A virocidal amphipathic {alpha}-helical peptide that inhibits hepatitis C virus infection in vitro

Abstract

An amphipathic alpha-helical peptide (C5A) derived from the membrane anchor domain of the hepatitis C virus (HCV) NS5A protein is virocidal for HCV at submicromolar concentrations in vitro. C5A prevents de novo HCV infection and suppresses ongoing infection by inactivating both extra- and intracellular infectious particles, and it is nontoxic in vitro and in vivo at doses at least 100-fold higher than required for antiviral activity. Mutational analysis indicates that C5A's amphipathic alpha-helical structure is necessary but not sufficient for its virocidal activity, which depends on its amino acid composition but not its primary sequence or chirality. In addition to HCV, C5A inhibits infection by selected flaviviruses, paramyxoviruses, and HIV. These results suggest a model in which C5A destabilizes viral membranes based on their lipid composition, offering a unique therapeutic approach to HCV and other viral infections.

Fig. 1.

Identification of antiviral peptides. A library consisting of 441 overlapping peptides (18-mers) covering the entire HCV polyprotein (H77 strain, genotype 1a) was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program and screened at a concentration of 20 μM for the ability to inhibit HCV infection (JFH-1) in a focus reduction assay in Huh-7.5.1 cells, as described in Materials and Methods. The number of fluorescent HCV-positive foci was counted and expressed as a percentage (%) of foci in cells inoculated with virus and 0.5% DMSO. The peptide that displayed the strongest inhibition (termed C5A in this paper) is highlighted. For all subsequent experiments, this peptide was resynthesized to 95% purity.

Fig. 2.

C5A is virocidal for HCV. (a) To determine the peptide concentration required to inhibit HCV infection by 50% (IC₅₀), >95% pure peptide stock solutions (3.6 mM in DMSO) were serially diluted in DMSO and tested for inhibitory activity as described in Materials and Methods. Peptide cytotoxic activity was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay according to the manufacturer's instructions (American Type Culture Collection Catalogue no. 30–1010K), as described in SI Text. The peptide concentration that reduced the cell growth by 50% was designated the LC₅₀. (b) C5A was either added to the cells for 4 h and removed before inoculation (−4 h); together with virus at the time of inoculation (0h) or 4 h postinoculation after virus had been removed by washing (+4 h). The −4 h and 0 h cells were washed after 4 h incubation and replaced with virus/peptide-free medium, whereas the peptide remained in the +4 h cultures throughout the experiment. Twenty-four and 72 h postinfection, cells were lysed for HCV RNA quantitation. (c) Peptide (18 μM in 0.5% DMSO) and 0.5% DMSO without peptide were added to complete medium containing 5 × 10⁵ FFU/ml of HCV and incubated at 37°C for 1 h, at which point the virus–peptide and virus–DMSO mixtures were analyzed for total HCV RNA and infectivity. (d) The l- and d-isomers of C5A (18 μM) or 0.5% DMSO were incubated with virus (1 × 10⁵ FFU/ml) for 4 h at 37°C, and 100-μl samples were analyzed by velocity sedimentation ultracentrifugation as described (15). (e) To examine the ability of C5A to permeabilize lipid membranes, a liposome dye release assay was performed as described in Materials and Methods.

Fig. 3.

C5A prevents initiation of HCV infection and suppresses established infection. (a) Huh-7.5.1 cells were inoculated with HCV (moi 0.1) and C5A (18 μM) or 0.5% DMSO. After adsorption for 4 h at 37°C, the inocula were removed, the cells were washed two times, overlaid with 120 μl of fresh growth medium, and incubated at 37°C. At the indicated time points, total cellular HCV RNA was measured. (b) Huh-7 cells were infected with HCV (moi 0.1). On day 3 postinfection, when ≈10% of the cells were HCV E2-positive, C5A (18 μM) or 0.5% DMSO were added, and the cells were incubated at 37°C and split 1:6 every 3–4 days when confluent. C5A and DMSO were replenished when the cells were split, at which point total cellular HCV RNA was quantitated. (c) Huh-7 cells were pretreated with 1% DMSO for 10 days to induce differentiation and growth arrest, at which time they were infected by HCV (moi 0.01). Fifteen days after infection, when >90% cells were HCV E2-positive, the l- and d-isomers of C5A (18 μM) were added, and the cells were replenished every day with medium containing fresh peptide. At the indicated time points, total cellular HCV RNA content was measured. For comparison, the infected cells were treated with 100 units/ml of recombinant human IFNα (PBL Biomedical Laboratories) and replenished daily as above.

Fig. 4.

C5A inhibits intracellular HCV particle infectivity. (a) To determine whether C5A enters the cells, a fluorescent C5A containing a dansyl group at its N terminus (18 μM) was incubated with Huh-7 cells for 4 h at 37°C, washed five times with PBS, fixed with 4% paraformaldehyde, immunostained with rabbit polyclonal anti-dansyl antibody (Molecular Probes), and analyzed by confocal fluorescence microscopy. (b) Huh-7 cells previously infected with JFH-1 for 10 days were washed four times, and treated with d-isomers of C5A (18 μM) or DMSO (0.5%). After 6 h, intracellular HCV infectivity, extracellular HCV infectivity, and cellular HCV RNA content were determined as described in Materials and Methods.