Gary Ewart

Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride.

A 63 residue peptide, p7, encoded by hepatitis C virus was synthesised and tested for ion channel activity in lipid bilayer membranes. Ion channels formed by p7 had a variable conductance: some channels had conductances as low as 14 pS. The reversal potential of currents flowing through the channels formed by p7 showed that they were permeable to potassium and sodium ions and less permeable to calcium ions. Addition of Ca2+ to solutions made channels formed by p7 less potassium‐ or sodium‐selective. Hexamethylene amiloride, a drug previously shown to block ion channels formed by Vpu encoded by HIV‐1, blocked channels formed by p7. In view of the increasing number of peptides encoded by viruses that have been shown to form ion channels, it is suggested that ion channels may play an important role in the life cycle of many viruses and that drugs that block these channels may prove to be useful antiviral agents.

SARS coronavirus E protein forms cation-selective ion channels.

Abstract Severe Acute Respiratory Syndrome (SARS) is caused by a novel coronavirus (SARS-CoV). Coronaviruses including SARS-CoV encode an envelope (E) protein, a small, hydrophobic membrane protein. We report that, in planar lipid bilayers, synthetic peptides corresponding to the SARS-CoV E protein forms ion channels that are more permeable to monovalent cations than to monovalent anions. Affinity-purified polyclonal antibodies recognizing the N-terminal 19 residues of SARS-CoV E protein were used to establish the specificity of channel formation by inhibiting the ion currents generated in the presence of the E protein peptides.

Amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by HIV-1 protein Vpu

Abstract The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels and enhances the process of virion budding and release. Mutagenesis studies have shown that the N-terminal transmembrane domain primarily controls both of these activities. Here we report that the Vpu ion channel is inhibited by the amiloride derivatives 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)amiloride but not by amiloride itself, nor by amantadine. Hexamethyleneamiloride also inhibits budding of virus-like particles from HeLa cells expressing HIV-1 Gag and Vpu proteins. These results confirm the link between Vpu ion channel activity and the budding process and also suggest that amiloride derivatives might have useful anti-HIV-1 properties.

Mutations in the white gene of Drosophila melanogaster affecting ABC transporters that determine eye colouration.

The white, brown and scarlet genes of Drosophila melanogaster encode proteins which transport guanine or tryptophan (precursors of the red and brown eye colour pigments) and belong to the ABC transporter superfamily. Current models envisage that the white and brown gene products interact to form a guanine specific transporter, while white and scarlet gene products interact to form a tryptophan transporter. In this study, we report the nucleotide sequence of the coding regions of five white alleles isolated from flies with partially pigmented eyes. In all cases, single amino acid changes were identified, highlighting residues with roles in structure and/or function of the transporters. Mutations in w(cf) (G589E) and w(sat) (F590G) occur at the extracellular end of predicted transmembrane helix 5 and correlate with a major decrease in red pigments in the eyes, while brown pigments are near wild-type levels. Therefore, those residues have a more significant role in the guanine transporter than the tryptophan transporter. Mutations identified in w(crr) (H298N) and w(101) (G243S) affect amino acids which are highly conserved among the ABC transporter superfamily within the nucleotide binding domain. Both cause substantial and similar decreases of red and brown pigments indicating that both tryptophan and guanine transport are impaired. The mutation identified in w(Et87) alters an amino acid within an intracellular loop between transmembrane helices 2 and 3 of the predicted structure. Red and brown pigments are reduced to very low levels by this mutation indicating this loop region is important for the function of both guanine and tryptophan transporters.

Hexamethylene amiloride blocks E protein ion channels and inhibits coronavirus replication

Abstract All coronaviruses encode a small hydrophobic envelope (E) protein, which mediates viral assembly and morphogenesis by an unknown mechanism. We have previously shown that the E protein from Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) forms cation-selective ion channels in planar lipid bilayers (Wilson, L., McKinlay, C., Gage, P., Ewart, G., 2004. SARS coronavirus E protein forms cation-selective ion channels. Virology 330(1), 322–331). We now report that three other E proteins also form cation-selective ion channels. These E proteins were from coronaviruses representative of taxonomic groups 1–3: human coronavirus 229E (HCoV-229E), mouse hepatitis virus (MHV), and infectious bronchitis virus (IBV), respectively. It appears, therefore, that coronavirus E proteins in general, belong to the virus ion channels family. Hexamethylene amiloride (HMA) – an inhibitor of the HIV-1 Vpu virus ion channel – inhibited the HCoV-229E and MHV E protein ion channel conductance in bilayers and also inhibited replication of the parent coronaviruses in cultured cells, as determined by plaque assay. Conversely, HMA had no antiviral effect on a recombinant MHV with the entire coding region of E protein deleted (MHVΔE). Taken together, the data provide evidence of a link between inhibition of E protein ion channel activity and the antiviral activity of HMA.

Ion Channels Formed by NB, an Influenza B Virus Protein

Abstract.The influenza B virus protein, NB, was expressed in Escherichia coli, either with a C-terminal polyhistidine tag or with NB fused to the C-terminus of glutathione S-transferase (GST), and purified by affinity chromatography. NB produced ion channel activity when added to artificial lipid bilayers separating NaCl solutions with unequal concentrations (150–500 mmcis, 50 mmtrans). An antibody to a peptide mimicking the 25 residues at the C-terminal end of NB, and amantadine at high concentration (2–3 mm), both depressed ion channel activity. Ion channels had a variable conductance, the lowest conductance observed being approximately 10 picosiemens. At a pH of 5.5 to 6.5, currents reversed at positive potentials indicating that the channel was more permeable to sodium than to chloride ions (PNa/PCl∼ 9). In asymmetrical NaCl solutions at a pH of 2.5, currents reversed closer to the chloride than to the sodium equilibrium potential indicating that the channel had become more permeable to chloride than to sodium ions (PCl/PNa∼ 4). It was concluded that, at normal pHs, NB forms cation-selective channels.

Sub-cellular localisation of the white/scarlet ABC transporter to pigment granule membranes within the compound eye of Drosophila melanogaster.

The white, scarlet, and browngenes of Drosophila melanogasterencode ABC transporters involved with the uptake and storage of metabolic precursors to the red and brown eye colour pigments. It has generally been assumed that these proteins are localised in the plasma membrane and transport precursor molecules from the heamolymph into the eye pigment cells. However, the immuno-electron microscopy experiments in this study reveal that the White and Scarlet proteins are located in the membranes of pigment granules within pigment cells and retinula cells of the compound eye. No evidence of their presence in the plasma membrane was observed. This result suggests that, rather than tranporting tryptophan into the cell across the plasma membrane, the White/Scarlet complex transports a metabolic intermediate (such as 3-hydroxy kynurenine) from the cytoplasm into the pigment granules. Other functional implications of this new finding are discussed.

Isoforms of Mammalian Cytochrome c Oxidase:Correlation with Human Cytochrome c Oxidase Deficiency

ABSTRACT: We have reviewed the structure, function, and biogenesis of mammalian cytochrome c oxidase, examined the tissue-specific expression of isoforms of cytochrome c oxidase subunits in different mammals, and attempted to correlate the data with our knowledge of cytochrome c oxidase deficiency, illustrated by one particular patient. Cytochrome c oxidase was isolated from bovine tissues, and individual subunits examined by SDS-PAGE, N-terminal peptide sequencing, and antibody binding. Isoforms of subunits VIa, VIIa, and VIII were identified, manifesting one pattern of expression in heart and skeletal muscle, and another in liver, kidney, and brain. In rat heart and liver, only one form of subunit VIIa was identified. Northern analysis of bovine and rat tissues suggested that the tissue-specific expression of subunits VIa and VIII is regulated transcriptionally in liver, kidney, and brain, and posttranscriptionally in heart and skeletal muscle. In humans, antibody binding documented isoforms of subunits VIa and VIIa, with the pattern of expression in heart and skeletal muscle differing from that in liver, kidney, and brain; our data suggested that both isoforms of subunit VIa may be expressed in human heart. In a patient with cytochrome c oxidase deficiency, the clinical, morphologic, and biochemical manifestations were much more severe in heart than in skeletal muscle. Antibody binding suggested partial assembly of the enzyme in heart. These and other data suggest considerably more variability in the tissue-specific expression of isoforms of cytochrome c oxidase subunits than previously recognized

A novel Hepatitis C virus p7 ion channel inhibitor, BIT225, inhibits bovine viral diarrhea virus in vitro and shows synergism with recombinant interferon-α-2b and nucleoside analogues

The novel small molecule, BIT225 (N-[5-(1-methyl-1H-pyrazol-4-yl)-napthalene-2-carbonyl]-guanidine: CAS No. 917909-71-8), was initially identified using a screening strategy designed to detect inhibitors of Hepatitis C virus (HCV) p7 ion channel activity. Here we report that BIT225 has potent stand-alone antiviral activity against the HCV model pestivirus bovine viral diarrhea virus (BVDV) with an IC(50) of 314nM. Combinations of BIT225 with recombinant interferon alpha-2b (rIFNalpha-2b) show synergistic antiviral action against BVDV and the synergy is further enhanced by addition of ribavirin. Synergy was also observed between BIT225 and two nucleoside analogues known to inhibit the HCV RNA-dependent RNA polymerase. BIT225 has successfully completed a phase Ia dose escalating, single dose safety trial in healthy volunteers and a phase Ib/IIa trial to evaluate the safety and pharmacokinetics of repeated dosing for selected doses of BIT225 in HCV-infected persons. A modest, but statistically significant drop in patient viral load was detected over the 7 days of dosing (ref. www.biotron.com.au). Given the critical role of the p7 protein in the HCV life cycle and pathogenicity, our data indicate that molecules like BIT225, representing a new class of antiviral compounds, may be developable for therapeutic use against HCV infection, either as monotherapy, or in combination with other HCV drugs.

Potential New Anti-Human Immunodeficiency Virus Type 1 Compounds Depress Virus Replication in Cultured Human Macrophages

ABSTRACT We report that the amiloride analogues 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)amiloride inhibit, at micromolar concentrations, the replication of human immunodeficiency virus type 1 (HIV-1) in cultured human blood monocyte-derived macrophages. These compounds also inhibit the in vitro activities of the HIV-1 Vpu protein and might represent lead compounds for a new class of anti-HIV-1 drugs.