The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine.

Hepatitis C virus (HCV) cannot be grown in vitro, making biochemical identification of new drug targets especially important. HCV p7 is a small hydrophobic protein of unknown function, yet necessary for particle infectivity in related viruses [Harada, T. et al., (2000) J. Virol. 74, 9498-9506]. We show that p7 can be cross-linked in vivo as hexamers. Escherichia coli expressed p7 fusion proteins also form hexamers in vitro. These and HIS-tagged p7 function as calcium ion channels in black lipid membranes. This activity is abrogated by Amantadine, a compound that inhibits ion channels of influenza [Hay, A.J. et al. (1985) EMBO J. 4, 3021-3024; Duff, K.C. and Ashley, R.H. (1992) Virology 190, 485-489] and has recently been shown to be active in combination with current HCV therapies.

High efficiency gene transfer into cultured primary rat and human hepatic stellate cells using baculovirus vectors.

BACKGROUND/AIMS: Gene transfer into hepatic stellate cells (HSC) is inefficient when using plasmid-based transfection methods; viral-based systems are therefore being developed. A baculovirus system has recently been shown to be useful for expressing genes in mammalian cells. The aim of this study was to determine if baculovirus vectors can infect and express target genes in rat and human HSC and to assess potential cytotoxic and modulatory effects of infection. METHODS: A recombinant baculovirus vector (AcCALacZ) carrying the LacZ gene was used to infect HSC. beta-Galactosidase assays and electron microscopy were used to determine efficiency of infection and gene expression. Counting of trypan blue negative cells was used to assess cytotoxic/cytostatic effects of infection. Measurement of protein content of cells and alpha-smooth muscle actin expression were performed to assess the effects of baculovirus on cell function/phenotype. RESULTS: Baculovirus infection of activated HSC was highly efficient (> 90%) and provided long-term LacZ gene expression (15 days) in the absence of cytotoxic, cytostatic or modulatory effects. Infection of freshly isolated cells was also observed but at lower levels (20%). CONCLUSIONS: Baculovirus vectors can therefore be used to deliver target genes to cultured rat and human HSC with high efficiency and longevity in the absence of detrimental effects on cell function.