No abstract

We show that hepatitis C virus (HCV) p7 protein forms ion channels in black lipid membranes. HCV p7 ion channels are inhibited by long-alkyl-chain iminosugar derivatives, which have antiviral activity against the HCV surrogate bovine viral diarrhea virus. HCV p7 presents a potential target for antiviral therapy.H epatitis C virus (HCV) is the major cause of chronic hepatitis with a significant risk of end-stage liver cirrhosis and hepatocellular carcinoma (1). HCV belongs to the family Flaviviridae, which consists of three genera: flaviviruses, pestiviruses, and hepaciviruses. In the absence of both a suitable small animal model and a reliable in vitro infectivity assay for HCV, potential antiviral drugs initially have been tested by using a related pestivirus, bovine viral diarrhea virus (BVDV) (2). BVDV in vitro infectivity assays were used to demonstrate that long-alkyl-chain iminosugar derivatives containing either the glucose analogue deoxynojirimycin (DNJ) or the galactose analogue deoxygalactonojirimycin (DGJ) are potent antiviral inhibitors (3).DNJ derivatives inhibit endoplasmic reticulum (ER) ␣-glucosidases I and II (4, 5), and this inhibition leads to the misfolding of many host-and virus-encoded glycoproteins, including the envelope glycoproteins of BVDV (6) and HCV (7). Previous experiments have shown that the antiviral effect of the longalkyl-chain derivative N-nonyl-DNJ (NN-DNJ) is more pronounced than that of the short-alkyl-chain derivative N-butyl-DNJ (NB-DNJ), although the latter achieves a more effective ER ␣-glucosidase inhibition in cellulo. In addition, long-alkylchain DGJ derivatives that are not recognized by and do not inhibit ER ␣-glucosidases also show potent antiviral activity (3). Therefore, ER ␣-glucosidase inhibition does not correlate directly with the observed antiviral effect and is ruled out as the sole antiviral mechanism.The additional mechanism of action apparently is associated with the length of the alkyl side chain, because the short-chain N-butyl-DGJ (NB-DGJ) shows no antiviral activity, whereas the long-alkyl-chain derivative NN-DGJ is a potent inhibitor (3).The predominant antiviral mechanism is proposed to be mediated directly or indirectly by an effect of the long-alkyl side chains on the membrane and͞or membrane proteins, because treatment with long-alkyl-chain iminosugars affects the dimerization of viral membrane glycoproteins and alters the membrane glycoprotein composition of secreted BVDV virions but does not influence either viral RNA replication or protein synthesis (3).We decided to investigate the small membrane-spanning protein p7 as a potential target of long-alkyl-chain iminosugar derivatives, because flaviviruses such as dengue virus and Japanese encephalitis virus (8), which do not contain p7, are not inhibited by long-alkyl-chain DGJ derivatives, whereas pestiviruses are (3). Pesti-and hepaciviruses both contain the p7 protein.Most functional data about p7 are derived from the pestivirus p7, a 70-aa protein very similar to HCV p7. Functional data hav...

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus but are mistargeted to lysosomes in sphingolipid storage disorders. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Niemann-Pick type C (NPC) disease is a disorder of intracellular transport where glycosphingolipids (GSLs) and cholesterol accumulate in endosomal compartments. The mechanisms of altered intracellular trafficking are not known but may involve the mistargeting and disrupted function of proteins associated with GSL membrane microdomains. Membrane microdomains were isolated by Triton X-100 and sucrose density gradient ultracentrifugation. High pressure liquid chromatography and mass spectrometric analysis of NPC1 ؊/؊ mouse brain revealed large increases in GSL. Sphingosine was also found to be a component of membrane microdomains, and in NPC liver and spleen, large increases in cholesterol and sphingosine were found. GSL and cholesterol levels were increased in mutant NPC1-null Chinese hamster ovary cells as well as U18666A and progesterone induced NPC cell culture models. However, inhibition of GSL synthesis in NPC cells with N-butyldeoxygalactonojirimycin led to marked decreases in GSL but only small decreases in cholesterol levels. Both annexin 2 and 6, membrane-associated proteins that are important in endocytic trafficking, show distorted distributions in NPC cells. Altered BODIPY lactosylceramide targeting, decreased endocytic uptake of a fluid phase marker, and mistargeting of annexin 2 (phenotypes associated with NPC) are reversed by inhibition of GSL synthesis. It is suggested that accumulating GSL is part of a mislocalized membrane microdomain and is responsible for the deficit in endocytic trafficking found in NPC disease.Increasing evidence suggests that the membranes of eukaryotic cells are not homogeneously fluid. They contain membrane microdomains, often referred to as lipid rafts and/or caveolae, enriched in glycosphingolipids (GSLs), 1 specific proteins, and cholesterol (1-3). Being operationally defined, i.e. by detergent insolubility and low buoyant density, the natural (in situ) existence of such membrane microdomains has been and continues to be controversial (4). However, they have recently been visualized by using electron microscopy as small structures covering as much as 35% of the cell surface (5). Although membrane microdomains on the cell surface tend to be transient (6), relatively stable microdomains have been visualized in the endocytic pathway (7) where they are proposed to play a role in protein and lipid sorting (8). Many GSL storage diseases, including Niemann-Pick type C (NPC), are often characterized by enlarged liver and spleen as well as severe neurodegeneration. In these diseases, cholesterol and GSLs accumulate in the endocytic pathway because of reduced breakdown. Hence, they may be a useful tool for determining the relative importance of GSLs in endocytic transport. Fibroblasts from patients suffering f...

Development of invariant natural killer T (iNKT) cells requires the presentation of lipid ligand(s) byCD1d molecules in the thymus. The glycosphingolipid (GSL) isoglobotrihexosylceramide (iGb3) has been proposed as the natural iNKT cell-selecting ligand in the thymus and to be involved in peripheral activation of iNKT cells by dendritic cells (DCs). However, there is no direct biochemical evidence for the presence of iGb3 in mouse or human thymus or DCs. Using a highly sensitive HPLC assay, the only tissue where iGb3 could be detected in mouse was the dorsal root ganglion (DRG). iGb3 was not detected in other mouse or any human tissues analyzed, including thymus and DCs. Even in mutant mice that store isoglobo-series GSLs in the DRG, we were still unable to detect these GSLs in the thymus. iGb3 is therefore unlikely to be a physiologically relevant iNKT cell-selecting ligand in mouse and humans. A detailed study is now warranted to better understand the nature of iNKT cell-selecting ligand(s) in vivo.glycosphingolipid ͉ thymus ͉ CD1d ͉ lipid presentation

The inhibition of ER (endoplasmic reticulum) alpha-glucosidases I and II by imino sugars, including NB-DNJ (N-butyl-deoxynojirimycin), causes the retention of glucose residues on N-linked oligosaccharides. Therefore, normal glycoprotein trafficking and processing through the glycosylation pathway is abrogated and glycoproteins are directed to undergo ERAD (ER-associated degradation), a consequence of which is the production of cytosolic FOS (free oligosaccharides). Following treatment with NB-DNJ, FOS were extracted from cells, murine tissues and human plasma and urine. Improved protocols for analysis were developed using ion-exchange chromatography followed by fluorescent labelling with 2-AA (2-aminobenzoic acid) and purification by lectin-affinity chromatography. Separation of 2-AA-labelled FOS by HPLC provided a rapid and sensitive method that enabled the detection of all FOS species resulting from the degradation of glycoproteins exported from the ER. The generation of oligosaccharides derived from glucosylated protein degradation was rapid, reversible, and time- and inhibitor concentration-dependent in cultured cells and in vivo. Long-term inhibition in cultured cells and in vivo indicated a slow rate of clearance of glucosylated FOS. In mouse and human urine, glucosylated FOS were detected as a result of transrenal excretion and provide unique and quantifiable biomarkers of ER-glucosidase inhibition.