report the isolation and characterization of conformational mAbs that recognize the extracellular and intracellular domains of GLUT4, including mAbs that are specific for the inward-open and outward-open states of GLUT4. mAbs against GLUT4 were generated using virus-like particles to present this complex membrane protein in its native conformation and using a divergent host species (chicken) for immunization to overcome immune tolerance. As a result, the isolated mAbs recognize conformational epitopes on native GLUT4 in cells, with apparent affinities as high as 1 pM and with specificity for GLUT4 across the human membrane proteome. Epitope mapping using shotgun mutagenesis alanine scanning across the 509 amino acids of GLUT4 identified the binding epitopes for mAbs specific for the states of GLUT4 and allowed the comprehensive identification of the residues that functionally control the GLUT4 inward-open and outward-open states. The mAbs identified here will be valuable molecular tools for monitoring GLUT4 structure, function, and trafficking, for differentiating GLUT4 conformational states, and for the development of novel therapeutics for the treatment of diabetes.

Abstract 74: Discovery of new therapeutic monoclonal antibodies to challenging GPCRs, ion channels and transporters

The objective of this work was to evaluate the ability to generate panels of monoclonal antibodies against a set of highly challenging targets including GPCRs (CB1, C5AR, CXCR5 and CGRPR), transporters (GLUT4), and ion channels (P2X3). Integral membrane proteins are important drug targets and monoclonal antibodies (MAbs) directed against them are highly sought for therapeutic purposes. However, the complex structure of multispan membrane protein targets makes the discovery of these MAbs especially challenging. To address this need, Integral Molecular has developed the MPS Discovery Engine® to enable the isolation, characterization, and engineering of monoclonal antibodies for GPCRs, ion channels, and transporters. MPS utilizes a collection of technologies to address each of the barriers to monoclonal antibody development against the native extracellular epitopes of multispan membrane proteins. These include, antigen engineering to attain high levels of surface expression, DNA and Lipoparticle immunization to present native epitopes to the immune system, diverse immunization host species to deal with highly conserved proteins, Lipoparticles (high concentration native membrane proteins) to enable phage display and microfludic B-cell isolation, and shotgun mutagenesis (comprehensive Alanine scanning) for epitope mapping. Using the MPS Discovery Engine® we were able to successfully generate large panels of antibodies to the targets that were able to bind to the native extracellular epitopes on cells by flow cytometry. A subset of the antibodies had antagonist activity. With this technology we have the ability to target intact, conformation specific, and functional antibodies to complex membrane proteins. Citation Format: Lewis J. Stafford, Ross Chambers, Sharon H. Willis, Moniquetta Hall, Brad Screnci, Manu Mabila, David Tucker, Trevor Barnes, Rachel Fong, Andrew Ettenger, Jennifer Pfaff, Chidananda Sulli, Nicholas Molino, Andrew Hudacek, Benjamin J. Doranz, Joseph Rucker. Discovery of new therapeutic monoclonal antibodies to challenging GPCRs, ion channels and transporters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 74. doi:10.1158/1538-7445.AM2017-74