Purification and characterization of an antimicrobial peptide produced by Pseudomonas sp strain 4B

An antimicrobial peptide produced by a bacterium isolated from the effluent pond of a bovine abattoir was purified and characterized. The strain was characterized by biochemical profiling and 16S rDNA sequencing as Pseudomonas sp. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A major band on SDS-PAGE suggested that the antimicrobial peptide has a molecular mass of about 30 kDa. The substance was inhibitory to a broad range of indicator strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, among other. The partially purified antimicrobial substance remained active over a wide temperature range and was resistant to all proteases tested. This substance showed different properties than other antimicrobials from Pseudomonas species, suggesting a novel antimicrobial peptide was characterized.

Modification of surface properties of Ti-16Si-4B powder alloy by plasma immersion ion implantation

Results of the surface modification of Ti-16Si-4B powder alloy by nitrogen ion implantation are presented, together with the experimental description of the preparation of that powder by high-energy ball milling and hot pressing. The phase structure, chemical composition and morphology of sample surfaces were observed by utilizing X-ray diffractometer (XRD), atomic force microscope (AFM) and scanning electron microscopy (SEM). A tribological characterization was carried out with a ball-on-disc tribometer and an SEM. Friction coefficient is compared with the one obtained for Ti-6Al-4V alloy and the wear scars characterized by SEM/EDS (energy dispersive spectroscopy). The concentration profile of the detected elements have been investigated using Auger electron spectroscopy (AES) depth profiling. Our results show that a shallow implanted layer of oxygen and nitrogen ions were obtained at the Ti-16Si -4B alloy surface, sufficient to modify slightly its tribological properties. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved.

Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B.

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic alpha-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic alpha-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.

Functional Characterization of a n NTPase Activity of the Hepatitis C Virus Nonstructural Protein 4B

Background: Nonstructural p rotein 4 B (NS4B) i s the m asterorganizer of hepatitis C virus (HCV) replication complexformation. It is a multispanning membrane protein that has beenreported to p ossess NTPase activity. This enzymatic functionhas been poorly studied so far and its role in the HCV life cycleis u nknown. T he present w ork-in-progress a ims at validatingand functionally c haracterizing this a ctivity a nd its r ole in t heviral life cycle.Methods: B ioinformatic analyses were performed to i dentifykey residues for site-directed mutagenesis, both in t he contextof s ubgenomic r eplicons a s well as recombinant v iruses.Mutants were investigated with respect to R NA replication andinfectious particle p roduction. In p arallel, expression andpurification of recombinant wild-type and mutant NS4B proteinsare being pursued to characterize enzymatic activity in vitro.Results: B ioinformatic a nalyses revealed t hat p redictedNTPase features are conserved only in H CV NS4B b ut n ot i nNS4B from other Flaviviridae f amily m embers. A laninesubstitutions were designed to target predicted key Walker A, Band C motifs. These substitutions affected RNA replication andinfectious virus production to v arying degrees. Optimization ofrecombinant protein production is i n progress both in b acterialas well as mammalian expression systems.Conclusions: These studies should yield new insights into thefunctions of this hitherto poorly characterized viral nonstructuralprotein and may reveal novel targets for antiviral intervention inthe future.

Analysis of hepatitis C virus resistance to silibinin in vitro and in vivo points to a novel mechanism involving nonstructural protein 4B.

Intravenous silibinin (SIL) is an approved therapeutic that has recently been applied to patients with chronic hepatitis C, successfully clearing hepatitis C virus (HCV) infection in some patients even in monotherapy. Previous studies suggested multiple antiviral mechanisms of SIL; however, the dominant mode of action has not been determined. We first analyzed the impact of SIL on replication of subgenomic replicons from different HCV genotypes in vitro and found a strong inhibition of RNA replication for genotype 1a and genotype 1b. In contrast, RNA replication and infection of genotype 2a were minimally affected by SIL. To identify the viral target of SIL we analyzed resistance to SIL in vitro and in vivo. Selection for drug resistance in cell culture identified a mutation in HCV nonstructural protein (NS) 4B conferring partial resistance to SIL. This was corroborated by sequence analyses of HCV from a liver transplant recipient experiencing viral breakthrough under SIL monotherapy. Again, we identified distinct mutations affecting highly conserved amino acid residues within NS4B, which mediated phenotypic SIL resistance also in vitro. Analyses of chimeric viral genomes suggest that SIL might target an interaction between NS4B and NS3/4A. Ultrastructural studies revealed changes in the morphology of viral membrane alterations upon SIL treatment of a susceptible genotype 1b isolate, but not of a resistant NS4B mutant or genotype 2a, indicating that SIL might interfere with the formation of HCV replication sites. CONCLUSION: Mutations conferring partial resistance to SIL treatment in vivo and in cell culture argue for a mechanism involving NS4B. This novel mode of action renders SIL an attractive candidate for combination therapies with other directly acting antiviral drugs, particularly in difficult-to-treat patient cohorts.

Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory.

Hepatitis C virus (HCV) is a positive-strand RNA virus that replicates its genome in a membrane-associated replication complex. Nonstructural protein 4B (NS4B) induces the specific membrane alteration, designated as membranous web (MW), that harbours this complex. HCV NS4B is an integral membrane protein predicted to comprise four transmembrane segments in its central part. The N-terminal part comprises two amphipathic alpha-helices of which the second has the potential to traverse the membrane bilayer, likely upon oligomerisation. The C-terminal part comprises a predicted highly conserved alpha-helix, a membrane-associated amphipathic alpha-helix and two reported palmitoylation sites. NS4B interacts with other viral nonstructural proteins and has been reported to bind viral RNA. In addition, it was found to harbour an NTPase activity. Finally, NS4B has recently been found to have a role in viral assembly. Much work needs to be done with respect to further dissecting these multiple functions as well as providing a refined membrane topology and complete structure of NS4B. Progress in this direction should yield important insights into the functional architecture of the HCV replication complex and may reveal new opportunities for antiviral intervention against a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide.

An amphipathic alpha-helix at the C terminus of hepatitis C virus nonstructural protein 4B mediates membrane association.

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is an integral membrane protein that has been only poorly characterized to date. It is believed to comprise a cytosolic N-terminal part, a central part harboring four transmembrane passages, and a cytosolic C-terminal part. Here, we describe an amphipathic alpha-helix at the C terminus of NS4B (amino acid residues 229 to 253) that mediates membrane association and is involved in the formation of a functional HCV replication complex.

Mapping the Membrane Topology of Hepatitis C Virus Nonstructural Protein 4B

BACKGROUND: Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is an integral membrane protein that has only poorly been characterized to date. In particular, a precise membrane topology is thus far elusive. Here, we explored a novel strategy to map the membrane topology of HCV NS4B. METHODS: Selective permeabilization of the plasma membrane, maleimide-polyethyleneglycol (mPEG) labeling of natural or engineered cysteine residues and immunoblot analyses were combined to map the membrane topology of NS4B. Cysteine substitutions were introduced at carefully selected positions within NS4B and their impact on HCV RNA replication and infectious virus production analyzed in cell culture. RESULTS: We established a panel of viable HCV mutants with cysteine substitutions at strategic positions within NS4B. These mutants are infectious and replicate to high levels in cell culture. In parallel, we adapted and optimized the selective permeabilization and mPEG labeling techniques to Huh-7 human hepatocellular carcinoma cells which can support HCV infection and replication. CONCLUSIONS: The newly established experimental tools and techniques should allow us to refine the membrane topology of HCV NS4B in a physiological context. The expected results should enhance our understanding of the functional architecture of the HCV replication complex and may provide new opportunities for antiviral intervention in the future.

Aminoterminal amphipathic α-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and virus production.

Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. In concert with other nonstructural proteins, it induces a specific membrane rearrangement, designated as membranous web, which serves as a scaffold for the HCV replicase. The N-terminal part of NS4B comprises a predicted and a structurally resolved amphipathic α-helix, designated as AH1 and AH2, respectively. Here, we report a detailed structure-function analysis of NS4B AH1. Circular dichroism and nuclear magnetic resonance structural analyses revealed that AH1 folds into an amphipathic α-helix extending from NS4B amino acid 4 to 32, with positively charged residues flanking the helix. These residues are conserved among hepaciviruses. Mutagenesis and selection of pseudorevertants revealed an important role of these residues in RNA replication by affecting the biogenesis of double-membrane vesicles making up the membranous web. Moreover, alanine substitution of conserved acidic residues on the hydrophilic side of the helix reduced infectivity without significantly affecting RNA replication, indicating that AH1 is also involved in virus production. Selective membrane permeabilization and immunofluorescence microscopy analyses of a functional replicon harboring an epitope tag between NS4B AH1 and AH2 revealed a dual membrane topology of the N-terminal part of NS4B during HCV RNA replication. Luminal translocation was unaffected by the mutations introduced into AH1, but was abrogated by mutations introduced into AH2. In conclusion, our study reports the three-dimensional structure of AH1 from HCV NS4B, and highlights the importance of positively charged amino acid residues flanking this amphipathic α-helix in membranous web formation and RNA replication. In addition, we demonstrate that AH1 possesses a dual role in RNA replication and virus production, potentially governed by different topologies of the N-terminal part of NS4B.

Amphipathic alpha-helix AH2 is a major determinant for the oligomerization of hepatitis C virus nonstructural protein 4B.

Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. It induces a specific membrane rearrangement, designated membranous web, that serves as a scaffold for the HCV replication complex. However, the mechanisms underlying membranous web formation are poorly understood. Based on fluorescence resonance energy transfer (FRET) and confirmatory coimmunoprecipitation analyses, we provide evidence for an oligomerization of NS4B in the membrane environment of intact cells. Several conserved determinants were found to be involved in NS4B oligomerization, through homotypic and heterotypic interactions. N-terminal amphipathic ?-helix AH2, comprising amino acids 42 to 66, was identified as a major determinant for NS4B oligomerization. Mutations that affected the oligomerization of NS4B disrupted membranous web formation and HCV RNA replication, implying that oligomerization of NS4B is required for the creation of a functional replication complex. These findings enhance our understanding of the functional architecture of the HCV replication complex and may provide new angles for therapeutic intervention. At the same time, they expand the list of positive-strand RNA virus replicase components acting as oligomers.