3,651 research outputs found
Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells
No full textBackground
Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa) and a truncated form (p25, 25 kDa). Here we report the expression and purification of HIV Nef from transgenic tobacco.
Results
We designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure.
Conclusion
We have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue
Formation of a Unique Cluster of G-Quadruplex Structures in the HIV-1 nef Coding Region: Implications for Antiviral Activity
No full textG-quadruplexes are tetraplex structures of nucleic acids that can form in G-rich sequences. Their presence and functional role have been established in telomeres, oncogene promoters and coding regions of the human chromosome. In particular, they have been proposed to be directly involved in gene regulation at the level of transcription. Because the HIV-1 Nef protein is a fundamental factor for efficient viral replication, infectivity and pathogenesis in vitro and in vivo, we investigated G-quadruplex formation in the HIV-1 nef gene to assess the potential for viral inhibition through G-quadruplex stabilization. A comprehensive computational analysis of the nef coding region of available strains showed the presence of three conserved sequences that were uniquely clustered. Biophysical testing proved that G-quadruplex conformations were efficiently stabilized or induced by G-quadruplex ligands in all three sequences. Upon incubation with a G-quadruplex ligand, Nef expression was reduced in a reporter gene assay and Nef-dependent enhancement of HIV-1 infectivity was significantly repressed in an antiviral assay. These data constitute the first evidence of the possibility to regulate HIV-1 gene expression and infectivity through G-quadruplex targeting and therefore open a new avenue for viral treatment. © 2013 Perrone et al
Nef-containing exosomes deliver Nef to macrophages.
No full textA—Size distribution of the extracellular vesicles secreted by HEK293 cells determined by EM; Inset–EM micrograph of the vesicles; bar– 200 nm. B—Western blot for the exosomal marker Alix and Nef in cells and exosomes (exNef); C—Western blot for the indicated amounts of rNef and in a typical preparation of exNef (10 μg of exosomal protein); D, E–Time-course of exosome uptake quantitated by confocal microscopy; F–Time-course of exosome uptake quantitated by fluorimetry; percentage of added exosomes that was taken up is shown; G–Cells were incubated with exosomes for 48 h, excess exosomes was washed out and cells incubated for the indicated periods of time in exosome-free medum; retained fluorscence of the exosome stain PKH67 was assessed using confocal microscopy; H–Visualisation of Nef-GFP inside the cells after exposure to exNef-GFP (5 μg/ml of exosomal protein) after staining with anti-GFP antibody. Scale bars– 10 μm.</p
Exploiting knowledge of immune selection in HIV-1 to detect HIV-specific CD8 T-cell responses
No full textSince HLA-restricted cytotoxic T-cell responses select specific polymorphisms in HIV-1 sequences and HLA diversity is relatively static in human populations, we investigated the use of peptide epitopes based on sites of HLA-associated adaptation in HIV-1 sequences to stimulate and detect T-cell responses ex vivo. These "HLA-optimised" peptides captured more HIV-1 Nef-specific responses compared with overlapping peptides of a single consensus sequence, in interferon-γ enzyme linked immunospot assays. Sites of immune selection can reveal more immunogenic epitopes in HLA-diverse populations and offer insights into the nature of HLA-epitope targeting, which could be applied in vaccine design
varioustoxins/NEF-Pipelines: 0.1.65
No full text<h1>NEF-Pipelines</h1>
<p>A set of command line (currently... there maybe a gui later!) tools for maniulating [NEF] or NMR Exchange Format files which can be used to move NMR meta data [peaks, shifts, sequences etc] between NMR data processing programs as storable and reuable workfloes. The programs provide basic tools for manipulating nef file file structures including:</p>
<ul>
<li><strong>entries: </strong>renaming</li>
<li><strong>molecular chains</strong>: listing, renaming and cloning molecular chains</li>
<li><strong>save-frames [tables]</strong>: deleting, inserting, listing and pretty printing (tabulate)</li>
<li><strong>headers</strong> creation / updating NEF headers with correct UUIDs and history</li>
<li><strong>streaming</strong>: NEF files into a pipeline</li>
<li><strong>testing</strong>: self testing of NEF pipelines</li>
</ul>
<p>The generic commands provided are</p>
<ul>
<li><strong>chains</strong> - carry out operations on chains</li>
<li><strong>entry</strong> - carry out operations on the nef file entry</li>
<li><strong>frames</strong> - carry out operations on frames in nef frames</li>
<li><strong>header</strong> - add a header to the stream</li>
<li><strong>save</strong> - save the entries in the strean to a file / files or stdout...</li>
<li><strong>stream</strong> - stream a nef file</li>
<li><strong>test</strong> - run the test suite</li>
</ul>
<p>It also provides tools for importing and exporing non NEF files from the following programs [transcoding / translators]</p>
<ul>
<li><strong>csv</strong> - read [rdcs]</li>
<li><strong>echidna</strong> - read echidna data [peaks]</li>
<li><strong>fasta</strong> - read and write fasta sequences</li>
<li><strong>mars</strong> - read and write mars [shifts and sequences]</li>
<li><strong>nmrpipe</strong> - read nmrpipe [peaks shifts & sequencess]</li>
<li><strong>nmrstar</strong> - read NMR-STAR [sequences & shifts]</li>
<li><strong>nmrview</strong> - read and write nmrview [peaks, sequences & shifts]</li>
<li><strong>pales</strong> - read and write pales/dc [rdcs]</li>
<li><strong>pdbx</strong> - read pdb [sequences]</li>
<li><strong>peaks</strong> - carry out operations on nef peaks</li>
<li><strong>rpf</strong> - write rpf shifts</li>
<li><strong>shifty</strong> - write shifty [shifts]</li>
<li><strong>simulate</strong> - simulate data</li>
<li><strong>sink</strong> - read the current stream and don't write anything</li>
<li><strong>sparky</strong> - read sparky files [shifts]</li>
<li><strong>talos</strong> - read and write talos files [shifts & restraints]</li>
<li><strong>xcamshift</strong> - write xcamshift for xplor [shifts]</li>
<li><strong>xeasy</strong> - read xeasy files [flya dialect: sequence]</li>
<li><strong>xplor</strong> - read xplor [sequences, dihedral & distance restraints]</li>
</ul>
Nef-isocyanide-Perkow access to novel pyrazolone derivations containing a cyclic ketene dithioacetal moiety
No full textAlkyl (E)-2-(3-Alkyl-5-oxo-1-phenyl-1,5-dihydro-4H-pyrazol-4-ylidene)-5-(alkylamino)-1,3-dithiole-4-carboxylates have been obtained by condensation of 2-pyrazolin-5-ones with carbon disulfide followed by ring formation with phosphorylated hydroxyketenimines [generated in situ from Nef-isocyanide-Perkow reaction] in the presence of Et3N. The structure of target compounds was confirmed by X-ray diffraction study. The good yields of the products, diastereoselectivity, and lack of activators or metal promoters are the main advantages of this method. </p
Zeolin–Nef accumulates to much higher amounts than zein–Nef or Nef-zein
No full text(A) Proteins were extracted with reducing buffer from young leaves of independent transgenic lines of tobacco expressing zeolin–Nef (lines L, N, and F), zein–Nef (16, 17, and 13), or Nef-zein (1 and 11) or from wild-type tobacco (wt). Analysis was by SDS–PAGE followed by protein blot using anti-γ-zein antiserum. (B) Different amounts of commercial Flag–BAP or total protein extracts from young leaves of tobacco plants expressing zeolin–Nef (transgenic line F, see A) were analysed by SDS–PAGE followed by protein blot using anti-Flag antibodies. (C) RNA was extracted from plants transformed as in (A) (three independent transgenic lines each, identified by numbers or letters) and hybridized with a probe corresponding to the zein portion of zeolin DNA. The image at the bottom shows ethidium bromide staining of 28S RNA in each lane, as a control for gel loading differences. In (A), (B), and (C), letters and numbers at the top identify the different independent transgenic lines. (D) Proteins were extracted with reducing buffer from young leaves of wild-type tobacco (lane 1) or tobacco expressing zeolin–Nef (lane 2) or zein–Nef (lane 3). Analysis was by SDS–PAGE followed by protein blot using anti-Nef antibody. The position of intact zeolin–Nef (arrowhead) is marked. (E) Shorter exposure of the protein blot shown in (D). Numbers on the left indicate the positions of molecular mass markers, in kilodaltons (A and D) or kilobase pairs (C).<p><b>Copyright information:</b></p><p>Taken from "The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin"</p><p></p><p>Journal of Experimental Botany 2008;59(10):2815-2829.</p><p>Published online 6 Jun 2008</p><p>PMCID:PMC2486477.</p><p></p
Présentation
No full textNef Frédéric. Présentation. In: Histoire Épistémologie Langage, tome 5, fascicule 2, 1983. La sémantique logique : Problèmes d'histoire et de méthode, sous la direction de Frédéric Nef. p. 3
Présentation
No full textNef Frédéric. Présentation. In: Histoire Épistémologie Langage, tome 5, fascicule 2, 1983. La sémantique logique : Problèmes d'histoire et de méthode, sous la direction de Frédéric Nef. p. 3
Natural HIV-1 Nef Polymorphisms Impair SERINC5 Downregulation Activity
No full textHIV-1 Nef enhances virion infectivity by counteracting host restriction factor SERINC5; however, the impact of natural Nef polymorphisms on this function is largely unknown. We characterize SERINC5 downregulation activity of 91 primary HIV-1 subtype B nef alleles, including isolates from 45 elite controllers and 46 chronic progressors. Controller-derived Nef clones display lower ability to downregulate SERINC5 (median 80% activity) compared with progressor-derived clones (median 96% activity) (p = 0.0005). We identify 18 Nef polymorphisms associated with differential function, including two CTL escape mutations that contribute to lower SERINC5 downregulation: K94E, driven by HLA-B( *)08, and H116N, driven by the protective allele HLA-B( *)57. HIV-1 strains encoding Nef K94E and/or H116N display lower infectivity and replication capacity in the presence of SERINC5. Our results demonstrate that natural polymorphisms in HIV-1 Nef can impair its ability to internalize SERINC5, indicating that variation in this recently described function may contribute to differences in viral pathogenesis
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