The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly ␣-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by 1 H and 13 C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal ␣-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism. Hepatitis C virus (HCV)8 infection is a major cause of human chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1). About 170 million individuals worldwide are chronically infected with HCV, and current therapy based on a combination of pegylated interferon and ribavirin is poorly tolerated and ineffective in 50% of patients. In this context, the ongoing search for new drugs and targets is very active, and the structural and functional characterization of the HCV viroporin p7 is essential for the molecular understanding of its role in HCV replication and for antiviral drug development.HCV is a highly variable enveloped positive-stranded RNA virus, and patient isolates are classified into seven genotypes and numerous subtypes (2, 3) within the genus Hepacivirus of the family Flaviviridae (4). The HCV genome encodes a polyprotein precursor,

We determined the structure in solution by 1H two‐dimensional NMR of Maurocalcine from the venom of Scorpio maurus. This toxin has been demonstrated to be a potent effector of ryanodyne‐sensitive calcium channel from skeletal muscles. This is the first description of a scorpion toxin which folds following the Inhibitor Cystine Knot fold (ICK) already described for numerous toxic and inhibitory peptides, as well as for various protease inhibitors. Its three dimensional structure consists of a compact disulfide‐bonded core from which emerge loops and the N‐terminus. A double‐stranded antiparallel β‐sheet comprises residues 20–23 and 30–33. A third extended strand (residues 9–11) is perpendicular to the β‐sheet. Maurocalcine structure mimics the activating segment of the dihydropyridine receptor II‐III loop and is therefore potentially useful for dihydropyridine receptor/ryanodine receptor interaction studies. Proteins 2000;40:436–442. © 2000 Wiley‐Liss, Inc.

The last decade has contributed to our understanding of the three-dimensional structure of the human immunodeficiency virus, type 1 (HIV-1) integrase (IN) and to the description of how the enzyme catalyzes the viral DNA integration into the host DNA. Recognition of the viral DNA termini by IN is sequence-specific, and that of the host DNA does not require particular sequence, although in physicochemical studies IN fails to discriminate between the two interactions. Here, such discrimination was allowed thanks to a model system using designed oligonucleotides and peptides as binding structures. Spectroscopic (circular dichroism, NMR, and fluorescence anisotropy) techniques and biochemical (enzymatic and filter binding) assays clearly indicated that the amphipathic helix ␣4, located at the catalytic domain surface, is responsible for the specific high affinity binding of the enzyme to viral DNA. Analogues of the ␣4 peptide having increased helicity and still bearing the biologically relevant lysines 156 and 159 on the DNA binding face, and oligonucleotides conserving an intact attachment site, are required to achieve high affinity complexes (K d of 1.5 nM). Data corroborate previous in vivo results obtained with mutated viruses.