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Dengue is a rapidly emerging, mosquito-borne viral infection, with an estimated 400 million infections occurring annually. To gain insight into dengue immunity, we characterized 145 human monoclonal antibodies (mAbs) and identified a previously unknown epitope, the envelope dimer epitope (EDE), that bridges two envelope protein subunits that make up the 90 repeating dimers on the mature virion. The mAbs to EDE were broadly reactive across the dengue serocomplex and fully neutralized virus produced in either insect cells or primary human cells, with 50% neutralization in the low picomolar range. Our results provide a path to a subunit vaccine against dengue virus and have implications for the design and monitoring of future vaccine trials in which the induction of antibody to the EDE should be prioritized.

Glioblastoma (GBM) is the most prevalent adult brain tumor, with virtually no cure, and with a median overall survival of 15 months from diagnosis despite of the treatment. SNARE pro- teins mediate membrane fusion events in cells and are essential for many cellular process- es including exocytosis and neurotransmission, intracellular trafficking and cell migration. Here we show that the blockade of the SNARE protein Syntaxin 1 (Stx1) function impairs GBM cell proliferation. We show that Stx1 loss-of-function in GBM cells, through ShRNA lentiviral transduction, a Stx1 dominant negative and botulinum toxins, dramatically reduces the growth of GBM after grafting U373 cells into the brain of immune compromised mice. In- terestingly, Stx1 role on GBM progression may not be restricted just to cell proliferation since the blockade of Stx1 also reduces in vitro GBM cell invasiveness suggesting a role in several processes relevant for tumor progression. Altogether, our findings indicate that the blockade of SNARE proteins may represent a novel therapeutic tool against GBM.

The major envelope fusion protein F of the budded virus of baculoviruses consists of two disulfide-linked subunits: an N-terminal F2 subunit and a C-terminal, membrane-anchored F1 subunit. There is one cysteine in F2 and there are 15 cysteines in F1, but their role in disulfide linking is largely unknown. In this study, the inter- and intra-subunit disulfide bonds of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) F protein were analysed by site-directed mutagenesis. Results indicated that in a functional F protein, an inter-subunit disulfide bond exists between amino acids C108 (F2) and C241 (F1). When C241 was mutated, an alternative disulfide bond was formed between C108 and C232, rendering F non-functional. No inter-subunit bridge was observed in a double C232/C241 mutant of F1. C403 was not involved in the formation of inter-subunit disulfide bonding, but mutation of this amino acid decreased viral infectivity significantly, suggesting that it might be involved in intra-subunit disulfide bonds. The influence of reductant [tris(2-carboxyethyl) phosphine (TCEP)] and free-thiol inhibitors [4-acetamido-4′-maleimidylstilbene 2,2′-disulfonic acid (AMS) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)] on the infectivity of HearNPV was tested. The results indicated that TCEP greatly decreased the infection of HzAm1 cells by HearNPV. In contrast, AMS and DTNB had no inhibitory effect on viral infectivity. The data suggested that free thiol/disulfide isomerization was not likely to play a role in viral entry and infectivity.

To infect a cell, the Paramyxoviridae family of enveloped viruses relies on the coordinated action of a receptor-binding protein (variably HN, H, or G) and a more conserved metastable fusion protein (F) to effect membrane fusion and allow genomic transfer. Upon receptor binding, HN (H or G) triggers F to undergo an extensive refolding event to form a stable postfusion state. Little is known about the intermediate states of the F refolding process. Here, a soluble form of parainfluenza virus 5 F was triggered to refold using temperature and was footprinted along the refolding pathway using fast photochemical oxidation of proteins (FPOP). Localization of the oxidative label to solvent-exposed side chains was determined by high-resolution MS/MS. Globally, metastable prefusion F is oxidized more extensively than postfusion F, indicating that the prefusion state is more exposed to solvent and is more flexible. Among the first peptides to be oxidatively labeled after temperature-induced triggering is the hydrophobic fusion peptide. A comparison of peptide oxidation levels with the values of solvent-accessible surface area calculated from molecular dynamics simulations of available structural data reveals regions of the F protein that lie at the heart of its prefusion metastability. The strong correlation between the regions of F that experience greater-than-expected oxidative labeling and epitopes for neutralizing antibodies suggests that FPOP has a role in guiding the development of targeted therapeutics. Analysis of the residue levels of labeled F intermediates provides detailed insights into the mechanics of this critical refolding event.

Priming of cytotoxic T lymphocyte (CTL) activity with exogenous antigen requires introduction of the antigen into the MHC class I presentation pathway of antigen-presenting cells. In the present study, we used fusogenic reconstituted envelopes (virosomes), derived from influenza virus, as a carrier system for delivery of a synthetic soluble peptide corresponding to a major murine CTL epitope of the influenza virus nucleoprotein (NP). Virosomes containing encapsulated NP-peptide efficiently sensitized target cells for recognition by influenza-specific CTLs generated through priming of mice with infectious virus. Intramuscular immunization of mice with peptide-containing virosomes induced a potent class I MHC-restricted CTL response against influenza-infected target cells. By contrast, an equal dose of NP-peptide encapsulated in fusion-inactivated virosomes did not induce CTL activity, indicating an essential role of the membrane fusion activity of the virosomes in the induction of the response. Likewise, NP-peptide encapsulated in liposomes, NP-peptide mixed with empty virosomes and NP-peptide in IFA failed to induce a CTL response. These results demonstrate that fusion-active virosomes represent a promising delivery system for induction of class I MHC-restricted CTL activity with non-replicating viral antigens. (C) 2000 Elsevier Science Ltd. All rights reserved.

Priming of cytotoxic T lymphocyte (CTL) activity with exogenous antigen requires introduction of the antigen into the MHC class I presentation pathway of antigen-presenting cells. In the present study, we used fusogenic reconstituted envelopes (virosomes), derived from influenza virus, as a carrier system for delivery of a synthetic soluble peptide corresponding to a major murine CTL epitope of the influenza virus nucleoprotein (NP). Virosomes containing encapsulated NP-peptide efficiently sensitized target cells for recognition by influenza-specific CTLs generated through priming of mice with infectious virus. Intramuscular immunization of mice with peptide-containing virosomes induced a potent class I MHC-restricted CTL response against influenza-infected target cells. By contrast, an equal dose of NP-peptide encapsulated in fusion-inactivated virosomes did not induce CTL activity, indicating an essential role of the membrane fusion activity of the virosomes in the induction of the response. Likewise, NP-peptide encapsulated in liposomes, NP-peptide mixed with empty virosomes and NP-peptide in IFA failed to induce a CTL response. These results demonstrate that fusion-active virosomes represent a promising delivery system for induction of class I MHC-restricted CTL activity with non-replicating viral antigens. (C) 2000 Elsevier Science Ltd. All rights reserved.

Cationic liposomes, consisting of synthetic amphiphiles and a so-called helper lipid, rapidly form complexes with DNA, known as lipoplexes. When incubated with cells in culture, the DNA can be delivered into the cell and becomes expressed. Because of these properties, lipoplexes are considered a useful alternative for viral vectors for in vivo genetherapy. Yet, many hurdles have still to be taken. These an illustrated in the present overview, which briefly describes the critical steps involved in overall gene delivery in vitro, ranging from lipoplex formation to nuclear penetration and delivery of the desired gene.

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 Dr 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 El homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the El trimer represents the fusion-active conformation of the viral spike.

Hemagglutinin, the membrane fusion protein of influenza virus,is known to mediate a low-pH-dependent fusion reaction between the viral envelope and the limiting membrane of the endosomal cell compartment following cellular uptake of the virus particles by receptor-mediated endocytosis. Here we exploited this activity of hemagglutinin to achieve efficient gene delivery to cultured cells. Hemagglutinin was reconstituted in the presence of the monocationic lipid dioleoyldimethylammonium chloride (DODAC) to permit plasmid binding to the virosome surface. Virosomes with 30 mol% DODAC exhibited a distinct binding capacity plasmid without causing aggregation. The virosome an activity was not affected by the cationic lipid DODAC demonstrated by low-phr-dependent lipid mixing with erythrocyte ghosts. Efficient cell transfection of BHK-21 cells was observed with virosomes containing 30 mol% DODAC and plasmid encoding for p-galactosidase (pCMV; beta-gal) associated to their surface. The transfection activity observed was dependent on the functional activity of hemagglutinin. Contrary to DNA/cationic lipid complexes the transfection was not dependent on the cationic lipid to DNA charge ratio. Importantly, transfection of BHK-21 cells with pCMV beta-gal by DODAC-containing virosomes did not show any significant signs of cytotoxicity that is commonly observed with DNA/cationic lipid complexes. Together with the high levels of expression of the transgene this highlights the potential of DODAC-containing virosomes as a novel approach in nonviral gene transfer.

Many viral fusion proteins only become activated under mildly acidic condition (pH 4.5–6.5) close to the pKa of histidine side-chain protonation. Analysis of the sequences and structures of influenza HA (haemagglutinin) and flaviviral envelope glycoproteins has led to the identification of a number of histidine residues that are not only fully conserved themselves but have local environments that are also highly conserved [Kampmann, Mueller, Mark, Young and Kobe (2006) Structure 14, 1481–1487]. Here, we summarize studies aimed at determining the role, if any, that protonation of these potential switch histidine residues plays in the low-pH-dependent conformational changes associated with fusion activation of a flaviviral envelope protein. Specifically, we report on MD (Molecular Dynamics) simulations of the DEN2 (dengue virus type 2) envelope protein ectodomain sE (soluble E) performed under varied pH conditions designed to test the histidine switch hypothesis of Kampmann et al. (2006).

Vesicular stomatitis virus (VSV) was used as a model virus to study the processes involved in photoinactivation by aluminum phthalocyanine tetrasulfonate (AIPcS(4),) or silicon phthalocyanine HOSiPcOSi(CH3)(2)(CH2)(3)N(CH3)(2) (Pc4) and red light. Previously a very rapid decrease in the intracellular viral RNA synthesis after photodynamic treatment was observed. This decrease was correlated to different steps in the replication cycle. Binding of VSV to host cells and internalization were only slightly impaired and could be visualized by electron microscopy. The capability of the virus to fuse with membranes in an acidic endosomal environment was studied using both pyrene-labeled liposomes and a hemolysis assay as a model. These tests indicate a rapid decrease of fusion capacity after AIPcS(4), treatment, which correlated with the decrease in RNA synthesis, For Pc4 treatment no such correlation was found. The fusion process is the first step in the replication cycle, affected by AIPcS(4) treatment, but also in vitro RNA polymerase activity was previously shown to be inhibited. Inactivation of VSV by Pc4 treatment is apparently caused by damage to a variety of viral components. Photodynamic treatment of virus suspensions with both sensitizers causes formation of 8-oxo-7,8-dihydroguanosine in viral RNA as measured by HPLC with electrochemical detection. This damage might be partly responsible for inhibition of the in vitro viral RNA polymerase activity by photodynamic treatment.

In this study, the mechanism by which an amphipathic negatively charged peptide consisting of 11 amino acids (WAE) induces fusion of liposomal phosphatidylcholine membranes is investigated. WAE-induced fusion, which only occurs when the peptide is covalently attached to the bilayer, shows a highly remarkable dependence on naturally occurring phosphatidylcholine species. The initial rate of fusion increased in the order l-palmitoyl 2-arachidonoyl PC (PAPC) > 1-palmitoyl 2-oleoyl PC (POPC) > 1-stearoyl 2-oleoyl PC (SOPC) > dioleoyl PC (DOPC) > egg yolk PC. interestingly, the susceptibility of the various PC species toward WAE-induced fusion matched a similar order of increase in intrinsic lipid headgroup spacing of the target membrane. The degree of spacing, in turn, was found to be related to the extent by which the fluorescence quantum yield of the Trp residue increased, which occurred upon the interaction of WAE with target membranes. Therefore, these results demonstrate an enhanced ability for WAE to engage in hydrophobic interactions when headgroup spacing increases. Thus, this latter parameter most likely regulates the degree of penetration of WAE into the target membrane. Apart from penetrating, WAE oligomerizes at the site of fusion as revealed by monitoring the self-quenching of the fluorescently derivatized lipid anchor to which WAE is attached. Clustering appears specifically related to the process of membrane fusion and nor membrane aggregation. This is indicated by the fact that fusion and clustering, but not aggregation, display the same strict temperature dependence. However, evidence is presented indicating that clustering is an accompanying event rather than a prerequisite for fusion. The notion that various biologically relevant fusion phenomena are accompanied by protein clustering and the specific PC-species-dependent regulation of membrane fusion emphasize the biological significance of the peptide in serving as a model for investigating mechanisms of protein-induced fusion.

Negatively charged albumins (NCAs, with the prototypes Suc-HSA and Aco-HSA), modified proteins with a potent anti-HIV-1 activity in the nanomolar concentration range, were studied for several aspects of their antiviral mechanism. In addition we investigated the antiviral activity of combinations and covalent conjugates of these NCAs and the reverse transcriptase inhibitor AZT.Addition of NCAs to HIV-l-infected target cells in time-of-addition experiments could be delayed for 30 min after infection before significant loss of activity occurred. Syncytium formation of HIV-infected and uninfected T-cells was inhibited by the NCAs at concentrations of 1-4 mu g/ml. The gp120-mediated virus/cell binding, however, was only inhibited by the NCAs at a 10-fold higher concentrations. The combined data are compatible with a preferential influence on virus/cell fusion.A subsynergistic activity against HIV-I was observed with the non-covalent mixture of Aco-HSA and AZT, When AZT was covalently coupled to the NCAs, and added one hour after infection of the target cells, the anti-HIV-l activity of NCA-AZTMP was diminished by only 2-6-fold, while this was diminished at least 120-fold for the NCAs, Furthermore the addition of NCA-AZTMP could be delayed up to at least 3h after infection without loss of antiviral activity. It is concluded that AZT that was coupled to the NCAs significantly contributes to the overall antiviral activity of the conjugates leading to complementary effects.These results highlight the potential of using NCA-AZTMP as dual-targeting preparations against HIV-I in which both the carrier and the coupled drug contribute to the therapeutic efficacy acting at a different level in the replication cycle.

The peptide HIVarg, corresponding to a sequence of 23 amino acid residues at the N-terminus of HIV-1 gp41, has the capacity to induce fusion of large unilamellar vesicles (LUV) consisting of negatively charged or zwitterionic phospholipids. In the present study, we further characterize this destabilization and fusion process using LUV consisting of phosphatidylcholine, phosphatidylethanolamine and cholesterol (molar ratio, 1:1:1). Evidence for fusion includes a demonstration of membrane lipid mixing as well as mixing of aqueous vesicle contents. Kinetic analysis of the overall process of vesicle aggregation and fusion revealed that the rate constant of the fusion step per se increased dramatically with the peptide-to-lipid molar ratio, indicating that the peptide acts as a true fusogen. The peptide caused the release of small molecules (ANTS/DPX), whereas large solutes (FITC-dextran, MWav 19,600) were partly retained. The estimated critical number of peptides per vesicle necessary to release vesicle contents, M = 2-4, indicates that leakage does not involve the formation of classical pores. Infrared spectroscopy of the peptide in the presence of liposomes demonstrated that the equilibrium conformation of the membrane-hound peptide is an antiparallel beta-structure. This finding supports the notion that the HIV fusion peptide in a beta-conformation has the capacity to perturb vesicle bilayers, inducing initial permeabilization and subsequent membrane fusion.

Artificial membranes (liposomes) can interact with the equatorial segment (ES) of human spermatozoa, provided that the acrosome reaction (AR) has occurred [Arts, Kuiken, Jager and Hoekstra (1993) fur. J. Biochem. 217, 1001-1009]. Using fluorescently labelled liposomes, this interaction can be seen as either punctate fluorescence in the ES (lip-ESp), reflecting only bound liposomes, or as diffuse fluorescence in this region (lip-ESd), indicating that the liposomes have fused with the ES membrane. Only equatorial segments that still contain constituents of the acrosomal matrix have the capacity to bind liposomes and eventually to fuse with them. Since the exposure of such intact equatorial segments is the exclusive result of induction of the AR under physiological conditions, these results imply that liposomes can be used for the rapid detection of acrosome-reacted spermatozoa. The lip-ESp and lip-ESd patterns were shown to be reflections of two distinct properties of the ES. Proteolytic treatment after AR completely inhibited the formation of a lip-ESd pattern, whereas formation of the lip-ESp pattern was only marginally inhibited by the proteolytic treatment. The same results were obtained using antisperm antibodies, which did not react with acrosome-intact spermatozoa. Proteolytic treatment of spermatozoa before AR induction had no effect on the fusion capacity of the ES after subsequent AR, which implies that the putative fusion protein is not accessible before AR. Thus fusion of liposomes with the ES of human spermatozoa is mediated by a sperm protein(s), whereas the lip-ESp pattern is not likely to represent the liposome-binding stage that precedes the fusion step.

We calculate the Gaussian curvature modulus (k) over bar of a systematically coarse-grained (CG) one-component lipid membrane by applying the method recently proposed by Hu et al. [Biophys. J., 2012, 102, 1403] to the MARTINI representation of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). We find the value (k) over bar /k = -1.04 +/- 0.03 for the elastic ratio between the Gaussian and the mean curvature modulus and deduce (k) over bar (m)/k(m) approximate to -0.98 +/- 0.09 for the monolayer elastic ratio, where the latter is based on plausible assumptions for the distance z(0) of the monolayer neutral surface from the bilayer midplane and the spontaneous lipid curvature K-0m. By also analyzing the lateral stress profile sigma(0)(z) of our system, two other lipid types and pertinent data from the literature, we show that determining K-0m and (k) over bar through the first and second moment of sigma(0)(z) gives rise to physically implausible values for these observables. This discrepancy, which we previously observed for a much simpler CG model, suggests that the moment conditions derived from simple continuum assumptions miss the effect of physically important correlations in the lipid bilayer.

Four new phospholipid derivatives containing a beta-nitrostyrene unit linked to the phosphate headgroup have been synthesized and converted into unilamellar vesicles. The vesicles were characterized by freeze-fracture transmission electron microscopy (FFEM), cryo-scanning electron microscopy (cryo-SEM), light scattering, and differential scanning calorimetry (DSC). At pH 11.5 and at T

SNARE proteins play a crucial role in intracellular trafficking by catalyzing membrane fusion, but assigning SNAREs to specific intracellular transport routes is challenging with current techniques. We developed a novel Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM)-based technique allowing visualization of real-time local interactions of fluorescently tagged SNARE proteins in live cells. We used FRET-FLIM to delineate the trafficking steps underlying the release of the inflammatory cytokine interleukin-6 (IL-6) from human blood-derived dendritic cells. We found that activation of dendritic cells by bacterial lipopolysaccharide leads to increased FRET of fluorescently labeled syntaxin 4 with VAMP3 specifically at the plasma membrane, indicating increased SNARE complex formation, whereas FRET with other tested SNAREs was unaltered. Our results revealed that SNARE complexing is a key regulatory step for cytokine production by immune cells and prove the applicability of FRET-FLIM for visualizing SNARE complexes in live cells with subcellular spatial resolution. DOI: http://dx.doi.org/10.7554/eLife.23525.001, eLife digest Many processes in living cells involve membranes coming together and fusing. For example, white blood cells known as dendritic cells rely on membrane fusion to fight off infections. When a dendritic cell detects a bacterial infection, it releases signaling molecules called cytokines to recruit other immune cells that help to eliminate the bacteria. The cytokines are contained in membrane-bound packages inside the cell, called vesicles, and are transported outside when these vesicles fuse with the membrane that surrounds the dendritic cell. Proteins called SNAREs drive the fusion of a cell’s membranes. These proteins, which are found on both membranes that will fuse, entwine to form a tight complex that pulls the membranes together. Mammals have over 30 different SNARE proteins, and many scientists believe that specific transport routes within cells use distinct pairs of SNAREs. However, to date, it has been difficult to assign specific pairs of SNAREs to specific transport routes with existing techniques. Verboogen et al. have now engineered human dendritic cells to add labels onto their SNAREs that fluoresce if the proteins interact. This approach meant that the interactions could be tracked via a microscope. The experiments showed that exposing dendritic cells to a bacterial compound that stimulates the release of cytokines caused two SNARE proteins called syntaxin 4 and VAMP3 to interact more at the cell membrane. This indicates that syntaxin 4 and VAMP3 are important for the release of cytokines from these cells. This finding was supported by an additional experiment in which Verboogen et al. switched off the gene for VAMP3 in the dendritic cells and found that this reduced the amount of cytokines that were released. This new microscope-based approach will be useful for identifying the specific pairs of SNARE proteins that are needed for the release and transport of molecules – like hormones and enzymes – that are important in health and disease. DOI: http://dx.doi.org/10.7554/eLife.23525.002

Since reticulocytes have a high demand for iron, which is required for heme biosynthesis, these cells are highly specialized in the endocytosis of the iron carrier transferrin (Tf). From the resulting endocytic vesicles (EVs), iron is released and the vesicles rapidly return to the cell membrane where they fuse, causing the release of the apotransferrin. Due to a lack of other intracellular compartments, the endocytic vesicles can be readily isolated. In this study, we have investigated the fusogenic properties of EVs, using liposomes as target membranes. Membrane fusion was monitored by a lipid mixing assay based on the relief of fluorescence self-quenching, using octadecylrhodamine B-chloride (R18). Application of this procedure was verified and solidified by analysis of the fusion event by an independent lipid mixing assay, after in situ labeling of EVs, and by determination of the mixing of aqueous contents. We demonstrate that the endocytic vesicles are particularly prone to fuse with target membranes that contain dioleoylphosphatidylethanolamine (DOPE). Relative to DOPE, bilayers composed of phosphatidylserine or phosphatidylcholine show a reduced fusion activity with EV. The specific and strong inhibition of fusion by cyclosporin A and a peptide known to interfere with the propensity of DOPE to adopt the hexagonal HII phase suggests that the mechanism of fusion involves the ability of this lipid to readily adopt non-bilayer phases. ATP, GTP, and/or cytosol are not necessary to obtain fusion. However, trypsin treatment of the endocytic vesicles inhibits fusion, indicating the involvement of (a) protein(s) in the fusion event.

Since reticulocytes have a high demand for iron, which is required for heme biosynthesis, these cells are highly specialized in the endocytosis of the iron carrier transferrin (Tf). From the resulting endocytic vesicles (EVs), iron is released and the vesicles rapidly return to the cell membrane where they fuse, causing the release of the apotransferrin. Due to a lack of other intracellular compartments, the endocytic vesicles can be readily isolated. In this study, we have investigated the fusogenic properties of EVs, using liposomes as target membranes. Membrane fusion was monitored by a lipid mixing assay based on the relief of fluorescence self-quenching, using octadecylrhodamine B-chloride (R18). Application of this procedure was verified and solidified by analysis of the fusion event by an independent lipid mixing assay, after in situ labeling of EVs, and by determination of the mixing of aqueous contents. We demonstrate that the endocytic vesicles are particularly prone to fuse with target membranes that contain dioleoylphosphatidylethanolamine (DOPE). Relative to DOPE, bilayers composed of phosphatidylserine or phosphatidylcholine show a reduced fusion activity with EV. The specific and strong inhibition of fusion by cyclosporin A and a peptide known to interfere with the propensity of DOPE to adopt the hexagonal HII phase suggests that the mechanism of fusion involves the ability of this lipid to readily adopt non-bilayer phases. ATP, GTP, and/or cytosol are not necessary to obtain fusion. However, trypsin treatment of the endocytic vesicles inhibits fusion, indicating the involvement of (a) protein(s) in the fusion event.

Low-pH-induced membrane fusion of Semliki Forest virus (SFV) in a model system is mediated by sphingolipids in the target membrane; ceramide is the sphingolipid minimally required (J. L. Nieva, R. Bron, J. Corver, and J. Wilschut, EMBO J. 13:2797-2804, 1994). Here, using various ceramide analogs, we demonstrate that sphingolipid-dependent fusion of SFV with cholesterol-containing liposomes exhibits remarkable molecular specificity, the 3-hydroxyl group and the 4,5-trans carbon-carbon double bond of the sphingosine backbone being critical for the sphingolipid to mediate the process. This observation supports the notion that sphingolipids act as a cofactor in SFV fusion, interacting directly with the viral fusion protein to induce its ultimate fusion-active conformation.

The functions and morphology of cellular membranes are intimately related and depend not only on their protein content but also on the repertoire of lipids that comprise them. In the absence of in vivo data on lipid asymmetry in endomembranes, it has been argued that motors, scaffolding proteins or integral membrane proteins rather than non-lamellar bilayer lipids such as diacylglycerol (DAG), are responsible for shaping of organelles, local membrane curvature and fusion. The effects of direct alteration of levels of such lipids remain predominantly uninvestigated. Diacylglycerol (DAG) is a well documented second messenger. Here we demonstrate two additional conserved functions of DAG: a structural role in organelle morphology, and a role in localised extreme membrane curvature required for fusion for which proteins alone are insufficient. Acute and inducible DAG depletion results in failure of the nuclear envelope (NE) to reform at mitosis and reorganisation of the ER into multi-lamellar sheets as revealed by correlative light and electron microscopy and 3D reconstructions. Remarkably, depleted cells divide without a complete NE, and unless rescued by 1,2 or 1,3 DAG soon die. Attenuation of DAG levels by enzyme microinjection into echinoderm eggs and embryos also results in alterations of ER morphology and nuclear membrane fusion. Our findings demonstrate that DAG is an in vivo modulator of organelle morphology in mammalian and echinoderm cells, indicating a fundamental role conserved across the deuterostome superphylum.

Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fc gamma-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st) virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles.

Liposomes consisting of negatively charged phospholipids interact almost exclusively with the equatorial segment (ES) of human spermatozoa provided the cells have undergone the acrosome reaction (AR) [Arts, Kuiken, Jager and Hoekstra (1993) Eur. J. Biochem. 217, 1001-1009]. Using fluorescently tagged liposomes, this interaction can be observed by fluorescence microscopy, showing either a diffuse fluorescence in the ES region (pattern ESd, presumably reflecting membrane-incorporated lipids as a result of fusion) or a punctate fluorescence (pattern ESp, representing adhering liposomes). These distribution patterns remain unchanged during prolonged incubation, up to 40 min. Not only do these observations suggest the existence of fairly specific liposomal binding sites, associated with the ES region, but also that a barrier to lipid lateral diffusion seems to exist in the ES membrane. Using liposomes that contain fluorescent lipid analogues in either both leaflets or in the inner leaflet only, we demonstrate that this putative barrier entails both membrane leaflets. Treatment with EDTA caused fluorescence to spread from the ES towards other membrane domains. Since only spermatozoa displaying pattern ESd were affected by the chelator, the randomization was not caused by EDTA-induced fusion activity. Therefore, this observation provides further evidence that in spermatozoa displaying pattern ESd the fluorescent lipid analogues were incorporated in the ES membrane as a result of fusion. Furthermore, these experiments support the view of the existence of a transmembranous block to lipid lateral diffusion in the ES, the stability of which may be governed by bivalent cations.

Liposomes consisting of negatively charged phospholipids interact almost exclusively with the equatorial segment (ES) of human spermatozoa provided the cells have undergone the acrosome reaction (AR) [Arts, Kuiken, Jager and Hoekstra (1993) Eur. J. Biochem. 217, 1001-1009]. Using fluorescently tagged liposomes, this interaction can be observed by fluorescence microscopy, showing either a diffuse fluorescence in the ES region (pattern ESd, presumably reflecting membrane-incorporated lipids as a result of fusion) or a punctate fluorescence (pattern ESp, representing adhering liposomes). These distribution patterns remain unchanged during prolonged incubation, up to 40 min. Not only do these observations suggest the existence of fairly specific liposomal binding sites, associated with the ES region, but also that a barrier to lipid lateral diffusion seems to exist in the ES membrane. Using liposomes that contain fluorescent lipid analogues in either both leaflets or in the inner leaflet only, we demonstrate that this putative barrier entails both membrane leaflets. Treatment with EDTA caused fluorescence to spread from the ES towards other membrane domains. Since only spermatozoa displaying pattern ESd were affected by the chelator, the randomization was not caused by EDTA-induced fusion activity. Therefore, this observation provides further evidence that in spermatozoa displaying pattern ESd the fluorescent lipid analogues were incorporated in the ES membrane as a result of fusion. Furthermore, these experiments support the view of the existence of a transmembranous block to lipid lateral diffusion in the ES, the stability of which may be governed by bivalent cations.

This paper presents a study on the membrane fusion activity of a 23-residue synthetic peptide, representing the N-terminus of gp41 of the human immunodeficiency virus type I (HIV-1; LAV(1a) strain), in a model system involving large unilamellar vesicles (LUV) composed of the negatively charged 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG). The peptide (HIVarg) induced fusion of POPG LUV as evidenced by (i) mixing of membrane lipids, (ii) mixing of aqueous vesicle contents, and (iii) an irreversible increase in vesicle size. Fusion could be induced only in the presence of millimolar concentrations of Ca2+ or Mg2+ needed for induction of vesicle aggregation; the divalent cations by themselves did not induce any fusion. The rate constant of the fusion reaction, as determined by simulation of the process according to a kinetic model, increased dramatically with the peptide-to-lipid molar ratio, indicating that the peptide was the mediator of the process. In the absence of divalent cations, the HIVarg peptide induced leakage of small molecules due to formation of pores in the membrane of single vesicles. Final extents and kinetics of this leakage process could be simulated adequately by model calculations for peptide-to-lipid ratios ranging from 1:25 to 1:750. Experiments, in which the order of peptide and Ca2+ addition to the vesicles was varied, indicated that the peptide is likely to adopt two different structures, one in the absence of Ca2+, primarily supporting leakage by formation of pores in separate vesicles, and one in the presence of Ca2+, primarily supporting fusion. Once a final structure had been established, it persisted even upon addition or removal of Ca2+. From the infrared spectroscopy of the peptide at equilibrium with POPG vesicles it is concluded that the structure formed in the absence of Ca2+, supporting leakage, represents predominantly an alpha-helix, whereas in the presence of Ca2+, i.e., under conditions supporting fusion, the peptide adopts mostly an extended antiparallel beta-structure.

International audience; At the synapse, vesicles stably dock at the active zone. However, in cellular membranes, proteins undergo a diffusive motion. It is not known how the motion of membrane proteins involved in vesicle exocytosis is compatible with both vesicle docking and the dynamic remodeling of the plasma membrane imposed by cycles of exocytosis and endocytosis. To address this question, we studied the motion of the presynaptic membrane protein syntaxin1A at both the population and single-molecule levels in primary cultures of rat spinal cord neurons. Syntaxin1A was rapidly exchanged between synaptic and extrasynaptic regions. Changes in syntaxin1A mobility were associated with interactions related to the formation of the exocytotic complex. Finally, we propose a reaction-diffusion model reconciling the observed diffusive properties of syntaxin at the population level and at the molecular level. This work allows us to describe the diffusive behavior and kinetics of interactions between syntaxin1A and its partners that lead to its transient stabilization at the synapse.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser(187) of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser(187) of SNAP-25 with Ala using "knock-in" technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects., Article, PLOS ONE. 6(9):e25158 (2011)

Contains fulltext : 95996.pdf (Publisher’s version ) (Open Access) Resistance mutations to the HIV-1 fusion inhibitor enfuvirtide emerge mainly within the drug's target region, HR1, and compensatory mutations have been described within HR2. The surrounding envelope (env) genetic context might also contribute to resistance, although to what extent and through which determinants remains elusive. To quantify the direct role of the env context in resistance to enfuvirtide and in viral infectivity, we compared enfuvirtide susceptibility and infectivity of recombinant viral pairs harboring the HR1-HR2 region or the full Env ectodomain of longitudinal env clones from 5 heavily treated patients failing enfuvirtide therapy. Prior to enfuvirtide treatment onset, no env carried known resistance mutations and full Env viruses were on average less susceptible than HR1-HR2 recombinants. All escape clones carried at least one of G36D, V38A, N42D and/or N43D/S in HR1, and accordingly, resistance increased 11- to 2800-fold relative to baseline. Resistance of full Env recombinant viruses was similar to resistance of their HR1-HR2 counterpart, indicating that HR1 and HR2 are the main contributors to resistance. Strictly X4 viruses were more resistant than strictly R5 viruses, while dual-tropic Envs featured similar resistance levels irrespective of the coreceptor expressed by the cell line used. Full Env recombinants from all patients gained infectivity under prolonged drug pressure; for HR1-HR2 viruses, infectivity remained steady for 3/5 patients, while for 2/5 patients, gains in infectivity paralleled those of the corresponding full Env recombinants, indicating that the env genetic context accounts mainly for infectivity adjustments. Phylogenetic analyses revealed that quasispecies selection is a step-wise process where selection of enfuvirtide resistance is a dominant factor early during therapy, while increased infectivity is the prominent driver under prolonged therapy.

A variety of approaches can be applied to investigate the multiple steps and interactions that occur during virus entry into the host cell. Single-virus tracking is a powerful real-time imaging technique that offers the possibility to monitor virus-cell binding, internalization, intracellular trafficking behavior, and the moment of membrane fusion of single virus particles in living cells. Here we describe the development and applications of a single-virus tracking assay based on the use of DiD-labeled dengue virus (DENV) in BS-C-1 cells. In addition - and using the same experimental setup - we present a binding and fusion assay that can be used to obtain a rapid insight into the relative extent of virus binding to the cell surface and membrane fusion. Details of virus labeling and characterization, microscopy setup, protocols, data analysis, and hints for troubleshooting are described throughout the paper. (C) 2011 Elsevier Inc. All rights reserved.

International audience; Proteins of the Argonaute family are small RNA carriers that guide regulatory complexes to their targets. The family comprises two major subclades. Members of the Ago subclade, which are present in most eukaryotic phyla, bind different classes of small RNAs and regulate gene expression at both transcriptional and post-transcriptional levels. Piwi subclade members appear to have been lost in plants and fungi and were mostly studied in metazoa, where they bind piRNAs and have essential roles in sexual reproduction. Their presence in ciliates, unicellular organisms harbouring both germline micronuclei and somatic macronuclei, offers an interesting perspective on the evolution of their functions. Here, we report phylogenetic and functional analyses of the 15 Piwi genes from Paramecium tetraurelia. We show that four constitutively expressed proteins are involved in siRNA pathways that mediate gene silencing throughout the life cycle. Two other proteins, specifically expressed during meiosis, are required for accumulation of scnRNAs during sexual reproduction and for programmed genome rearrangements during development of the somatic macronucleus. Our results indicate that Paramecium Piwi proteins have evolved to perform both vegetative and sexual functions through mechanisms ranging from post-transcriptional mRNA cleavage to epigenetic regulation of genome rearrangements.

International audience; A distinctive feature of HCV is that its life cycle depends on lipoprotein metabolism. Viral morphogenesis and secretion follow the very low-density lipoprotein (VLDL) biogenesis pathway and, consequently, infectious HCV in the serum is associated with triglyceride-rich lipoproteins (TRL). Lipoprotein lipase (LPL) hydrolyzes TRL within chylomicrons and VLDL but, independently of its catalytic activity, it has a bridging activity, mediating the hepatic uptake of chylomicrons and VLDL remnants. We previously showed that exogenously added LPL increases HCV binding to hepatoma cells by acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate, while simultaneously decreasing infection levels. We show here that LPL efficiently inhibits cell infection with two HCV strains produced in hepatoma cells or in primary human hepatocytes transplanted into uPA-SCID mice with fully functional human ApoB-lipoprotein profiles. Viruses produced in vitro or in vivo were separated on iodixanol gradients into low and higher density populations, and the infection of Huh 7.5 cells by both virus populations was inhibited by LPL. The effect of LPL depended on its enzymatic activity. However, the lipase inhibitor tetrahydrolipstatin restored only a minor part of HCV infectivity, suggesting an important role of the LPL bridging function in the inhibition of infection. We followed HCV cell entry by immunoelectron microscopy with anti-envelope and anti-core antibodies. These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets. In the presence of LPL, HCV was retained at the cell surface. We conclude that LPL efficiently inhibits HCV infection by acting on TRL associated with HCV particles through mechanisms involving its lipolytic function, but mostly its bridging function. These mechanisms lead to immobilization of the virus at the cell surface. HCV-associated lipoproteins may therefore be a promising target for the development of new therapeutic approaches.

The aggregation of beta-cyclodextrin vesicles can be induced by an adamantyl-substituted zwitterionic guanidiniocarbonylpyrrole carboxylate guest molecule (1). Upon addition of 1 to the cyclodextrin vesicles at neutral pH, the vesicles aggregate (but do not fuse), as shown by using UV/Vis and fluorescence spectroscopy, dynamic light scattering, zeta-potential measurements, cryogenic transmission electron microscopy, and atomic force microscopy. Aggregation of the vesicles is induced by a twofold supramolecular interaction. First, the adamantyl group of 1 forms an inclusion complex with beta-cyclodextrin. Second, at neutral pH the guanidiniocarbonylpyrrole carboxylate zwitterion dimerizes through the formation of hydrogen-bonded ion pairs. Because the dimerization of 1 depends on the zwitterionic protonation state of 1, the aggregation of the cyclodextrin vesicles is also pH dependent; the cyclodextrin vesicles do not interact at pH 5 or 9, at which 1 is either cationic or anionic and, therefore, not self-complementary. These observations are consistent with molecular recognition of the vesicles through a combination of two different supramolecular interactions, that is, host guest inclusion and dimerization of zwitterions, at the bilayer membrane surface.

Two distinct envelope fusion proteins (EFPs) (GP64 and F) have been identified in members of the Baculoviridae family of viruses. F proteins are found in group II nucleopolyhedroviruses (NPVs) of alphabaculoviruses and in beta- and deltabaculoviruses, while GP64 occurs only in group I NPVs of alphabaculoviruses. It was proposed that an ancestral baculovirus acquired the gp64 gene that conferred a selective advantage and allowed it to evolve into group I NPVs. The F protein is a functional analogue of GP64, as evidenced from the rescue of gp64 -null Autographa californica multicapsid nucleopolyhedrovirus (MNPV) (AcMNPV) by F proteins from group II NPVs or from betabaculoviruses. However, GP64 failed to rescue an F-null Spodoptera exigua MNPV (SeMNPV) (group II NPV). Here, we report the successful generation of an infectious gp64 -rescued group II NPV of Helicoverpa armigera (vHaBacΔF-gp64). Viral growth curve assays and quantitative real-time PCR (Q-PCR), however, showed substantially decreased infectivity of vHaBacΔF-gp64 compared to the HaF rescue control virus vHaBacΔF-HaF. Electron microscopy further showed that most vHaBacΔF-gp64 budded viruses (BV) in the cell culture supernatant lacked envelope components and contained morphologically aberrant nucleocapsids, suggesting the improper BV envelopment or budding of vHaBacΔF-gp64. Bioassays using pseudotyped viruses with a reintroduced polyhedrin gene showed that GP64-pseudotyped Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) significantly delayed the mortality of infected H. armigera larvae.

The interaction of clathrin with large unilamellar vesicles of various lipid compositions has been examined at neutral pH. Clathrin induces leakage of contents of vesicles that contain the acidic phospholipid phosphatidylserine. Leakage is greatly enhanced by the presence of a relatively minor amount of cholesterol, but is inhibited by phosphatidylcholine. Resonance energy transfer measurements between tryptophan residues of the protein and a fluorescent lipid analog, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine incorporated into the liposomal bilayer, suggests a dynamic interaction of clathrin with the bilayer at neutral pH. This interaction includes a (partial) penetration of the protein into the lipid bilayer, as revealed by hydrophobic photoaffinity labeling with 3-(trifluoromethyl)-3-(m-[I-125]iodophenyl)-diazirine. The interaction of clathrin with lipid vesicles at neutral pH is inhibited when the protein is pretreated with trypsin or with the reducing agent dithiothreitol, suggesting that structural requirements govern clathrin-membrane interaction at these conditions. The physiological relevance of the present observations in light of vesiculation and endosomal maturation is discussed.

The influence of a transmembrane pH gradient on the Ca2+-induced fusion of phospholipid vesicles, containing free fatty acids, has been investigated. Large unilamellar vesicles composed of an equimolar mixture of cardiolipin, dioleoylphosphatidylcholine, and cholesterol, containing 20 mol % oleic acid, were employed. Fusion was measured using a kinetic assay for lipid mixing, based on fluorescence resonance energy transfer. At pH 7.5, but not at pH 6.0, in the absence of a pH gradient, oleic acid stimulates the fusion of the vesicles by shifting the Ca2+ threshold concentration required for aggregation and fusion of the vesicles from about 13 mM to 10 mM. In the presence of a pH gradient (at an external pH of 7.5 and a vesicle interior pH of 10.5), the vesicles exhibit fusion characteristics similar to vesicles that do not contain oleic acid at all, consistent with an effective sequestration of the fatty acid to the inner monolayer of the vesicle bilayer induced by the imposed pH gradient. The kinetics of the fusion process upon simultaneous generation of the pH gradient across the vesicle bilayer and initiation of the fusion reaction show that the inward movement of oleic acid in response to the pH gradient is extremely fast, occurring well within 1 s. Conversely, dissipation of an imposed pH gradient, by addition of a proton ionophore during the course of the fusion process, results in a rapid enhancement of the rate of fusion due to reequilibration of the oleic acid between the two bilayers leaflets.

Background Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles. Results Peroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extra-cellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies. Conclusion Our results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.

International audience; DNA/cationic lipid (lipoplexes), DNA/cationic polymer (polyplexes) and DNA/cationic polymer/cationic lipid (lipopolyplexes) electrostatic complexes are proposed as non-viral nucleic acids delivery systems. These DNA-nanoparticles are taken up by the cells through endocytosis processes, but the low capacity of DNA to escape from endosomes is regarded as the major limitations of their transfection efficiency. Here, we present a current report on a particular class of carriers including the polymers, peptides and lipids, which is based on the exploitation of the imidazole ring as an endosome destabilization device to favour the nucleic acids delivery in the cytosol. The imidazole ring of histidine is a weak base that has the ability to acquire a cationic charge when the pH of the environment drops bellow 6. As it has been demonstrated for poly(histidine), this phenomena can induce membrane fusion and/or membrane permeation in an acidic medium. Moreover, the accumulation of histidine residues inside acidic vesicles can induce a proton sponge effect, which increases their osmolarity and their swelling. The proof of concept has been shown with polylysine partially substituted with histidine residues that has caused a dramatic increase by 3-4.5 orders of magnitude of the transfection efficiency of DNA/polylysine polyplexes. Then, several histidine-rich polymers and peptides as well as lipids with imidazole, imidazolinium or imidazolium polar head have been reported to be efficient carriers to deliver nucleic acids including genes, mRNA or SiRNA in vitro and in vivo. More remarkable, histidylated carriers are often weakly cytotoxic, making them promising chemical vectors for nucleic acids delivery.

The interaction of the non-enveloped plant viruses TMV (rod-shaped) and CCMV (spherical) and of their coat proteins in several well-defined aggregation states, with artificial membranes was investigated to study the early stages of the cellular infection process. Information about the separate steps in the interaction mechanisms was obtained by employing three assays, performed as a function of vesicle size, net membrane charge, pH and ionic strength. The assays allow to discriminate between aggregation of vesicles (turbidity assay) and membrane destabilization (vesicle leakage assay and lipid mixing assay). The aggregation of the vesicles is a result of electrostatic interactions between the viral material and vesicles surface (cross-linking), while the destabilization of the membrane is a result of penetration or bilayer disruption by hydrophobic protein domains. TMV virions and its coat protein, and CCMV virons, due to their net negative charge, predominantly interact with positively charged membranes. The coat protein of CCMV was found to interact with negatively charged membranes, an interaction that can be assigned to its basical N-terminal sequence. Changing the aggregational state of the viral coat proteins yielded most significant interactions in case of TMV coat protein aggregated in the disk form and CCMV coat protein aggregated in empty capsids with oppositely charged membranes. These protein aggregates are found to be the best compromise between efficiency (capacity of the protein to bridge vesicles and destabilize their membranes) and concentration of protein aggregates. The results are discussed with respect to previously proposed biological models of the early stages of plant virus infection.

We have investigated the fusion behavior of a mixed vesicle system consisting of vesicles prepared from the simple synthetic surfactants di(n-dodecyl)phosphate (DDP) or di(n-tetradecyl)phosphate (DTP) and vesicles prepared from the phospholipids phosphatidylserine (PS) or dioleoylphosphatidylcholine (DOPC). Fusion between the vesicles, induced by Ca2+, was determined by a resonance energy transfer assay for lipid mixing, sucrose density gradient analysis, and electron microscopy. We demonstrate that synthetic surfactant vesicles can specifically engage in asymmetric fusion events, provided that the incubation temperature is kept below the gel-liquid crystalline phase-transition temperature (T(c)) of the synthetic amphiphile (29 and 48-degrees-C for DDP and DTP, respectively) and that the physical state of the target membrane is fluid. Asymmetric fusion of DDP or DTP vesicles was most efficient with PS vesicles, but it also occurred with zwitterionic PC vesicles. In the latter case, fusion proceeded spontaneously, but the process was markedly accelerated upon addition of Ca2+. Furthermore, in contrast to a massive transformation of bilayer into nonbilayer hexagonal H(II) tubular structures, as occurs upon symmetric Ca2+-induced fusion of DDP vesicles, asymmetric fusion with phospholipid bilayers predominantly leads to the formation of larger vesicles. This indicates that both PS and DOPC stabilize the DDP bilayer structure in the fusion product.

Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments., Author Summary Dengue virus (DENV) is the most common arthropod-borne infection worldwide with 50–100 million cases annually. Despite its high clinical impact, little is known about the infectious cell entry pathway of the virus. Previous studies have shown conflicting evidence about whether the virus fuses directly with the cell plasma membrane or enters cells by receptor-mediated endocytosis. In this manuscript, we dissect the cell entry pathway of DENV by tracking single fluorescently labeled DENV particles in living cells expressing various fluorescent cellular markers, using real-time multi-color fluorescence microscopy. We show that DENV particles are delivered to pre-existing clathrin-coated pits by diffusion along the cell surface. Following clathrin-mediated uptake, the majority of DENV particles are transported to early endosomes, which mature into late endosomes, where membrane fusion occurs. This is the first study that describes the cell entry process of DENV at the single particle level and therefore provides unique mechanistic and kinetic insights into the route of entry, endocytic trafficking behavior, and membrane fusion properties of individual DENV particles in living cells. This paper opens new avenues in flavivirus biology and will lead toward a better understanding of the critical determinants in DENV infection.

F proteins of group II nucleopolyhedroviruses (NPVs) are envelope fusion proteins essential for virus entry and egress. An F-null Helicoverpa armigera single nucleocapsid NPV (HearNPV) bacmid, HaBacF, was constructed. This bacmid could not produce infectious budded virus (BV) when transfected into HzAM1 cells, showing that F protein is essential for cell-to-cell transmission of BVs. When HaBacF was pseudotyped with the homologous F protein (HaBacF-HaF, positive control) or with the heterologous F protein from Spodoptera exigua multinucleocapsid NPV (SeMNPV) (HaBacF-SeF), infectious BVs were produced with similar kinetics. In the late phase of infection, the BV titre of HaBacF-SeF virus was about ten times lower than that of HaBacF-HaF virus. Both pseudotyped viruses were able to fuse HzAM1 cells in a similar fashion. The F proteins of both HearNPV and SeMNPV were completely cleaved into F1 and F2 in the BVs of vHaBacF-HaF and vHaBacF-SeF, respectively, but the cleavage of SeF in vHaBacF-SeF-infected HzAM1 cells was incomplete, explaining the lower BV titre of vHaBacF-SeF. Polyclonal antisera against HaF1 and SeF1 specifically neutralized the infection of vHaBacF-HaF and vHaBacF-SeF, respectively. HaF1 antiserum showed some cross-neutralization with vHaBacF-SeF. These results demonstrate that group II NPV F proteins can be functionally replaced with a homologue of other group II NPVs, suggesting that the interaction of F with other viral or host proteins is not absolutely species-specific.

International audience; Glycoprotein G of the vesicular stomatitis virus triggers membrane fusion via a low pH-induced structural rearrangement. Despite the equilibrium between the pre- and postfusion states, the structure of the prefusion form, determined to 3.0 angstrom resolution, shows that the fusogenic transition entails an extensive structural reorganization of G. Comparison with the structure of the postfusion form suggests a pathway for the conformational change. In the prefusion form, G has the shape of a tripod with the fusion loops exposed, which point toward the viral membrane, and with the antigenic sites located at the distal end of the molecule. A large number of G glycoproteins, perhaps organized as in the crystals, act cooperatively to induce membrane merging.

The F protein is the major glycoprotein present in the envelopes of budded virus (BV) of members of the family Baculoviridae. The F protein mediates low-pH-activated fusion with insect cell membranes. Baculovirus F proteins are synthesized as a precursor (F(0)) and cleaved post-translationally into two disulfide-bonded subunits, F(1) (C-terminal, large subunit) and F(2) (N-terminal, small subunit). Recently, N-linked glycosylation of the F(1) and F(2) subunits of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was demonstrated [Long, G., Westenberg, M., Wang, H., Vlak, J. M.Hu, Z. (2006). J Gen Virol 87, 839-846]. Sequence analysis frequently predicts that one or more N-linked glycosylation sites are present in the F(2) subunit of baculovirus F proteins. N-glycans on envelope fusion proteins are usually required for proper conformational integrity and biological function, such as infectivity. This study examined the importance of N-linked glycosylation of the F(2) subunit of HearNPV by site-directed mutagenesis. The only putative N-linked glycosylation site in F(2) was eliminated by mutating asparagine (N(104)) to glutamine (Q), resulting in the mutant HearNPV(fN104Q). When inserted into an f-null HearNPV and a gp64-null bacmid of Autographa californica multiple nucleopolyhedrovirus, infectious BV could be retrieved that contained unglycosylated F(2). The virulence of HearNPV(fN104Q) was enhanced, as BV was produced earlier after infection and yielded larger plaques than f-null HearNPV repaired with the wild-type f gene. HearNPV(fN104Q) BV also induced much more efficient low-pH-activated syncytium formation. These results indicate that N-linked glycosylation of the HearNPV baculovirus F(2) subunit is not essential for viral infectivity and suggest that it is involved in BV production and fusogenicity.

Group II nucleopolyhedroviruses (NPVs), e.g. Helicoverpa armigera (Hear) NPV and Spodoptera exigua (Se) MNPV (multiple NPV), lack a GP64-like protein that is present in group I NPVs, e.g. Autographa californica (Ac)MNPV, but have an unrelated envelope fusion protein named F. Three AcMNPV viruses were constructed by introducing AcMNPV gp64, HearNPV f or SeMNPV f genes, respectively, into a gp64-negative AcMNPV bacmid. Sf21 cells were incubated with different amounts of inactivated budded virus to occupy receptors and were subsequently infected with a fixed amount of infectious virus to compete for attachment. The results suggest that GP64 and F act on their own and use different receptors, while the two different F proteins exploit the same receptor. Additionally, gp64-null AcMNPV pseudotyped with baculovirus F was, in contrast to GP64, unable to transduce mammalian cells, indicating that mammalian cells do not possess baculovirus F protein receptors despite the structural similarity of baculovirus F to vertebrate viral fusion proteins.