18 research outputs found

    Identification of NS2 determinants stimulating intrinsic HCV NS2 protease activity

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    Hepatitis C Virus NS2-NS3 cleavage is mediated by NS2 autoprotease (NS2pro) and this cleavage is important for genome replication and virus assembly. Efficient NS2-NS3 cleavage relies on the stimulation of an intrinsic NS2pro activity by the NS3 protease domain. NS2pro activation depends on conserved hydrophobic NS3 surface residues and yet unknown NS2-NS3 surface interactions. Guided by an in silico NS2-NS3 precursor model, we experimentally identified two NS2 surface residues, F103 and L144, that are important for NS2pro activation by NS3. When analyzed in the absence of NS3, a combination of defined amino acid exchanges, namely F103A and L144I, acts together to increase intrinsic NS2pro activity. This effect is conserved between different HCV genotypes. For mutation L144I its stimulatory effect on NS2pro could be also demonstrated for two other mammalian hepaciviruses, highlighting the functional significance of this finding. We hypothesize that the two exchanges stimulating the intrinsic NS2pro activity mimic structural changes occurring during NS3-mediated NS2pro activation. Introducing these activating NS2pro mutations into a NS2-NS5B replicon reduced NS2-NS3 cleavage and RNA replication, indicating their interference with NS2-NS3 surface interactions pivotal for NS2pro activation by NS3. Data from chimeric hepaciviral NS2-NS3 precursor constructs, suggest that NS2 F103 is involved in the reception or transfer of the NS3 stimulus by NS3 P115. Accordingly, fine-tuned NS2-NS3 surface interactions are a salient feature of HCV NS2-NS3 cleavage. Together, these novel insights provide an exciting basis to dissect molecular mechanisms of NS2pro activation by NS3

    Crystal Structure of a Mirror-Image L-RNA Aptamer (Spiegelmer) in Complex with the Natural L-Protein Target CCL2

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    We report the crystal structure of a 40mer mirror-image RNA oligonucleotide completely built from nucleotides of the non-natural L-chirality in complex with the pro-inflammatory chemokine L-CLL2 (monocyte chemoattractant protein-1), a natural protein composed of regular L-amino acids. The L-oligonucleotide is an L-aptamer (a Spiegelmer) identified to bind L-CCL2 with high affinity, thereby neutralizing the chemokine’s activity. CCL2 plays a key role in attracting and positioning monocytes; its overexpression in several inflammatory diseases makes CCL2 an interesting pharmacological target. The PEGylated form of the L-aptamer, NOX-E36 (emapticap pegol), already showed promising efficacy in clinical Phase II studies conducted in diabetic nephropathy patients. The structure of the L-oligonucleotide·L-protein complex was solved and refined to 2.05 Å. It unveils the L-aptamer’s intramolecular contacts and permits a detailed analysis of its structure–function relationship. Furthermore, the analysis of the intermolecular drug–target interactions reveals insight into the selectivity of the L-aptamer for certain related chemokines

    Peptide NMHRYPNQ of the cellular prion protein (PrP(C)) inhibits aggregation and is a potential key for understanding prion-prion interactions.

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    Pathogenesis of transmissible spongiform encephalopathies is correlated with a conversion of the normal cellular form of the prion protein (PrP(C)) into the abnormal isoform (scrapie form of PrP). Contact of the normal PrP with its abnormal isoform, the scrapie form of PrP, induces the transformation. Knowledge of molecules that inhibit such contacts leads to an understanding of the mechanism of the aggregation, and these molecules may serve as leads for drugs against transmissible spongiform encephalopathies. Therefore, we screened a synthetic octapeptide library of the globular domain of the human PrP(C) for binding affinity to PrP(C). Two fragments with binding affinity, 149YYRENMHR156 and 153NMHRYPNQ160, were identified with K(d) values of 21 and 25 microM, respectively. A 10-fold excess of peptide 153NMHRYPNQ160 inhibits aggregation of the PrP by 99%. NMR and mass spectrometry showed that the binding region of the peptide 153NMHRYPNQ160 is located at helix 3 of the PrP

    A Nanomolar Multivalent Ligand as Entry Inhibitor of the Hemagglutinin of Avian Influenza

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    Influenza virus attaches itself to sialic acids on the surface of epithelial cells of the upper respiratory tract of the host using its own protein hemagglutinin. Species specificity of influenza virus is determined by the linkages of the sialic acids. Birds and humans have α2–3 and α2–6 linked sialic acids, respectively. Viral hemagglutinin is a homotrimeric receptor, and thus, tri- or oligovalent ligands should have a high binding affinity. We describe the <i>in silico</i> design, chemical synthesis and binding analysis of a trivalent glycopeptide mimetic. This compound binds to hemagglutinin H5 of avian influenza with a dissociation constant of <i>K</i><sub>D</sub> = 446 nM and an inhibitory constant of <i>K</i><sub>I</sub> = 15 μM. <i>In silico</i> modeling shows that the ligand should also bind to hemagglutinin H7 of the virus that causes the current influenza outbreak in China. The trivalent glycopeptide mimetic and analogues have the potential to block many different influenza viruses

    A Nanomolar Multivalent Ligand as Entry Inhibitor of the Hemagglutinin of Avian Influenza

    No full text
    Influenza virus attaches itself to sialic acids on the surface of epithelial cells of the upper respiratory tract of the host using its own protein hemagglutinin. Species specificity of influenza virus is determined by the linkages of the sialic acids. Birds and humans have α2–3 and α2–6 linked sialic acids, respectively. Viral hemagglutinin is a homotrimeric receptor, and thus, tri- or oligovalent ligands should have a high binding affinity. We describe the <i>in silico</i> design, chemical synthesis and binding analysis of a trivalent glycopeptide mimetic. This compound binds to hemagglutinin H5 of avian influenza with a dissociation constant of <i>K</i><sub>D</sub> = 446 nM and an inhibitory constant of <i>K</i><sub>I</sub> = 15 μM. <i>In silico</i> modeling shows that the ligand should also bind to hemagglutinin H7 of the virus that causes the current influenza outbreak in China. The trivalent glycopeptide mimetic and analogues have the potential to block many different influenza viruses

    A Conserved NS3 Surface Patch Orchestrates NS2 Protease Stimulation, NS5A Hyperphosphorylation and HCV Genome Replication

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    <div><p>Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. The HCV RNA genome is translated into a single polyprotein. Most of the cleavage sites in the non-structural (NS) polyprotein region are processed by the NS3/NS4A serine protease. The vital NS2-NS3 cleavage is catalyzed by the NS2 autoprotease. For efficient processing at the NS2/NS3 site, the NS2 cysteine protease depends on the NS3 serine protease domain. Despite its importance for the viral life cycle, the molecular details of the NS2 autoprotease activation by NS3 are poorly understood. Here, we report the identification of a conserved hydrophobic NS3 surface patch that is essential for NS2 protease activation. One residue within this surface region is also critical for RNA replication and NS5A hyperphosphorylation, two processes known to depend on functional replicase assembly. This dual function of the NS3 surface patch prompted us to reinvestigate the impact of the NS2-NS3 cleavage on NS5A hyperphosphorylation. Interestingly, NS2-NS3 cleavage turned out to be a prerequisite for NS5A hyperphosphorylation, indicating that this cleavage has to occur prior to replicase assembly. Based on our data, we propose a sequential cascade of molecular events: in uncleaved NS2-NS3, the hydrophobic NS3 surface patch promotes NS2 protease stimulation; upon NS2-NS3 cleavage, this surface region becomes available for functional replicase assembly. This model explains why efficient NS2-3 cleavage is pivotal for HCV RNA replication. According to our model, the hydrophobic surface patch on NS3 represents a module critically involved in the temporal coordination of HCV replicase assembly.</p></div

    Serial crystallography on in vivo grown microcrystals using synchrotron radiation

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    Crystal structure determinations of biological macromolecules are limited by the availability of sufficiently sized crystals and by the fact that crystal quality deteriorates during data collection owing to radiation damage. Exploiting a micrometre-sized X-ray beam, high-precision diffractometry and shutterless data acquisition with a pixel-array detector, a strategy for collecting data from many micrometre-sized crystals presented to an X-ray beam in a vitrified suspension is demonstrated. By combining diffraction data from 80 Trypanosoma brucei procathepsin B crystals with an average volume of 9 µm3, a complete data set to 3.0 Å resolution has been assembled. The data allowed the refinement of a structural model that is consistent with that previously obtained using free-electron laser radiation, providing mutual validation. Further improvements of the serial synchrotron crystallography technique and its combination with serial femtosecond crystallography are discussed that may allow the determination of high-resolution structures of micrometre-sized crystals

    A conserved hydrophobic NS3 surface patch consisting of residues Y105, P115 and L127 is important for NS2-activation by NS3.

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    <p><b>(A)</b> Location of the residues I3, Y105, P115 and L127 in the NS3 structure of the genotype 1b. The surface residues are shown in green stick representation. The residues T4, L104, I114 and L126 are depicted in red stick representation. The overall NS3 structure is shown in grey surface representation. Carbon and backbone ribbon are colored in yellow for the protease domain and blue for the helicase domain, respectively. An enlargement of the NS3 surface patch consisting of I3, Y105, P115 and L127 is shown on the right. The figure was generated using Pymol version 1.10 and the coordinates of PDB code 1CU1 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004736#ppat.1004736.ref030" target="_blank">30</a>]. <b>(B)</b> Effect of alanine substitutions in NS3 of HCV strain BK on NS2-NS3 cleavage efficiency. Plasmids expressing either wild type (WT) or mutant FLAG-NS2-NS3(1–172)GST/BK were transfected into Huh7/T7 cells and the cleavage activity was analyzed by Western blot analysis. <b>(C)</b> Quantification of NS2-NS3 cleavage from Western blots related to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004736#ppat.1004736.g002" target="_blank">Fig. 2B</a> using ImageJ software. <b>(D)</b> The hydrophobic character of the NS3 surface area is important for the NS2 protease stimulation by NS3. The cleavage efficiencies of the indicated BK NS2-NS3 polyprotein fragments carrying either charged (arginine) or hydrophobic (phenylalanine) NS3 amino acid substitutions were determined by Western blot analysis. Quantification of these Western blots is presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004736#ppat.1004736.s003" target="_blank">S3 Fig.</a><b>(E)</b> The importance of hydrophobic NS3 surface residues Y105, P115 and L127 for NS2-activation by NS3 is conserved in HCV genotype 2a (JFH1). Plasmids expressing either wild type (WT) or mutant FLAG-NS2-NS3(1–172)GST/JFH1 were transfected into Huh-7/T7 cells and NS2-NS3 cleavage was analyzed by Western blot analysis. Blots shown are representative from three different experiments. <b>(F)</b> Quantification of NS2-NS3 cleavage from Western blots related to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004736#ppat.1004736.g002" target="_blank">Fig. 2E</a>.</p
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