Domain III from class II fusion proteins functions as a dominant-negative inhibitor of virus membrane fusion

Liao, Maofu; Kielian, Margaret

doi:10.1083/jcb.200507075

Cited by 120 publications

(210 citation statements)

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“…The proteins used in this study were N-terminally hexahistidine-tagged SFV domain III with the stem region and N-terminally hexahistidine-tagged DV domain III, previously termed SFV HDIIIS and DV HDIII (27). For simplicity, these proteins are referred to here as SFV DIII and DV DIII, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…To measure the interaction of SFV DIII with cell-bound virus (27), radiolabeled WT SFV or G91D or H230A mutant viruses were bound to cells for 90 min on ice with shaking and treated at pH 5.5 for 1 min at 37°C in the presence of 2 M SFV or DV DIII. Low-pH treatment was performed on ice for 0, 5, 10, or 30 s (see Fig.…”

Section: Methodsmentioning

confidence: 99%

“…For simplicity, these proteins are referred to here as SFV DIII and DV DIII, respectively. The proteins were generated by expression in Escherichia coli strain BL21(DE3), refolding in vitro, and purification on a Superdex G-75 gel filtration column (GE Healthcare) (27).…”

Section: Methodsmentioning

confidence: 99%

“…Given the important movement and packing of DIII during E1's rearrangement to the final homotrimer, we explored the use of exogenous DIII as a fusion inhibitor (27). We found that alphavirus or dengue virus DIII proteins can specifically bind to E1 or E during the low-pH-triggered fusion reaction.…”

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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: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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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“…Recombinant DIII has previously been shown to block both virus binding and E-mediated fusion in a number of flaviviruses (Chu et al, 2005;Hung et al, 2004;Liao & Kielian, 2005). To assess the relative ability of our MBP-DIII constructs to prevent virus entry, we performed infection in their presence and quantified virus infection by measuring viral non-structural protein 1 (NS1) expression in an in-cell Western assay.…”

Section: Antiviral Activity Of Mbp-diii Proteinsmentioning

confidence: 99%

Residues in domain III of the dengue virus envelope glycoprotein involved in cell-surface glycosaminoglycan binding

Watterson

Kobe

Young

2012

Journal of General Virology

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The dengue virus (DENV) envelope (E) protein mediates virus entry into cells via interaction with a range of cell-surface receptor molecules. Cell-surface glycosaminoglycans (GAGs) have been shown to play an early role in this interaction, and charged oligosaccharides such as heparin bind to the E protein. We have examined this interaction using site-directed mutagenesis of a recombinant form of the putative receptor-binding domain III of the DENV-2E protein expressed as an MBP (maltose-binding protein)-fusion protein. Using an ELISA-based GAG-binding assay, cell-based binding analysis and antiviral-activity assays, we have identified two critical residues, K291 and K295, that are involved in GAG interactions. These studies have also demonstrated differential binding between mosquito and human cells. INTRODUCTIONDengue virus (DENV) belongs to the medically important genus Flavivirus. Spread by the mosquito vector Aedes aegypti, DENV has re-emerged as the most globally significant arthropod-borne viral pathogen in terms of annual incidence and morbidity. Antiviral and vaccine development against DENV is therefore a priority (Mackenzie et al., 2004). There are four distinct serotypes of DENV (DENV-1-DENV-4) and reinfection with a heterologous serotype is associated with an increased risk of severe disease, thereby complicating effective vaccine strategies (WHO, 2009).Flaviviruses are small (50 nm), positive-strand RNA, enveloped viruses with a smooth, spherical morphology. The mature virion comprises three structural proteins: a capsid protein (C), which associates with the viral RNA genome; a membrane protein (M), which is derived from a structural precursor (prM); and the glycosylated envelope protein (E), which is the major surface protein and mediates both cell attachment and fusion of the viral and cellular membranes. The atomic structure of E has been solved for tick-borne encephalitis virus (TBEV), DENV-2, DENV-3, West Nile virus (WNV) and Japanese encephalitis virus (JEV) by X-ray crystallography of recombinant proteins comprising the first approximately 400 residues of E, referred to as the soluble E ectodomain (sE) (Kanai et al., 2006;Modis et al., 2003Modis et al., , 2005Rey et al., 1995; JEV E, PDB accession no. 3P54). This recombinant lacks the Cterminal membrane-spanning anchors and the associated 'stem' region. These structural studies demonstrated a conserved global fold across the entire family, suggesting a shared mode of action with respect to the two functional roles of receptor binding and membrane fusion. The structures also revealed that the E glycoprotein forms a head-to-tail homodimer and is composed of three distinct domains, referred to as domains I (DI), II (DII) and III (DIII), that reflect the earlier determined antigenic domains (Guirakhoo et al., 1989). DIII consists of the Cterminal region (residues 292-395) of sE, which adopts an immunoglobulin-like fold of seven anti-parallel b-sheets linked by flexible loops (Huang et al., 2008;Mukherjee et al., 2006;Volk et al., 2004 Volk et a...

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In VivoAnalysis of Membrane Fusion

Palfreyman

Jørgensen

2015

Encyclopedia of Life Sciences

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Membranes provide a barrier that allows chemical reactions to be isolated from the environment. The plasma membrane, for example, delineates self from nonself, and thus must have played an essential role in the evolution of life. Yet under numerous circumstances it is equally important that membranes be breached. Numerous forces oppose the spontaneous fusion of membranes; thus, specialized proteins have evolved to fuse membranes. The most well-understood fusion proteins are the viral fusion proteins and the SNARE proteins used in the secretory pathway. In addition, recent discoveries have lead to models for the fusion of organelles such as mitochondria and peroxisomes, as well as for cell-cell fusion. Despite the diverse structures of fusion proteins, it is possible that they function to drive membranes through a series of common lipid intermediates. Here we review the mechanisms of fusion for biological membranes, and highlight the similarities and differences in these processes.

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Domain III from class II fusion proteins functions as a dominant-negative inhibitor of virus membrane fusion

Cited by 120 publications

References 49 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

Residues in domain III of the dengue virus envelope glycoprotein involved in cell-surface glycosaminoglycan binding

In VivoAnalysis of Membrane Fusion

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