Single Vesicle Analysis Reveals Nanoscale Membrane Curvature Selective Pore Formation in Lipid Membranes by an Antiviral α‑Helical Peptide Seyed R. Tabaei, † Michael Rabe, † Vladimir P. Zhdanov, †,‡ Nam-Joon Cho,* ,§ and Fredrik Hö ö k* ,† † Department of Applied Physics, Chalmers University of Technology, Gothenburg, Sweden ‡ Boreskov Institute of Catalysis, Russian Academy of Sciences, Novosibirsk, Russia § School of Materials Science and Engineering, Nanyang Technological University, Singapore * S Supporting Information ABSTRACT: Using tethered sub-100 nm lipid vesicles that mimic enveloped viruses with nanoscale membrane curvature, we have in this work designed a total internal reflection fluorescence microscopy-based single vesicle assay to inves- tigate how an antiviral amphipathic α-helical (AH) peptide interacts with lipid membranes to induce membrane curvature- dependent pore formation and membrane destabilization. Based on a combination of statistics from single vesicle imaging, binding kinetics data, and theoretical analysis, we propose a mechanistic model that is consistent with the experimentally observed peptide association and pore formation kinetics at medically relevant peptide concentrations (10 nM to 1 μM) and unusually low peptide-to-lipid (P/L) ratio (∼1/1000). Importantly, the preference of the AH peptide to selectively rupture virions with sub-100 nm diameters appears to be related to membrane strain-dependent pore formation rather than to previously observed nanoscale membrane curvature facilitated binding of AH peptides. Compared to other known proteins and peptides, the combination of low effective P/L ratio and high specificity for nm-sized membrane curvature lends this particular AH peptide great potential to serve as a framework for developing a highly specific and potent antiviral agent for prophylactic and therapeutic applications while avoiding toxic side effects against host cell membranes. KEYWORDS: Single molecule fluorescence microscopy, lipid vesicles, peptide nucleation, pore formation, membrane curvature, antiviral peptides T he rapid emergence of drug-resistant strains among viruses places a great burden on antiviral drug discovery and development. A principle challenge is the predominant selection of antiviral targets that are encoded by the viral genome whose replication machinery is error prone and therefore generates a diversity of mutant strains that can evade administered drugs. For lipid-enveloped viruses, such as HIV, hepatitis C, and influenza, an alternative targeting approach exists. All of these viruses feature a conserved structural component that is critical for the virus life cycle and yet not encoded within the virus genome. This Achilles’ heel is the virus particle’s lipid envelope which is derived from host cell membranes. Identification of agents that interfere with the envelope coating thus represents an important strategy toward the development of broad-spectrum antiviral agents. In the course of studying the membrane-associating proper- ties of an amphipathic α-helical (AH) peptide derived from the N-terminus of the hepatitis C virus (HCV) NS5A protein that is necessary for HCV genome replication, one such potential antiviral agent was discovered. 1 The peptide, here referred to as AH peptide, was observed to rupture both suspended and surface-immobilized lipid vesicles, which in these initial studies served as models for the lipid envelopes of virus particles. 2,3 Through further characterization efforts, the vesicle lysing property was shown to be dependent on vesicle size, and the size range leading to efficient rupture (complete lysis for vesicles with diameter of <75 nm) corresponded well with several medically important viruses including HIV, HCV, herpes simplex, and dengue. 3 Despite the simplicity of lipid vesicles made of a single lipid component, in vitro studies confirmed this hypothesis by demonstrating that the AH peptide could act as an antiviral agent capable of disrupting small-scale lipid-enveloped virions. 3-5 Recently, results very similar to those observed for AH peptide induced rupture of adsorbed POPC lipid vesicles were obtained for vesicles with a composition mimicking that of HIV, 6 which further supports a membrane curvature selective mode of action of this AH peptide. This surprising selectivity against highly curved Received: August 9, 2012 Revised: October 18, 2012 Published: October 24, 2012 Letter pubs.acs.org/NanoLett © 2012 American Chemical Society 5719 dx.doi.org/10.1021/nl3029637 | Nano Lett. 2012, 12, 5719-5725