Hepatitis C virus (HCV) infection is a common cause of chronic hepatitis and is currently treated with alpha interferon (IFN-␣)-based therapies.However, the underlying mechanism of IFN-␣ therapy remains to be elucidated. To identify the cellular proteins that mediate the antiviral effects of IFN-␣, we created a HEK293-based cell culture system to inducibly express individual interferon-stimulated genes (ISGs) and determined their antiviral effects against HCV. By screening 29 ISGs that are induced in Huh7 cells by IFN-␣ and/or up-regulated in HCV-infected livers, we discovered that viperin, ISG20, and double-stranded RNA-dependent protein kinase (PKR) noncytolytically inhibited the replication of HCV replicons. Mechanistically, inhibition of HCV replication by ISG20 and PKR depends on their 3-5 exonuclease and protein kinase activities, respectively. Moreover, our work, for the first time, provides strong evidence suggesting that viperin is a putative radical S-adenosyl-L-methionine (SAM) enzyme. In addition to demonstrating that the antiviral activity of viperin depends on its radical SAM domain, which contains conserved motifs to coordinate [4Fe-4S] cluster and cofactor SAM and is essential for its enzymatic activity, mutagenesis studies also revealed that viperin requires an aromatic amino acid residue at its C terminus for proper antiviral function. Furthermore, although the N-terminal 70 amino acid residues of viperin are not absolutely required, deletion of this region significantly compromises its antiviral activity against HCV. Our findings suggest that viperin represents a novel antiviral pathway that works together with other antiviral proteins, such as ISG20 and PKR, to mediate the IFN response against HCV infection. Hepatitis C virus (HCV) is the sole member of the genusHepacivirus in the family Flaviviridae (43). It establishes persistent infections in the vast majority of infected individuals and is the only known positive-stranded RNA virus that causes persistent life-long infections in humans. Currently, HCV chronically infects more than 170 million people worldwide. Although the initial infection is largely asymptomatic, prolonged infection carries a high risk of chronic hepatitis, cirrhosis, and primary hepatocellular carcinoma (2).Although it has been elegantly demonstrated that HCV can evade the host cellular innate defense response through proteolytic cleavage of RIG-I/MDA5 adaptor protein MAVS and Tolllike receptor 3 adaptor protein TRIF (7,22,25,42,44,48,69), microarray studies performed with liver samples obtained from HCV transiently infected chimpanzees and chronically infected humans revealed that the induction of interferon (IFN)-stimulated genes (ISGs) in HCV-infected livers is a hallmark of the virus infection (5,6,33,39,58,61). These discoveries suggest that the HCV-infected liver is a constant battlefield between the virus and host innate immunity defense systems, and thus IFN-mediated innate responses induced by HCV may play an important role in shaping the pathogenesis and clini...

Glycoprotein B (gB) of herpes simplex virus type 1 is an envelope protein that is essential for viral growth. We previously reported the isolation of two gB-null viruses, which form gB-free virions in nonpermissive cells. In the present study, these gB-free virions were shown to bind to the cell surface at the same rate as the wild-type virus. They failed, however, to form plaques and to synthesize virus-specific proteins upon infection. Their plating efficiency was significantly enhanced by treatment with polyethylene glycol, a membrane fusion agent. Therefore, gB is required in a stage after viral attachment but before the expression of the virus-specific proteins. A gB-null syncytial virus was isolated, which contained a gB defect and a syncytial mutation in another genetic locus. It caused complete fusion of gB-transformed cells but no fusion on untransformed cells, indicating the essential role of gB in virus-induced cell fusion. Mutations located at two independent sites in the cytoplasmic domain of gB were transferred to viral DNA and shown to confer a syncytial phenotype to the virus. A transient-expression assay was developed to determine the ability of a set of plasmids containing addition and nonsense mutations in the gB gene to complement the cell-fusion defect in the gB-null syncytial virus. Mutations in plasmids, including those located in the extracytoplasmic domain of gB, were identified that reduced the fusion activity of gB. Therefore, gB contains different functional regions responsible for fusion induction and its inhibition.

"Noncompensated n-p codoping" is established as an enabling concept for enhancing the visible-light photoactivity of TiO2 by narrowing its band gap. The concept embodies two crucial ingredients: the electrostatic attraction within the n-p dopant pair enhances both the thermodynamic and kinetic solubilities, and the noncompensated nature ensures the creation of tunable intermediate bands that effectively narrow the band gap. The concept is demonstrated using first-principles calculations, and is validated by direct measurements of band gap narrowing using scanning tunneling spectroscopy, dramatically redshifted optical absorbance, and enhanced photoactivity manifested by efficient electron-hole separation in the visible-light region. This concept is broadly applicable to the synthesis of other advanced functional materials that demand optimal dopant control.

Microbial conversion of inorganic mercury (IHg) to methylmercury (MeHg) is a significant environmental concern because of the bioaccumulation and biomagnification of toxic MeHg in the food web. Laboratory incubation studies have shown that, despite the presence of large quantities of IHg in cell cultures, MeHg biosynthesis often reaches a plateau or a maximum within hours or a day by an as yet unexplained mechanism. Here we report that mercuric Hg(II) can be taken up rapidly by cells of Desulfovibrio desulfuricans ND132, but a large fraction of the Hg(II) is unavailable for methylation because of strong cellular sorption. Thiols, such as cysteine, glutathione, and penicillamine, added either simultaneously with Hg(II) or after cells have been exposed to Hg(II), effectively desorb or mobilize the bound Hg(II), leading to a substantial increase in MeHg production. The amount of thiol-desorbed Hg(II) is strongly correlated to the amount of MeHg produced (r = 0.98). However, cells do not preferentially take up Hg(II)-thiol complexes, but Hg(II)-ligand exchange between these complexes and the cell-associated proteins likely constrains Hg(II) uptake and methylation. We suggest that, aside from aqueous chemical speciation of Hg(II), binding and exchange of Hg(II) between cells and complexing ligands such as thiols and naturally dissolved organics in solution is an important controlling mechanism of Hg(II) bioavailability, which should be considered when predicting MeHg production in the environment.

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of an in vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC 50 of ϳ0.5 M, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.Hepatitis C virus (HCV), 1 a positive strand RNA virus of the Flaviviridae family, is the major etiological agent of post-transfusion and sporadic non-A, non-B hepatitis (1). An estimated 2-3% of the world population is chronically infected with HCV, which causes significant liver disease, cirrhosis, and can eventually lead to the development of hepatocellular carcinoma. In infected cells, translation of the viral RNA yields a 3011-residue polyprotein chain (2-4), which is subsequently cleaved to generate envelope and core proteins, for assembly of new virus particles and nonstructural enzymes essential for viral replication (5-7). Studies using recombinant NS5B polymerase have provided direct evidence for RNA-dependent RNA polymerase activity (8, 9), and this catalytic activity has been confirmed to be required for infectivity in chimpanzees (10).NS5B polymerase contains a hydrophobic C-terminal domain thought to be responsible for anchoring the protein to mammalian cell membranes. Removal of the C-terminal 21 residues has been reported to facilitate protein isolation from Escherichia coli without compromising RdRp activity (11). The HCV RdRp initiates RNA synthesis preferentially from the 3Ј terminus of the template RNA (12, 13-15) but lacks specificity for HCV RNA in vitro, because it readily utilizes heterologous nonviral templates (8). Based on crystallographic studies of the enzyme containing C-terminal truncations (16, 17), the hydrophobic tail present in the full-length ...

No abstract

Due to their unique antimicrobial properties silver nanocrystallites have garnered substantial attention and are used extensively for biomedical applications as an additive to wound dressings, surgical instruments and bone substitute materials. They are also released into unintended locations such as the environment or biosphere. Therefore it is imperative to understand the potential interactions, fate and transport of nanoparticles with environmental biotic systems. Numerous factors including the composition, size, shape, surface charge, and capping molecule of nanoparticles are known to influence cell cytotoxicity. Our results demonstrate that the physical/chemical properties of the silver nanoparticles including surface charge, differential binding and aggregation potential, which are influenced by the surface coatings, are a major determining factor in eliciting cytotoxicity and in dictating potential cellular interactions. In the present investigation, silver nanocrystallites with nearly uniform size and shape distribution but with different surface coatings, imparting overall high negativity to high positivity, were synthesized. These nanoparticles included poly(diallyldimethylammonium) chloride-Ag, biogenic-Ag, colloidal-Ag (uncoated), and oleate-Ag with zeta potentials +45 ± 5, -12 ± 2, -42 ± 5, and -45 ± 5 mV, respectively; the particles were purified and thoroughly characterized so as to avoid false cytotoxicity interpretations. A systematic investigation on the cytotoxic effects, cellular response, and membrane damage caused by these four different silver nanoparticles was carried out using multiple toxicity measurements on mouse macrophage (RAW-264.7) and lung epithelial (C-10) cell lines. Our results clearly indicate that the cytotoxicity was dependent on various factors such as surface charge and coating materials used in the synthesis, particle aggregation, and the cell-type for the different silver nanoparticles that were investigated. Poly(diallyldimethylammonium)-coated Ag nanoparticles were found to be the most toxic, followed by biogenic-Ag and oleate-Ag nanoparticles, whereas uncoated or colloidal silver nanoparticles were found to be the least toxic to both macrophage and lung epithelial cells. Also, based on our cytotoxicity interpretations, lung epithelial cells were found to be more resistant to the silver nanoparticles than the macrophage cells, regardless of the surface coating.

Microbial methylation and demethylation are two competing processes controlling the net production and bioaccumulation of neurotoxic methylmercury (MeHg) in natural ecosystems. Although mercury (Hg) methylation by anaerobic microorganisms and demethylation by aerobic Hg-resistant bacteria have both been extensively studied, little attention has been given to MeHg degradation by anaerobic bacteria, particularly the iron-reducing bacterium Geobacter bemidjiensis Bem. Here we report, for the first time, that the strain G. bemidjiensis Bem can mediate a suite of Hg transformations, including Hg(II) reduction, Hg(0) oxidation, MeHg production and degradation under anoxic conditions. Results suggest that G. bemidjiensis utilizes a reductive demethylation pathway to degrade MeHg, with elemental Hg(0) as the major reaction product, possibly due to the presence of genes encoding homologues of an organomercurial lyase (MerB) and a mercuric reductase (MerA). In addition, the cells can strongly sorb Hg(II) and MeHg, reduce or oxidize Hg, resulting in both time and concentration-dependent Hg species transformations. Moderate concentrations (10-500 μM) of Hg-binding ligands such as cysteine enhance Hg(II) methylation but inhibit MeHg degradation. These findings indicate a cycle of Hg methylation and demethylation among anaerobic bacteria, thereby influencing net MeHg production in anoxic water and sediments.