Rapid 2H NMR Transverse Relaxation of Perdeuterated Lipid Acyl Chains of Membrane with Bound Viral Fusion Peptide Supports Large-Amplitude Motions of These Chains That Can Catalyze Membrane Fusion

ABSTRACT Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic pH in the endocytic pathway. Fusion is mediated by the spike protein E1 subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion. We have characterized four monoclonal antibodies (MAbs) specific for the acid conformation of E1. These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptide. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map the binding site, we selected for virus mutants resistant to one of the MAbs, E1a-1. One virus isolate, SFV 4-2, showed reduced binding of three acid-specific MAbs including E1a-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 mutant was fully infectious, formed the E1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Decreased binding of MAb E1a-1 was observed under a wide range of assay conditions, strongly suggesting that the E1 G157R mutation directly affects the MAb binding site. These data thus localize an E1 region that is normally hidden in the neutral pH structure and becomes exposed as part of the reorganization of the spike protein to its fusion-active conformation.

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Characterization of a Highly Conserved Domain within the Severe Acute Respiratory Syndrome Coronavirus Spike Protein S2 Domain with Characteristics of a Viral Fusion Peptide

Journal of Virology ◽

10.1128/jvi.00079-09 ◽

2009 ◽

Vol 83 (15) ◽

pp. 7411-7421 ◽

Cited By ~ 102

Author(s):

Ikenna G. Madu ◽

Shoshannah L. Roth ◽

Sandrine Belouzard ◽

Gary R. Whittaker

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Membrane Fusion ◽

Cleavage Site ◽

Critical Role ◽

Helical Structure ◽

Fusion Peptide ◽

Proteolytic Cleavage ◽

Viral Fusion ◽

Fusion Peptides ◽

Pseudotyped Virion

ABSTRACT Many viral fusion proteins are primed by proteolytic cleavage near their fusion peptides. While the coronavirus (CoV) spike (S) protein is known to be cleaved at the S1/S2 boundary, this cleavage site is not closely linked to a fusion peptide. However, a second cleavage site has been identified in the severe acute respiratory syndrome CoV (SARS-CoV) S2 domain (R797). Here, we investigated whether this internal cleavage of S2 exposes a viral fusion peptide. We show that the residues immediately C-terminal to the SARS-CoV S2 cleavage site SFIEDLLFNKVTLADAGF are very highly conserved across all CoVs. Mutagenesis studies of these residues in SARS-CoV S, followed by cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for residues L803, L804, and F805 in membrane fusion. Mutation of the most N-terminal residue (S798) had little or no effect on membrane fusion. Biochemical analyses of synthetic peptides corresponding to the proposed S2 fusion peptide also showed an important role for this region in membrane fusion and indicated the presence of α-helical structure. We propose that proteolytic cleavage within S2 exposes a novel internal fusion peptide for SARS-CoV S, which may be conserved across the Coronaviridae.

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An Antibody Directed against the Fusion Peptide of Junín Virus Envelope Glycoprotein GPC Inhibits pH-Induced Membrane Fusion

Journal of Virology ◽

10.1128/jvi.02700-09 ◽

2010 ◽

Vol 84 (12) ◽

pp. 6119-6129 ◽

Cited By ~ 22

Author(s):

Joanne York ◽

Jody D. Berry ◽

Ute Ströher ◽

Qunnu Li ◽

Heinz Feldmann ◽

...

Keyword(s):

Cell Membrane ◽

Membrane Fusion ◽

Host Cell ◽

Envelope Glycoprotein ◽

Fusion Peptide ◽

Intermediate Form ◽

Viral Fusion ◽

Virus Envelope ◽

Host Cell Membrane ◽

Helix Bundle

ABSTRACT The arenavirus envelope glycoprotein (GPC) initiates infection in the host cell through pH-induced fusion of the viral and endosomal membranes. As in other class I viral fusion proteins, this process proceeds through a structural reorganization in GPC in which the ectodomain of the transmembrane fusion subunit (G2) engages the host cell membrane and subsequently refolds to form a highly stable six-helix bundle structure that brings the two membranes into apposition for fusion. Here, we describe a G2-directed monoclonal antibody, F100G5, that prevents membrane fusion by binding to an intermediate form of the protein on the fusion pathway. Inhibition of syncytium formation requires that F100G5 be present concomitant with exposure of GPC to acidic pH. We show that F100G5 recognizes neither the six-helix bundle nor the larger trimer-of-hairpins structure in the postfusion form of G2. Rather, Western blot analysis using recombinant proteins and a panel of alanine-scanning GPC mutants revealed that F100G5 binding is dependent on an invariant lysine residue (K283) near the N terminus of G2, in the so-called fusion peptide that inserts into the host cell membrane during the fusion process. The F100G5 epitope is located in the internal segment of the bipartite GPC fusion peptide, which also contains four conserved cysteine residues, raising the possibility that this fusion peptide may be highly structured. Collectively, our studies indicate that F100G5 identifies an on-path intermediate form of GPC. Binding to the transiently exposed fusion peptide may interfere with G2 insertion into the host cell membrane. Strategies to effectively target fusion peptide function in the endosome may lead to novel classes of antiviral agents.

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Membrane Interface-Interacting Sequences within the Ectodomain of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein: Putative Role during Viral Fusion

Journal of Virology ◽

10.1128/jvi.74.17.8038-8047.2000 ◽

2000 ◽

Vol 74 (17) ◽

pp. 8038-8047 ◽

Cited By ~ 127

Author(s):

Tatiana Suárez ◽

William R. Gallaher ◽

Aitziber Agirre ◽

Félix M. Goñi ◽

José L. Nieva

Keyword(s):

Human Immunodeficiency Virus ◽

Membrane Fusion ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Fusion Peptide ◽

Viral Fusion ◽

Membrane Interface ◽

Immunodeficiency Virus ◽

Transmembrane Anchor

ABSTRACT We have identified a region within the ectodomain of the fusogenic human immunodeficiency virus type 1 (HIV-1) gp41, different from the fusion peptide, that interacts strongly with membranes. This conserved sequence, which immediately precedes the transmembrane anchor, is not highly hydrophobic according to the Kyte-Doolittle hydropathy prediction algorithm, yet it shows a high tendency to partition into the membrane interface, as revealed by the Wimley-White interfacial hydrophobicity scale. We have investigated here the membrane effects induced by NH2-DKWASLWNWFNITNWLWYIK-CONH2(HIVc), the membrane interface-partitioning region at the C terminus of the gp41 ectodomain, in comparison to those caused by NH2-AVGIGALFLGFLGAAGSTMGARS-CONH2(HIVn), the fusion peptide at the N terminus of the subunit. Both HIVc and HIVn were seen to induce membrane fusion and permeabilization, although lower doses of HIVc were required for comparable effects to be detected. Experiments in which equimolar mixtures of HIVc and HIVn were used indicated that both peptides may act in a cooperative way. Peptide-membrane and peptide-peptide interactions underlying those effects were further confirmed by analyzing the changes in fluorescence of peptide Trp residues. Replacement of the first three Trp residues by Ala, known to render a defective gp41 phenotype unable to mediate both cell-cell fusion and virus entry, also abrogated the HIVc ability to induce membrane fusion or form complexes with HIVn but not its ability to associate with vesicles. Hydropathy analysis indicated that the presence of two membrane-partitioning stretches separated by a collapsible intervening sequence is a common structural motif among other viral envelope proteins. Moreover, sequences with membrane surface-residing residues preceding the transmembrane anchor appeared to be a common feature in viral fusion proteins of several virus families. According to our experimental results, such a feature might be related to their fusogenic function.

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The Coronavirus Spike Protein Is a Class I Virus Fusion Protein: Structural and Functional Characterization of the Fusion Core Complex

Journal of Virology ◽

10.1128/jvi.77.16.8801-8811.2003 ◽

2003 ◽

Vol 77 (16) ◽

pp. 8801-8811 ◽

Cited By ~ 605

Author(s):

Berend Jan Bosch ◽

Ruurd van der Zee ◽

Cornelis A. M. de Haan ◽

Peter J. M. Rottier

Keyword(s):

Fusion Protein ◽

Membrane Fusion ◽

Fusion Proteins ◽

Limited Proteolysis ◽

Fusion Peptide ◽

Spike Protein ◽

Class I ◽

Heptad Repeat ◽

Viral Fusion ◽

Viral Fusion Protein

ABSTRACT Coronavirus entry is mediated by the viral spike (S) glycoprotein. The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit. The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it. Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins. We investigated the role of these regions in coronavirus membrane fusion. Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in Escherichia coli. When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex. Both on their own and within the complex, the peptides were highly alpha helical. Electron microscopic analysis of the complex revealed a rod-like structure ∼14.5 nm in length. Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion. In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity. Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion. Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein.

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Understanding (coupled) large amplitude motions: the interplay of microwave spectroscopy, spectral modeling, and quantum chemistry

Physical Sciences Reviews ◽

10.1515/psr-2020-0037 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Ha Vinh Lam Nguyen ◽

Isabelle Kleiner

Keyword(s):

Fourier Transform ◽

Quantum Chemistry ◽

Large Amplitude ◽

Environmental Chemistry ◽

Broad Band ◽

Microwave Spectroscopy ◽

Rotational Spectrum ◽

Spectral Modeling ◽

Rotational Spectra ◽

Large Amplitude Motions

AbstractA large variety of molecules contain large amplitude motions (LAMs), inter alia internal rotation and inversion tunneling, resulting in tunneling splittings in their rotational spectrum. We will present the modern strategy to study LAMs using a combination of molecular jet Fourier transform microwave spectroscopy, spectral modeling, and quantum chemical calculations to characterize such systems by the analysis of their rotational spectra. This interplay is particularly successful in decoding complex spectra revealing LAMs and providing reference data for fundamental physics, astrochemistry, atmospheric/environmental chemistry and analytics, or fundamental researches in physical chemistry. Addressing experimental key aspects, a brief presentation on the two most popular types of state-of-the-art Fourier transform microwave spectrometer technology, i.e., pulsed supersonic jet expansion–based spectrometers employing narrow-band pulse or broad-band chirp excitation, will be given first. Secondly, the use of quantum chemistry as a supporting tool for rotational spectroscopy will be discussed with emphasis on conformational analysis. Several computer codes for fitting rotational spectra exhibiting fine structure arising from LAMs are discussed with their advantages and drawbacks. Furthermore, a number of examples will provide an overview on the wealth of information that can be drawn from the rotational spectra, leading to new insights into the molecular structure and dynamics. The focus will be on the interpretation of potential barriers and how LAMs can act as sensors within molecules to help us understand the molecular behavior in the laboratory and nature.

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