A Stable Prefusion Intermediate of the Alphavirus Fusion Protein Reveals Critical Features of Class II Membrane Fusion

Martin, Carolyn; Sosa, Hernando; Kielian, Margaret

doi:10.1016/j.chom.2008.10.012

Cited by 42 publications

(101 citation statements)

References 32 publications

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“…This agrees with the postfusion trimer structure, which demonstrates packing of DIII into the groove formed between two E1 proteins in the trimer (14). While it is possible that dimers are generated during the core trimer assembly process, our electron microscopy studies of DI/DII membrane interactions have not detected dimers (40,49). Thus, our current and previous data strongly support the idea that the initial DIII target is a membrane-inserted extended E1 trimer.…”

Section: Discussionsupporting

confidence: 79%

“…In the presence of excess DIII, the E1 intermediate exhibited neither SDS nor trypsin resistance, presumably reflecting a complete block in fold-back of endogenous DIII. Previous in vitro experiments showed that DIII binds to trimers of E1 DI/II proteins but not to monomers (40). This agrees with the postfusion trimer structure, which demonstrates packing of DIII into the groove formed between two E1 proteins in the trimer (14).…”

Section: Discussionsupporting

confidence: 75%

“…Although G91D E1 inserts into target membranes and forms the extended trimer, intratrimer interactions of the fusion loops may be disrupted. It is also possible that cooperative interfusion loop interactions normally help to stabilize the core trimer and are disrupted in the mutant (40). While we cannot distinguish among these possibilities at this point, our data demonstrate an important role of the fusion loops in trimer stability and the reversibility of the initial extended E1 trimer in the G91D mutant.…”

Section: Discussionmentioning

confidence: 56%

“…No cross-inhibition of alphaviruses by dengue DIII (or vice versa) is observed. Using an in vitro reconstitution approach, we showed that a truncated form of E1 containing domains I and II and the linker region (DI/II) could form stable trimers on target membranes at low pH (40). These core trimers act as an efficient target for DIII binding, whereas monomeric DI/II does not stably bind DIII.…”

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confidence: 99%

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The Interaction of Alphavirus E1 Protein with Exogenous Domain III Defines Stages in Virus-Membrane Fusion

Roman-Sosa

Kielian

2011

J Virol

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Alphaviruses such as Semliki Forest virus (SFV) are enveloped viruses that infect cells through a low-pHtriggered membrane fusion reaction mediated by the transmembrane fusion protein E1. E1 drives fusion by insertion of its hydrophobic fusion loop into the cell membrane and refolding to a stable trimeric hairpin. In this postfusion conformation, the immunoglobulin-like domain III (DIII) and the stem region pack against the central core of the trimer. Membrane fusion and infection can be specifically inhibited by exogenous DIII, which binds to an intermediate in the E1 refolding pathway. Here we characterized the properties of the E1 target for interaction with exogenous DIII. The earliest target for DIII binding was an extended membraneinserted E1 trimer, which was not detectable by assays for the stable postfusion hairpin. DIII binding provided a tool to detect this extended trimer and to define a series of SFV fusion-block mutants. DIII binding studies showed that the mutants were blocked in distinct steps in fusion protein refolding. Our results suggested that formation of the initial extended trimer was reversible and that it was stabilized by the progressive fold-back of the DIII and stem regions.The alphaviruses are members of a genus of small spherical enveloped viruses with positive-sense RNA genomes (reviewed in reference 23). Alphaviruses include a number of medically important pathogens such as Eastern equine encephalitis virus and the emerging pathogen chikungunya virus, which has caused recent epidemics in India (10, 41, 43). Although human infections by pathogenic alphaviruses are increasing, to date there are no vaccines or antiviral therapies available for use in treatment of patients. Well-characterized alphaviruses such as Semliki Forest virus (SFV) and Sindbis virus have been used extensively to study the structure, entry, replication, and biogenesis of this important group of viruses (23).The alphavirus particle contains an inner core of the viral RNA in a complex with the capsid protein (23). This is surrounded by a lipid membrane containing the transmembrane E2 and E1 proteins, organized as trimers of E2 and E1 (E2/E1) heterodimers and arranged with T ϭ 4 icosahedral symmetry. Alphaviruses infect host cells by binding to receptors at the plasma membrane followed by uptake via clathrin-mediated endocytosis (reviewed in reference 18). The low-pH environment of the endosome then triggers the fusion of the viral and endosome membranes to deliver the nucleocapsid into the cytosol. Endocytic uptake and virus infection are blocked by expression of dominant-negative versions of host proteins involved in endocytosis (e.g., see references 7 and 42), whereas fusion and virus infection are inhibited by neutralizing the low pH of endocytic vesicles (e.g., see references 9 and 16). During entry, the E2 protein binds the virus receptor(s) while E1 mediates membrane fusion.The structures of the E2/E1 heterodimer and the prefusion and postfusion structures of the E1 protein provide important information ab...

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Section: Discussionsupporting

confidence: 79%

Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 56%

mentioning

confidence: 99%

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The Interaction of Alphavirus E1 Protein with Exogenous Domain III Defines Stages in Virus-Membrane Fusion

Roman-Sosa

Kielian

2011

J Virol

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“…Sanchez-San Martin et al reported that the E1 envelope from Semliki Forest virus, was found to associate with membranes in an in vitro assay in its truncated (soluble) form by forming clusters of five and six trimers (49). Of more importance, using a combination of x-ray crystallography and cryo-EM, Gibbons et al found that the same E1 protein in Semliki Forest virus forms a ring of five trimers along the fivefold axis in an early step of membrane fusion, before the formation of a fusion pore or channel (48).…”

Section: Bivalent Antibody Bindingmentioning

confidence: 99%

Modeling the Role of Epitope Arrangement on Antibody Binding Stoichiometry in Flaviviruses

Ripoll

Khavrutskii

Wallqvist

et al. 2016

Biophysical Journal

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Cryo-electron-microscopy (cryo-EM) structures of flaviviruses reveal significant variation in epitope occupancy across different monoclonal antibodies that have largely been attributed to epitope-level differences in conformation or accessibility that affect antibody binding. The consequences of these variations for macroscopic properties such as antibody binding and neutralization are the results of the law of mass action-a stochastic process of innumerable binding and unbinding events between antibodies and the multiple binding sites on the flavivirus in equilibrium-that cannot be directly imputed from structure alone. We carried out coarse-grained spatial stochastic binding simulations for nine flavivirus antibodies with epitopes defined by cryo-EM or x-ray crystallography to assess the role of epitope spatial arrangement on antibody-binding stoichiometry, occupancy, and neutralization. In our simulations, all epitopes were equally competent for binding, representing the upper limit of binding stoichiometry that results from epitope spatial arrangement alone. Surprisingly, our simulations closely reproduced the relative occupancy and binding stoichiometry observed in cryo-EM, without having to account for differences in epitope accessibility or conformation, suggesting that epitope spatial arrangement alone may be sufficient to explain differences in binding occupancy and stoichiometry between antibodies. Furthermore, we found that there was significant heterogeneity in binding configurations even at saturating antibody concentrations, and that bivalent antibody binding may be more common than previously thought. Finally, we propose a structure-based explanation for the stoichiometric threshold model of neutralization.

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An automated pipeline to screen membrane protein 2D crystallization

Kim

Vink

et al. 2010

J Struct Funct Genomics

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Electron crystallography relies on electron cryomicroscopy of two-dimensional (2D) crystals and is particularly well suited for studying the structure of membrane proteins in their native lipid bilayer environment. To obtain 2D crystals from purified membrane proteins, the detergent in a protein-lipid-detergent ternary mixture must be removed, generally by dialysis, under conditions favoring reconstitution into proteoliposomes and formation of well-ordered lattices. To identify these conditions a wide range of parameters such as pH, lipid composition, lipid-to-protein ratio, ionic strength and ligands must be screened in a procedure involving four steps: crystallization, specimen preparation for electron microscopy, image acquisition, and evaluation. Traditionally, these steps have been carried out manually and, as a result, the scope of 2D crystallization trials has been limited. We have therefore developed an automated pipeline to screen the formation of 2D crystals. We employed a 96-well dialysis block for reconstitution of the target protein over a wide range of conditions designed to promote crystallization. A 96-position magnetic platform and a liquid handling robot were used to prepare negatively stained specimens in parallel. Robotic grid insertion into the electron microscope and computerized image acquisition ensures rapid evaluation of the crystallization screen. To date, 38 2D crystallization screens have been conducted for 15 different membrane proteins, totaling over 3000 individual crystallization experiments. Three of these proteins have yielded diffracting 2D crystals. Our automated pipeline outperforms traditional 2D crystallization methods in terms of throughput and reproducibility.

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A Stable Prefusion Intermediate of the Alphavirus Fusion Protein Reveals Critical Features of Class II Membrane Fusion

Cited by 42 publications

References 32 publications

The Interaction of Alphavirus E1 Protein with Exogenous Domain III Defines Stages in Virus-Membrane Fusion

The Interaction of Alphavirus E1 Protein with Exogenous Domain III Defines Stages in Virus-Membrane Fusion

Modeling the Role of Epitope Arrangement on Antibody Binding Stoichiometry in Flaviviruses

An automated pipeline to screen membrane protein 2D crystallization

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