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Results for “Membrane Glycoproteins”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets—the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity—and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30–40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Overexpression of human ACE2 protein in mouse fibroblasts stably transfected with the intact ACE2 gene

Infection by SARS-CoV-2 is dependent on binding of the viral spike protein to angiotensin converting enzyme 2 (ACE2), a membrane glycoprotein expressed on epithelial cells in the human upper respiratory tract. Recombinant ACE2 protein has potential application for anti-viral therapy. Here we co-transfected mouse fibroblasts (A9 cells) with a cloned fragment of human genomic DNA containing the intact ACE2 gene and an unlinked neomycin phosphotransferase gene, and then selected stable neomycin-resistant transfectants. Transfectant clones expressed ACE2 protein at levels that were generally proportional to the number of ACE2 gene copies integrated in the cell genome, ranging up to approximately 50 times the level of ACE2 present of Vero-E6 cells. Cells overexpressing ACE2 were hypersensitive to infection by spike-pseudotyped vesicular stomatitis virus (VSV-S), and adsorption of VSV-S to these cells occurred at an accelerated rate compared to Vero-E6 cells. In conclusion, the transfectant cell clones described here therefore have favorable attributes as feedstocks for large-scale production of recombinant human ACE2 protein.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2023

The Polymorphic Membrane Protein G Has a Neutral Effect and the Plasmid Glycoprotein 3 an Antagonistic Effect on the Ability of the Major Outer Membrane Protein to Elicit Protective Immune Responses against a Chlamydia muridarum Respiratory Challenge

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen. The number of chlamydial infections continuous to increase and there is an urgent need for a safe and efficacious vaccine. To assess the ability of the Chlamydia muridarum polymorphic membrane protein G (PmpG) and the plasmid glycoprotein 3 (Pgp3) as single antigens, and in combination with the major outer-membrane protein (MOMP) to induce protection, BALB/c mice were immunized utilizing CpG-1826 and Montanide ISA 720 VG as adjuvants. Following vaccination with MOMP, significant humoral and cell-mediated immune responses were observed, while immunization with PmpG, or Pgp3, elicited weaker immune responses. Weaker immune responses were induced with MOMP+Pgp3 compared with MOMP alone. Following the intranasal challenge with C. muridarum, mice vaccinated with MOMP showed robust protection against body-weight loss, inflammatory responses in the lungs and number of Chlamydia recovered from the lungs. PmpG and Pgp3 elicited weaker protective responses. Mice immunized with MOMP+PmpG, were no better protected than animals vaccinated with MOMP only, while Pgp3 antagonized the protection elicited by MOMP. In conclusion, PmpG and Pgp3 elicited limited protective immune responses in mice against a respiratory challenge with C. muridarum and failed to enhance the protection induced by MOMP alone. The virulence of Pgp3 may result from its antagonistic effect on the immune protection induced by MOMP.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Visualization of conformational changes and membrane remodeling leading to genome delivery by viral class-II fusion machinery

Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKV’s complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoprotein’s post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Hyperfusogenic Mutations Destabilize the Postfusion Six-Helix Bundle of the Measles Virus Fusion Glycoprotein

Fusion of the host membrane and viral envelope by class I viral fusion proteins is driven by the assembly of a postfusion six-helix bundle formed through antiparallel interactions between N-terminal (HR1) and C-terminal (HR2) heptad-repeat regions. Although mutations in these regions of the measles virus (MeV) fusion (F) glycoprotein are known to promote neuropathogenic and hyperfusogenic phenotypes, their effects on postfusion core stability have not been systematically examined. Here, we combine peptide biophysics and X-ray crystallography to interrogate how mutations within the HR2 domain, present in native neuropathogenic MeV isolates (e.g., L454W and N462K) and laboratory-generated hyperfusogenic variants (e.g., L454M and T461A), influence postfusion 6HB assembly. Circular dichroism (CD) spectroscopy reveals that, with few exceptions, these mutations decrease postfusion core stability, despite their association with enhanced fusion activity. We also report the first crystal structure of the wild-type MeV postfusion core as well as structures of six hyperfusogenic variants, enabling high-resolution comparison of the molecular basis of destabilization. Structural analysis shows that these effects arise from localized perturbations to steric packing, hydrogen bonding networks, and helix-stabilizing interactions within HR2, while the overall 6HB architecture remains conserved. Together, these results indicate that hyperfusogenic mutations are not associated with stabilization of the postfusion state and are instead consistent with models in which hyperfusogenicity arises from a reduction in the energetic barrier to fusion, potentially through effects on prefusion stability or triggering efficiency. These findings establish key sequence-structure–stability relationships governing coiled-coil assembly and provide a framework for the design of HR1- and HR2-based fusion inhibitors.

Genetics↗
DOE OSTI2025

Cell surface crowding is a tunable energetic barrier to cell-cell fusion

Abstract Cell-cell fusion is fundamental to processes such as muscle formation and viral infection. An essential step in fusion is close membrane apposition, but cell membranes are crowded with proteins, glycoproteins, and glycolipids, which must be cleared before a fusion pore can be nucleated. Here, we find that cell surface crowding reduces fusogenicity independent of how fusion is driven. We estimate that crowding presents an energetic barrier to membrane apposition on the scale of ~$${100k}_{{\mbox{B}}}T$$ 100 k B T , greater than that of bare membrane fusion alone. We show that increasing cell surface crowding reduces fusion efficiency of PEG-mediated and fusogen-mediated cell-cell fusion, as well as synthetic membranes under force. Interestingly, we find that differentiating myoblasts naturally decrease their surface crowding prior to fusion. In this work, we show that cell surface crowding presents an underappreciated biophysical barrier that may be tuned developmentally and could be targeted externally to control tissue-specific cell-cell fusion.

Science & Technology - Other Topics↗
DOE OSTI2020

A glycoprotein B-neutralizing antibody structure at 2.8 Å uncovers a critical domain for herpesvirus fusion initiation

Members of the Herpesviridae, including the medically important alphaherpesvirus varicella-zoster virus (VZV), induce fusion of the virion envelope with cell membranes during entry, and between cells to form polykaryocytes in infected tissues. The conserved glycoproteins, gB, gH and gL, are the core functional proteins of the herpesvirus fusion complex. gB serves as the primary fusogen via its fusion loops, but functions for the remaining gB domains remain unexplained. As a pathway for biological discovery of domain function, our approach used structure-based analysis of the viral fusogen together with a neutralizing antibody. We report here a 2.8 Å cryogenic-electron microscopy structure of native gB recovered from VZV-infected cells, in complex with a human monoclonal antibody, 93k. This high-resolution structure guided targeted mutagenesis at the gB-93k interface, providing compelling evidence that a domain spatially distant from the gB fusion loops is critical for herpesvirus fusion, revealing a potential new target for antiviral therapies. Herpesvirus virions have an outer lipid membrane dotted with glycoproteins that enable fusion with cell membranes to initiate entry and establish infection. Here the authors elucidate the structural mechanism of a neutralizing antibody derived from a patient infected by the herpesvirus varicella-zoster virus and targeted to its fusogen, glycoprotein-B.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Anti-Ebola virus mAb 3A6 protects highly viremic animals from fatal outcome via binding GP(1,2) in a position elevated from the virion membrane

Abstract Monoclonal antibodies (mAbs) against Ebola virus (EBOV) glycoprotein (GP 1,2 ) are the standard of care for Ebola virus disease (EVD). Anti-GP 1,2 mAbs targeting the stalk and membrane proximal external region (MPER) potently neutralize EBOV in vitro and are protective in a mouse model of EVD. However, their neutralization mechanism is poorly understood because they target a GP 1,2 epitope that has evaded structural characterization. Using X-ray crystallography and cryo-electron tomography of mAb 3A6 complexed with its stalk–MPER epitope, we reveal a previously undescribed mechanism in which 3A6 binds to a conformation of GP 1,2 that is lifted from the virion membrane. We further show that in both domestic guinea pig and rhesus monkey EVD models, 3A6 provides therapeutic benefit at high-viremia advanced disease stages and at the lowest dose yet demonstrated for any anti-EBOV mAb-based monotherapy. The findings reported here can guide design of next-generation highly potent anti-EBOV therapeutics and vaccines.

Science & Technology - Other Topics↗
DOE OSTI2023

Targeted mutagenesis of the herpesvirus fusogen central helix captures transition states

Herpesviruses remain a burden for animal and human health, including the medically important varicella-zoster virus (VZV). Membrane fusion mediated by conserved core glycoproteins, the fusogen gB and the heterodimer gH-gL, enables herpesvirus cell entry. The ectodomain of gB orthologs has five domains and is proposed to transition from a prefusion to postfusion conformation but the functional relevance of the domains for this transition remains poorly defined. Here we describe structure-function studies of the VZV gB DIII central helix targeting residues 526 EHV 528 . Critically, a H527P mutation captures gB in a prefusion conformation as determined by cryo-EM, a loss of membrane fusion in a virus free assay, and failure of recombinant VZV to spread in cell monolayers. Importantly, two predominant cryo-EM structures of gB[H527P] are identified by 3D classification and focused refinement, suggesting they represented gB conformations in transition. These studies reveal gB DIII as a critical element for herpesvirus gB fusion function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Epstein-Barr virus gH/gL has multiple sites of vulnerability for virus neutralization and fusion inhibition

Epstein-Barr virus (EBV) is nearly ubiquitous in adults. EBV causes infectious mononucleosis and is associated with B cell lymphomas, epithelial cell malignancies, and multiple sclerosis. The EBV gH/gL glycoprotein complex facilitates fusion of virus membrane with host cells and is a target of neutralizing antibodies. Here, in this study, we examined the sites of vulnerability for virus neutralization and fusion inhibition within EBV gH/gL. We developed a panel of human monoclonal antibodies (mAbs) that targeted five distinct antigenic sites on EBV gH/gL and prevented infection of epithelial and B cells. Structural analyses using X-ray crystallography and electron microscopy revealed multiple sites of vulnerability and defined the antigenic landscape of EBV gH/gL. One mAb provided near-complete protection against viremia and lymphoma in a humanized mouse EBV challenge model. Our findings provide structural and antigenic knowledge of the viral fusion machinery, yield a potential therapeutic antibody to prevent EBV disease, and emphasize gH/gL as a target for herpesvirus vaccines and therapeutics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Engineered molecular sensors for quantifying cell surface crowding

Cells mediate interactions with the extracellular environment through a crowded assembly of transmembrane proteins, glycoproteins and glycolipids on their plasma membrane. The extent to which surface crowding modulates the biophysical interactions of ligands, receptors, and other macromolecules is poorly understood due to the lack of methods to quantify surface crowding on native cell membranes. In this work, we demonstrate that physical crowding on reconstituted membranes and live cell surfaces attenuates the effective binding affinity of macromolecules such as IgG antibodies in a surface crowding-dependent manner. We combine experiment and simulation to design a crowding sensor based on this principle that provides a quantitative readout of cell surface crowding. Our measurements reveal that surface crowding decreases IgG antibody binding by 2 to 20 fold in live cells compared to a bare membrane surface. Our sensors show that sialic acid, a negatively charged monosaccharide, contributes disproportionately to red blood cell surface crowding via electrostatic repulsion, despite occupying only ~1% of the total cell membrane by mass. We also observe significant differences in surface crowding for different cell types and find that expression of single oncogenes can both increase and decrease crowding, suggesting that surface crowding may be an indicator of both cell type and state. Our high-throughput, single-cell measurement of cell surface crowding may be combined with functional assays to enable further biophysical dissection of the cell surfaceome.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Dissecting the structural heterogeneity of proteins by native mass spectrometry

Abstract A single gene yields many forms of proteins via combinations of posttranscriptional/posttranslational modifications. Proteins also fold into higher‐order structures and interact with other molecules. The combined molecular diversity leads to the heterogeneity of proteins that manifests as distinct phenotypes. Structural biology has generated vast amounts of data, effectively enabling accurate structural prediction by computational methods. However, structures are often obtained heterologously under homogeneous states in vitro. The lack of native heterogeneity under cellular context creates challenges in precisely connecting the structural data to phenotypes. Mass spectrometry (MS) based proteomics methods can profile proteome composition of complex biological samples. Most MS methods follow the “bottom‐up” approach, which denatures and digests proteins into short peptide fragments for ease of detection. Coupled with chemical biology approaches, higher‐order structures can be probed via incorporation of covalent labels on native proteins that are maintained at the peptide level. Alternatively, native MS follows the “top‐down” approach and directly analyzes intact proteins under nondenaturing conditions. Various tandem MS activation methods can dissect the intact proteins for in‐depth structural elucidation. Herein, we review recent native MS applications for characterizing heterogeneous samples, including proteins binding to mixtures of ligands, homo/hetero‐complexes with varying stoichiometry, intrinsically disordered proteins with dynamic conformations, glycoprotein complexes with mixed modification states, and active membrane protein complexes in near‐native membrane environments. We summarize the benefits, challenges, and ongoing developments in native MS, with the hope to demonstrate an emerging technology that complements other tools by filling the knowledge gaps in understanding the molecular heterogeneity of proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Regions of hepatitis C virus E2 required for membrane association

Hepatitis C virus (HCV) uses a hybrid entry mechanism. Current structural data suggest that upon exposure to low pH and Cluster of Differentiation 81 (CD81), the amino terminus of envelope glycoprotein E2 becomes ordered and releases an internal loop with two invariant aromatic residues into the host membrane. Here, we present the structure of an amino-terminally truncated E2 with the membrane binding loop in a bent conformation and the aromatic side chains sequestered. Comparison with three previously reported E2 structures with the same Fab indicates that this internal loop is flexible, and that local context influences the exposure of hydrophobic residues. Biochemical assays show that the amino-terminally truncated E2 lacks the baseline membrane-binding capacity of the E2 ectodomain. Thus, the amino terminal region is a critical determinant for both CD81 and membrane interaction. These results provide new insights into the HCV entry mechanism.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Structural insights into the modulation of coronavirus spike tilting and infectivity by hinge glycans

Abstract Coronavirus spike glycoproteins presented on the virion surface mediate receptor binding, and membrane fusion during virus entry and constitute the primary target for vaccine and drug development. How the structure dynamics of the full-length spikes incorporated in viral lipid envelope correlates with the virus infectivity remains poorly understood. Here we present structures and distributions of native spike conformations on vitrified human coronavirus NL63 (HCoV-NL63) virions without chemical fixation by cryogenic electron tomography (cryoET) and subtomogram averaging, along with site-specific glycan composition and occupancy determined by mass spectrometry. The higher oligomannose glycan shield on HCoV-NL63 spikes than on SARS-CoV-2 spikes correlates with stronger immune evasion of HCoV-NL63. Incorporation of cryoET-derived native spike conformations into all-atom molecular dynamic simulations elucidate the conformational landscape of the glycosylated, full-length spike that reveals a role of hinge glycans in modulating spike bending. We show that glycosylation at N1242 at the upper portion of the stalk is responsible for the extensive orientational freedom of the spike crown. Subsequent infectivity assays implicated involvement of N1242-glyan in virus entry. Our results suggest a potential therapeutic target site for HCoV-NL63.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The N-terminus of varicella-zoster virus glycoprotein B has a functional role in fusion

Varicella-zoster virus (VZV) is a medically important alphaherpesvirus that induces fusion of the virion envelope and the cell membrane during entry, and between cells to form polykaryocytes within infected tissues during pathogenesis. All members of the Herpesviridae , including VZV, have a conserved core fusion complex composed of glycoproteins, gB, gH and gL. The ectodomain of the primary fusogen, gB, has five domains, DI-V, of which DI contains the fusion loops needed for fusion function. We recently demonstrated that DIV is critical for fusion initiation, which was revealed by a 2.8Å structure of a VZV neutralizing mAb, 93k, bound to gB and mutagenesis of the gB-93k interface. To further assess the mechanism of mAb 93k neutralization, the binding site of a non-neutralizing mAb to gB, SG2, was compared to mAb 93k using single particle cryogenic electron microscopy (cryo-EM). The gB-SG2 interface partially overlapped with that of gB-93k but, unlike mAb 93k, mAb SG2 did not interact with the gB N-terminus, suggesting a potential role for the gB N-terminus in membrane fusion. The gB ectodomain structure in the absence of antibody was defined at near atomic resolution by single particle cryo-EM (3.9Å) of native, full-length gB purified from infected cells and by X-ray crystallography (2.4Å) of the transiently expressed ectodomain. Both structures revealed that the VZV gB N-terminus (aa72-114) was flexible based on the absence of visible structures in the cryo-EM or X-ray crystallography data but the presence of gB N-terminal peptides were confirmed by mass spectrometry. Notably, N-terminal residues 109 KSQD 112 were predicted to form a small α-helix and alanine substitution of these residues abolished cell-cell fusion in a virus-free assay. Importantly, transferring the 109 AAAA 112 mutation into the VZV genome significantly impaired viral propagation. These data establish a functional role for the gB N-terminus in membrane fusion broadly relevant to the Herpesviridae .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Molecular Dynamics Investigation of Lipid-Specific Interactions with a Fusion Peptide

The HIV-1 fusion peptide, which is a short hydrophobic peptide from the gp41 coat glycoprotein that participates in the infection of a cell, interacts with model lipid bilayer membranes in a concentration-dependent manner. The interaction of the peptide with the bilayer also strongly depends on the lipid composition. Here, molecular dynamics simulations were performed to investigate lipid-specific interactions that arise shortly after the binding of a less-fusogenic variant of the HIV-1 fusion peptide to a lipid bilayer composed of a mixture of dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol. The impact of peptide concentration was also studied. An improved understanding was gained of the lipid-specific interactions experienced by the FP. New insight was also gained into how the peptide concentration changes these interactions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Structure of the Core Postfusion Porcine Endogenous Retrovirus Fusion Protein

Retroviral elements from endogenous retroviruses have functions in mammalian physiology. The best-known examples are the envelope proteins that function in placenta development and immune suppression. Porcine endogenous retroviruses (PERVs) are an understudied class of endogenous retroviruses that infect cultured human cells, raising concern regarding porcine xenografts. The PERV envelope glycoprotein has also been proposed as a possible swine syncytin with a role in placental development. Despite the growing interest in PERVs, their envelope glycoproteins remain poorly characterized. Here, we successfully determined the postfusion crystal structure of the PERV core fusion ectodomain. The PERV fusion protein structure reveals a conserved class I viral fusion protein six-helix bundle. Biophysical experiments demonstrated that the thermodynamic stability of the PERV fusion protein secondary structure was the same at physiological and acidic pHs. A conserved surface analysis highlights the high degree of sequence conservation among retroviral fusogens in the chain reversal region that facilitates the large-scale conformational change required for membrane fusion. Further structural alignment of class I viral fusogens revealed a phylogenetic clustering that shows evolution into various lineages that correlate with virus mechanisms of cell entry. Our work indicates that structural dendrograms can be used to qualitatively infer insights into the fusion mechanisms of newly discovered class I viral fusogen structures.

59 BASIC BIOLOGICAL SCIENCES↗