scholarly journals In Vitro Reconstitution Reveals Key Intermediate States of Trimer Formation by the Dengue Virus Membrane Fusion Protein

2010 ◽  
Vol 84 (11) ◽  
pp. 5730-5740 ◽  
Author(s):  
Maofu Liao ◽  
Claudia Sánchez-San Martín ◽  
Aihua Zheng ◽  
Margaret Kielian
Keyword(s):  
Dengue Virus ◽  
Membrane Fusion ◽  
Transmembrane Protein ◽  
Fusion Reaction ◽  
Low Ph ◽  
Viral Envelope ◽  
Interaction Sites ◽  
E Protein ◽  
Hairpin Formation

ABSTRACT The flavivirus dengue virus (DV) infects cells through a low-pH-triggered membrane fusion reaction mediated by the viral envelope protein E. E is an elongated transmembrane protein with three domains and is organized as a homodimer on the mature virus particle. During fusion, the E protein homodimer dissociates, inserts the hydrophobic fusion loop into target membranes, and refolds into a trimeric hairpin in which domain III (DIII) packs against the central trimer. It is clear that E refolding drives membrane fusion, but the steps in hairpin formation and their pH requirements are unclear. Here, we have used truncated forms of the DV E protein to reconstitute trimerization in vitro. Protein constructs containing domains I and II (DI/II) were monomeric and interacted with membranes to form core trimers. DI/II-membrane interaction and trimerization occurred efficiently at both neutral and low pH. The DI/II core trimer was relatively unstable and could be stabilized by binding exogenous DIII or by the formation of mixed trimers containing DI/II plus E protein with all three domains. The mixed trimer had unoccupied DIII interaction sites that could specifically bind exogenous DIII at either low or neutral pH. Truncated DV E proteins thus reconstitute hairpin formation and define properties of key domain interactions during DV fusion.

scholarly journals pH-dependence of intermediate steps of membrane fusion induced by the influenza fusion peptide

2006 ◽  
Vol 396 (3) ◽  
pp. 557-563 ◽  
Author(s):  
Ding-Kwo Chang ◽  
Shu-Fang Cheng
Keyword(s):  
Membrane Fusion ◽  
Self Assembly ◽  
Temporal Order ◽  
Fusion Reaction ◽  
Ph Dependence ◽  
Fusion Peptide ◽  
Low Ph ◽  
Fusion Event ◽  
Insertion Depth ◽  
Hairpin Formation

Membrane fusion mediated by the influenza-virus fusion protein is activated by low pH via a cascade of reactions. Some processes among them are irreversible, such as helix hairpin formation of the ectodomain, whereas others are reversible, such as exposure of the fusion peptide. Using this property, we attempted to dissect, in temporal order, different stages of the fusion reaction involving the fusion peptide by an acidic–neutral–acidic pH cycle. The fluorescence-quenching data indicated that both insertion depth and self-assembly are pH-reversible. In addition, lipid mixing assay was demonstrated to be arrested by neutral pH. By contrast, membrane leakage was shown to be irreversible with respect to pH. Our results, along with those from other studies on the pH-dependence of membrane fusion, are used to build a model for the virus-mediated fusion event from the perspective of pH-reversibility.

scholarly journals Antiviral activity of silymarin and baicalein against dengue virus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zhao Xuan Low ◽  
Brian Ming OuYong ◽  
Pouya Hassandarvish ◽  
Chit Laa Poh ◽  
Babu Ramanathan
Keyword(s):  
Antiviral Activity ◽  
Dengue Virus ◽  
Viral Entry ◽  
Dengue Infection ◽  
Antiviral Agents ◽  
Antiviral Drug ◽  
Vero Cells ◽  
Viral Envelope ◽  
E Protein

AbstractDengue is an arthropod-borne viral disease that has become endemic and a global threat in many countries with no effective antiviral drug available currently. This study showed that flavonoids: silymarin and baicalein could inhibit the dengue virus in vitro and were well tolerated in Vero cells with a half-maximum cytotoxic concentration (CC50) of 749.70 µg/mL and 271.03 µg/mL, respectively. Silymarin and baicalein exerted virucidal effects against DENV-3, with a selective index (SI) of 10.87 and 21.34, respectively. Baicalein showed a better inhibition of intracellular DENV-3 progeny with a SI of 7.82 compared to silymarin. Baicalein effectively blocked DENV-3 attachment (95.59%) to the Vero cells, while silymarin prevented the viral entry (72.46%) into the cells, thus reducing viral infectivity. Both flavonoids showed promising antiviral activity against all four dengue serotypes. The in silico molecular docking showed that silymarin could bind to the viral envelope (E) protein with a binding affinity of − 8.5 kcal/mol and form hydrogen bonds with the amino acids GLN120, TRP229, ASN89, and THR223 of the E protein. Overall, this study showed that silymarin and baicalein exhibited potential anti-DENV activity and could serve as promising antiviral agents for further development against dengue infection.

scholarly journals Membrane fusion mutants of Semliki Forest virus.

1984 ◽  
Vol 98 (1) ◽  
pp. 139-145 ◽  
Author(s):  
M C Kielian ◽  
S Keränen ◽  
L Kääriäinen ◽  
A Helenius
Keyword(s):  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Endocytic Pathway ◽  
Wild Type ◽  
New Class ◽  
Weak Bases ◽  
Ph Probes

Previous reports have indicated that the entry of Semliki Forest virus (SFV) into cells depends on a membrane fusion reaction catalyzed by the viral spike glycoproteins and triggered by the low pH prevailing in the endosomal compartment. In this study the in vitro pH-dependent fusion of SFV with nuclease-filled liposomes has been used to select for a new class of virus mutants that have a pH-conditional defect. The mutants obtained had a threshold for fusion of pH 5.5 as compared with the wild-type threshold of 6.2, when assayed by polykaryon formation, fusion with liposomes, or fusion at the plasma membrane. They were fully capable of infecting cells under standard infection conditions but were more sensitive to lysosomotropic agents that increase the pH in acidic vacuoles of the endocytic pathway. The mutants were, moreover, able to penetrate and infect baby hamster kidney-21 cells at 20 degrees C, indicating that the endosomes have a pH below 5.5. The results confirm the involvement of pH-triggered fusion in SFV entry, emphasize the central role played by acidic endosomal vacuoles in this reaction, shed further light on the mechanism of SFV inhibition by lysosomotropic weak bases, and demonstrate the usefulness of mutant viruses as biological pH probes of the endocytic pathway.

scholarly journals The Antimalarial Compound Atovaquone Inhibits Zika and Dengue Virus Infection by Blocking E Protein-Mediated Membrane Fusion

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1475
Author(s):  
Mizuki Yamamoto ◽  
Takeshi Ichinohe ◽  
Aya Watanabe ◽  
Ayako Kobayashi ◽  
Rui Zhang ◽  
...  
Keyword(s):  
Virus Infection ◽  
Dengue Virus ◽  
Membrane Fusion ◽  
Zika Virus ◽  
Mammalian Cells ◽  
Renilla Luciferase ◽  
E Proteins ◽  
E Protein ◽  
Viral Lifecycle

Flaviviruses bear class II fusion proteins as their envelope (E) proteins. Here, we describe the development of an in vitro quantitative mosquito-cell-based membrane-fusion assay for the E protein using dual split proteins (DSPs). The assay does not involve the use of live viruses and allows the analysis of a membrane-fusion step independent of other events in the viral lifecycle, such as endocytosis. The progress of membrane fusion can be monitored continuously by measuring the activities of Renilla luciferase derived from the reassociation of DSPs during cell fusion. We optimized the assay to screen an FDA-approved drug library for a potential membrane fusion inhibitor using the E protein of Zika virus. Screening results identified atovaquone, which was previously described as an antimalarial agent. Atovaquone potently blocked the in vitro Zika virus infection of mammalian cells with an IC90 of 2.1 µM. Furthermore, four distinct serotypes of dengue virus were also inhibited by atovaquone with IC90 values of 1.6–2.5 µM, which is a range below the average blood concentration of atovaquone after its oral administration in humans. These findings make atovaquone a likely candidate drug to treat illnesses caused by Zika as well as dengue viruses. Additionally, the DSP assay is useful to study the mechanism of membrane fusion in Flaviviruses.

scholarly journals Furin Processing and Proteolytic Activation of Semliki Forest Virus

2003 ◽  
Vol 77 (5) ◽  
pp. 2981-2989 ◽  
Author(s):  
Xinyong Zhang ◽  
Martin Fugère ◽  
Robert Day ◽  
Margaret Kielian
Keyword(s):  
Conformational Changes ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Protein Fusion ◽  
Proteolytic Activation ◽  
Control Virus

ABSTRACT The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed “p62,” which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.

scholarly journals Low-pH-Dependent Fusion of Sindbis Virus with Receptor-Free Cholesterol- and Sphingolipid-Containing Liposomes

1999 ◽  
Vol 73 (10) ◽  
pp. 8476-8484 ◽  
Author(s):  
Jolanda M. Smit ◽  
Robert Bittman ◽  
Jan Wilschut
Keyword(s):  
Sindbis Virus ◽  
Core Protein ◽  
Fusion Reaction ◽  
Low Ph ◽  
Viral Envelope ◽  
Cell Entry ◽  
Receptor Interaction ◽  
Neutral Ph ◽  
Virus Fusion ◽  
Entry Mechanism

ABSTRACT There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH or uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60 to 85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid catalyzing the fusion process itself. Under low-pH conditions, the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated, with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike.

scholarly journals Discovery of Dengue Virus NS4B Inhibitors

2015 ◽  
Vol 89 (16) ◽  
pp. 8233-8244 ◽  
Author(s):  
Qing-Yin Wang ◽  
Hongping Dong ◽  
Bin Zou ◽  
Ratna Karuna ◽  
Kah Fei Wan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dengue Virus ◽  
Transmembrane Protein ◽  
Selective Inhibition ◽  
Denv Infection ◽  
Viral Pathogens ◽  
Replication Complex ◽  
First Time

ABSTRACTThe four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV bothin vitroandin vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC50], 10 to 80 nM) but not DENV-1 and -4 (EC50, >20 μM). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial “hit” (compound 1), leading to compound 14a, which has goodin vivopharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2- and -3-infected patients.IMPORTANCEDengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions in the world where it was not previously endemic. While there are several promising vaccine candidates in clinical trials, approved vaccines or antivirals are not yet available. Here we describe the identification and characterization of a spiropyrazolopyridone as a novel inhibitor of DENV by targeting the viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) bothin vitroandin vivo. Our results validate, for the first time, that NS4B inhibitors could potentially be developed for antiviral therapy for treatment of DENV infection in humans.

scholarly journals Computational approach for selection of epitope-based dengue vaccine targets

2018 ◽  
Vol 14 (4) ◽  
pp. 605-618
Author(s):  
Phuc Nguyen ◽  
Ly Le
Keyword(s):  
Amino Acids ◽  
T Cell ◽  
Dengue Virus ◽  
Rational Design ◽  
Protein Sequences ◽  
Computational Approach ◽  
Amino Acid Sequences ◽  
Universal Vaccine ◽  
E Protein

High antigenic variability in the envelope (E) protein of different virus strains has been a major obstacle in designing effective vaccines for Dengue virus (DENV). To maintain their biological function, some parts of viral proteins remain stable during evolution thus one possible approach to solve this problem is to recognize specific regions within different protein sequences of E that have the tendency to stay constant through evolution. These regions may possess some special attributes to become a vaccine candidate against dengue virus. In this study, a computational approach was utilized to identify and analyze highly conserved amino acid sequences of the DENV E protein. Sequences of 9 amino acids or more were specifically focused due to their immune-relevant as T-cell determinants. Different bioinformatics tools were responsible for revealing conserved regions in the DENV E protein and constructing the phylogenetic tree from the sequence database. The tools also predicted immunogenicity of the identified vaccine targets. Ultimately, two peptide regions of at least 9 amino acids were chosen due to their high conserved attribute in more than 95% of all collected DENV sequences. Moreover, both of them was found to be immune-relevant by their correspondence to known or putative HLA-restricted T cell determinants. The conserved attribute of these sequences through the entire analysis of this study supports their potential as candidates for further in vitro experiments for rational design a universal vaccine which has longer and broader impact.

scholarly journals Type- and Subcomplex-Specific Neutralizing Antibodies against Domain III of Dengue Virus Type 2 Envelope Protein Recognize Adjacent Epitopes

2007 ◽  
Vol 81 (23) ◽  
pp. 12816-12826 ◽  
Author(s):  
Soila Sukupolvi-Petty ◽  
S. Kyle Austin ◽  
Whitney E. Purtha ◽  
Theodore Oliphant ◽  
Grant E. Nybakken ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Surface Display ◽  
Viral Envelope ◽  
Yeast Surface Display ◽  
Neutralizing Activity ◽  
Denv Serotypes ◽  
E Protein ◽  
Lateral Ridge ◽  
Domain Iii

ABSTRACT Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in animals. Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and cross-reactive antibodies with different neutralizing potentials.

scholarly journals pH-induced alterations in the fusogenic spike protein of Semliki Forest virus.

1985 ◽  
Vol 101 (6) ◽  
pp. 2284-2291 ◽  
Author(s):  
M Kielian ◽  
A Helenius
Keyword(s):  
Membrane Fusion ◽  
Conformational Changes ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Viral Envelope ◽  
Acidic Ph ◽  
Binding Assays ◽  
Protease Digestion ◽  
Nonionic Detergent

The spike glycoproteins of Semliki Forest virus mediate membrane fusion between the viral envelope and cholesterol-containing target membranes under conditions of mildly acidic pH (pH less than 6.2). The fusion reaction is critical for the infectious cycle, catalyzing virus penetration from the acidic endosome compartment. To define the role of the viral spike glycoproteins in the fusion reaction, conformational changes in the spikes at acid pH were studied using protease digestion and binding assays to liposomes and nonionic detergent. A method was also developed to prepare fragments of both transmembrane subunit glycopolypeptides of the spike, E1 and E2, which lacked the hydrophobic anchor peptides. Unlike the intact spikes the fragments were monomeric and therefore useful for obtaining information on conformational changes in individual subunits. The results showed that both E1 and E2 undergo irreversible conformational changes at the pH of fusion, that the conformational change of E1 depends, in addition to acidic pH, on the presence of cholesterol, and that no major changes in the solubility properties of the spikes takes place. On the basis of these findings it was concluded that fusion involves both subunits of the spike and that E1 confers the stereo-specific sterol requirement. The results indicated, moreover, that acid-induced fusion of Semliki Forest virus differs in important respects from that of influenza virus, another well-defined model system for protein-mediated membrane fusion.

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