Sendai-viral HN and F glycoproteins as probes of plasma-membrane protein catabolism in HTC cells. Studies with fusogenic reconstituted Sendai-viral envelopes.
Ontology highlight
ABSTRACT:
SUBMITTER: Earl RT
PROVIDER: S-EPMC1147633 | biostudies-other | 1987 Feb
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The catabolism of (14)C-labelled plasma glycoprotein in rats was studied after injecting homologous plasma protein labelled in the N-acetylglucosamine and sialic acid moieties. In normal animals the catabolism was approximately described by a four-compartment model. The fractional rate of catabolism of the plasma-protein amino sugar was found to be 0.0305hr.(-1), corresponding to the degradation of 2.75mumoles/hr. The (14)C label was eliminated from the animals largely as carbon dioxide with a small proportion appearing in the urine. Freely circulating amino sugars or glycopeptides did not appear in the plasma as a result of the catabolic processes, and there was no evidence that the protein-bound amino sugars were reutilized in biosynthetic processes. A study of the distribution of (14)C label in the carcasses of animals 24hr. after injection provided evidence that the gastrointestinal tract accounted for 25-38% of the total catabolic pool; the lungs, kidneys, spleen and liver also appeared to contribute to catabolism. Studies were conducted with rats that had been treated with turpentine to induce an inflammatory reaction; the results could not be analysed kinetically, since the metabolism of plasma proteins in these animals did not appear to be in a steady state. The injected plasma protein disappeared from the intravascular pool more quickly than in normal animals, but there were no significant differences in the rates of excretion of the (14)C label.
| S-EPMC1184937 | biostudies-other
Project description:Gene therapy for cystic fibrosis (CF) is making encouraging progress into clinical trials. However, further improvements in transduction efficiency are desired. To develop a novel gene transfer vector that is improved and truly effective for CF gene therapy, a simian immunodeficiency virus (SIV) was pseudotyped with envelope proteins from Sendai virus (SeV), which is known to efficiently transduce unconditioned airway epithelial cells from the apical side. This novel vector was evaluated in mice in vivo and in vitro directed toward CF gene therapy. Here, we show that (i) we can produce relevant titers of an SIV vector pseudotyped with SeV envelope proteins for in vivo use, (ii) this vector can transduce the respiratory epithelium of the murine nose in vivo at levels that may be relevant for clinical benefit in CF, (iii) this can be achieved in a single formulation, and without the need for preconditioning, (iv) expression can last for 15 months, (v) readministration is feasible, (vi) the vector can transduce human air-liquid interface (ALI) cultures, and (vii) functional CF transmembrane conductance regulator (CFTR) chloride channels can be generated in vitro. Our data suggest that this lentiviral vector may provide a step change in airway transduction efficiency relevant to a clinical programme of gene therapy for CF.
| S-EPMC2889732 | biostudies-literature
Project description:Reconstituted Sendai-viral envelopes (RSVE) were fused with hepatoma tissue-culture (HTC) cells, thereby introducing viral membrane glycoproteins into the plasma membrane [Earl, Billett, Hunneyball & Mayer (1987) Biochem. J. 241, 801-807]. Fractionation of homogenized cells on Nycodenz gradients shows that much of the viral 125I-labelled HN and F proteins were rapidly sequestered into a dense fraction distinct from fractions containing plasma membrane, lysosomes and mitochondria. Electron microscopy (results not shown) indicates that the dense fraction contains nuclear residues, multivesicular structures, dense bodies and fibrous structures. Both the dense fraction and a hexosaminidase-enriched fraction contain trichloroacetic acid-insoluble radioactivity, including intact 125I-labelled viral proteins. The viral proteins are progressively transferred from the dense fraction to the hexosaminidase-enriched fraction; the transfer is retarded by 50 micrograms of leupeptin/ml. Trichloroacetic acid-soluble radiolabel is progressively released into the culture medium as the proteins are degraded. Within 5 h after transplantation of viral HN and F proteins into recipient cells, a proportion (approx. 45%) of the 125I-labelled glycoproteins cannot be extracted by sequentially treating cells with digitonin (1 mg/ml), Triton X-100 (1%, w/v) and 0.3 M-KI. HN and F proteins in the non-extractable residue are tightly associated with nuclear-intermediate-filament (vimentin) material, as shown by Western blots and electron microscopy. The viral proteins are progressively transferred out of the nuclear-intermediate-filament residue; the transfer is slowed when cells are cultured with leupeptin. The data are consistent with the notion that transplanted viral HN and F proteins are sequestered to a perinuclear site in tight association with intermediate filaments before transfer into the autophagolysosomal system for degradation.
| S-EPMC1147634 | biostudies-other
Project description:In the assembly of paramyxoviruses, interactions between viral proteins are presumed to be specific. The focus of this study is to elucidate the protein-protein interactions during the final stage of viral assembly that result in the incorporation of the viral envelope proteins into virions. To this end, we examined the specificity of HN incorporation into progeny virions by transiently transfecting HN cDNA genes into Sendai virus (SV)-infected cells. SV HN expressed from cDNA was efficiently incorporated into progeny Sendai virions, whereas Newcastle disease virus (NDV) HN was not. This observation supports the theory of a selective mechanism for HN incorporation. To identify the region on HN responsible for the selective incorporation, we constructed chimeric SV and NDV HN cDNAs and evaluated the incorporation of expressed proteins into progeny virions. Chimera HN that contained the SV cytoplasmic domain fused to the transmembrane and external domains of the NDV HN was incorporated to SV particles, indicating that amino acids in the cytoplasmic domain are responsible for the observed specificity. Additional experiments using the chimeric HNs showed that 14 N-terminal amino acids are sufficient for the specificity. Further analysis identified five consecutive amino acids (residues 10 to 14) that were required for the specific incorporation of HN into SV. These residues are conserved among all strains of SV as well as those of its counterpart, human parainfluenza virus type 1. These results suggest that this region near the N terminus of HN interacts with another viral protein(s) to lead to the specific incorporation of HN into progeny virions.
| S-EPMC110485 | biostudies-literature
Project description:Enveloped viruses, such as SARS-CoV-2, infect cells via fusion of their envelope with the host membrane. By employing molecular simulations to characterize viral envelopes, researchers can gain insights into key determinants of infection. Here, the Frontera supercomputer is leveraged for large-scale modeling and analysis of authentic viral envelopes, whose lipid compositions are complex and realistic. Visual Molecular Dynamics (VMD) with support for MPI is employed, overcoming previous computational limitations and enabling investigation into virus biology at an unprecedented scale. The techniques applied here to an authentic HIV-1 envelope at two levels of spatial resolution (29 million particles and 280 million atoms) are broadly applicable to the study of other viruses. The authors are actively employing these techniques to develop and characterize an authentic SARS-CoV-2 envelope. A general framework for carrying out scalable analysis of simulation trajectories on Frontera is presented, expanding the utility of the machine in humanity's ongoing fight against infectious diseases.
| S-EPMC7839976 | biostudies-literature
Project description:A lentiviral vector (LV) pseudotype derived from the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of a murine respirovirus (Sendai virus) facilitates efficient targeting of murine lung in vivo. Since targeting of the human lung will depend upon the availability and distribution of receptors used by F/HN, we investigated transduction of primary human airway cells differentiated at the air-liquid interface (ALI). We observed targeting of human basal, ciliated, goblet, and club cells, and using a combination of sialidase enzymes and lectins, we showed that transduction is dependent on the availability of sialylated glycans, including α2,3 sialylated N-acetyllactosamine (LacNAc). Transduction via F/HN was 300-fold more efficient than another hemagglutinin-based LV pseudotype derived from influenza fowl plague virus (HA Rostock), despite similar efficiency reported in murine airways in vivo. Using specific glycans to inhibit hemagglutination, we showed this could be due to a greater affinity of F/HN for α2,3 sialylated LacNAc. Overall, these results highlight the importance of identifying the receptors used in animal and cell-culture models to predict performance in the human airways. Given the reported prevalence of α2,3 sialylated LacNAc on human pulmonary cells, these results support the suitability of the F/HN pseudotype for human lung gene therapy applications.
| S-EPMC9304433 | biostudies-literature
Project description:Caenorhabditis elegans proteins AFF-1 and EFF-1 [C. elegans fusion family (CeFF) proteins] are essential for developmental cell-to-cell fusion and can merge insect cells. To study the structure and function of AFF-1, we constructed vesicular stomatitis virus (VSV) displaying AFF-1 on the viral envelope, substituting the native fusogen VSV glycoprotein. Electron microscopy and tomography revealed that AFF-1 formed distinct supercomplexes resembling pentameric and hexameric "flowers" on pseudoviruses. Viruses carrying AFF-1 infected mammalian cells only when CeFFs were on the target cell surface. Furthermore, we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinella spiralis and the chordate Branchiostoma floridae could also fuse mammalian cells. Thus, FF proteins are part of an ancient family of cellular fusogens that can promote fusion when expressed on a viral particle.
| S-EPMC3084904 | biostudies-literature
Project description:Paramyxoviruses are enveloped negative-strand RNA viruses that are significant human and animal pathogens. Most paramyxoviruses infect host cells via the concerted action of a tetrameric attachment protein (variously called HN, H, or G) that binds either sialic acid or protein receptors on target cells and a trimeric fusion protein (F) that merges the viral envelope with the plasma membrane at neutral pH. F initially folds to a metastable prefusion conformation that becomes activated via a cleavage event during cellular trafficking. Upon receptor binding, the attachment protein, which consists of a globular head anchored to the membrane via a helical tetrameric stalk, triggers a major conformation change in F which results in fusion of virus and host cell membranes. We recently proposed a model for F activation in which the attachment protein head domains move following receptor binding to expose HN stalk residues critical for triggering F. To test the model in the context of wild-type viral glycoproteins, we used a restricted-diversity combinatorial Fab library and phage display to rapidly generate synthetic antibodies (sAbs) against multiple domains of the paramyxovirus parainfluenza 5 (PIV5) pre- and postfusion F and HN. As predicted by the model, sAbs that bind to the critical F-triggering region of the HN stalk do not disrupt receptor binding or neuraminidase (NA) activity but are potent inhibitors of fusion. An inhibitory prefusion F-specific sAb recognized a quaternary antigenic site and may inhibit fusion by preventing F refolding or by blocking the F-HN interaction. Importance: The paramyxovirus family of negative-strand RNA viruses cause significant disease in humans and animals. The viruses bind to cells via their receptor binding protein and then enter cells by fusion of their envelope with the host cell plasma membrane, a process mediated by a metastable viral fusion (F) protein. To understand the steps in viral membrane fusion, a library of synthetic antibodies to F protein and the receptor binding protein was generated in bacteriophage. These antibodies bound to different regions of the F protein and the receptor binding protein, and the location of antibody binding affected different processes in viral entry into cells.
| S-EPMC4178754 | biostudies-literature
Project description:Defective viral genomes (DVGs) are generated during virus replication. DVGs bearing complementary ends are strong inducers of dendritic cell (DC) maturation and of the expression of antiviral and pro-inflammatory cytokines by triggering signaling of the RIG-I family of intracellular pattern recognition receptors. Our data show that DCs stimulated with virus containing DVGs have an enhanced ability to activate human T cells and can induce adaptive immunity in mice. In addition, we describe the generation of a short Sendai virus (SeV)-derived DVG RNA (DVG-324) that maintains strong immunostimulatory activity in vitro and in vivo. DVG-324 induced high levels of Ifnb expression when transfected into cells and triggered fast expression of pro-inflammatory cytokines and mobilization of dendritic cells when injected into the footpad of mice. Importantly, DVG-324 enhanced the production of antibodies to a prototypic vaccine after a single intramuscular immunization in mice. Notably, the pro-inflammatory cytokine profile induced by DVG-324 was different from that induced by poly I:C, the only viral RNA analog currently used as an immunostimulant in vivo, suggesting a distinct mechanism of action. SeV-derived oligonucleotides represent novel alternatives to be harnessed as potent adjuvants for vaccination.
| S-EPMC4406099 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2022-12-15 | GSE179476 | GEO