These interfacial sequences are followed by the TMD (residues 684C704), which is mostly hydrophobic according to the Kyte-Doolittle hydropathy index (36). that mimic lipid composition and biophysical properties of the viral envelope. The data revealed that peptides endowed with virucidal activity were membrane active and induced permeabilization and fusion of virus-like lipid vesicles. In addition, they modulated lipid packing and miscibility of laterally segregated liquid domains, two properties that depend on the high cholesterol content of the viral membrane. Thus, the overall experimental evidence is consistent with a pattern of HIV inhibition that involves direct alteration of the physical chemistry of Mitoquinone the virus membrane. Furthermore, the sequence-dependent effects observed might guide the development of new virucidal peptides. Introduction Among the major drug classes approved for human immunodeficiency virus (HIV) antiretroviral therapy, entry inhibitors are unique at blocking the function of an extracellular target, the envelope glycoprotein (Env) (1, 2, 3). The Env subunits gp120 (surface) and gp41 (trans-membrane) are responsible for receptor/coreceptor binding and virus-cell fusion, respectively (4, 5, 6). Upon activation of the fusion process, gp41 ectodomain trimers refold into low-energy 6-helix bundles that pull cell and virus membranes into close contact. The conformational transition undergone by gp41 trimers constitutes the target Mitoquinone for the clinically approved fusion inhibitor Enfuvirtide (7), which blocks 6-helix bundle completion and hence membrane merger (8). Development of alternative fusion inhibitors displaying broad and sustained antiretroviral activity against HIV-1 remains a pursued goal to date (9, 10). It has been recently proposed that membrane-targeting virucidal compounds may comprise broad-spectrum inhibitors of enveloped virus entry (10, 11, 12). Arguably, compounds interfering with fusion by acting on the lipid component of the viral envelope could provide the basis for escape-proof antiviral therapies (13, 14). In addition, as opposed to host cell membranes GABPB2 that are subject to ongoing synthesis, degradation, and influx and efflux of their components, static viral membranes lacking repairing mechanisms cannot escape the major perturbations induced by virucide activity. In this regard, a series of?works support the Mitoquinone possibility that peptides derived from hydrophobic-at-interface envelope glycoprotein sequences may comprise new generic antivirals (15, 16, 17, 18, 19). Along this same line of thinking, in recent reviews it has been contended that these virucidal peptides could block viral entry by changing directly the physical chemistry of the viral membrane upon partitioning (20, Mitoquinone 21). We have recently established a synthetic virus-like (VL) mixture, which includes the main seven lipid species found in the HIV-1 membrane (22) and exhibits the same order degree and phase behavior (termed VL-4 in our previous report (23)). Here, to get new insights into the molecular mechanisms governing antiviral activity by membrane-partitioning peptides, we first compare the inhibitory potencies of several membrane-proximal external region-transmembrane domain (MPER-TMD)-derived peptides that possess distinct interfacial hydrophobicity patterns (20, 24), and then establish their membrane-restructuring capacities using the VL mixture as a surrogate of the HIV membrane. Our experimental data support a virucide-like activity that alters the physical chemistry of the HIV lipid envelope for peptides combining the hydrophobic-at-interface C-terminal MPER sequence with the N-terminal hydrophobic section of the TMD. We speculate that these sequences may serve as platforms for further development of antiretroviral peptides. Materials and Methods 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoylphosphatidylserine (POPS), cholesterol (Chol), egg sphingomyelin (SM, containing 86% were synthesized in C-terminal carboxamide form by solid-phase methods using Fmoc chemistry, purified by reverse-phase high-performance liquid chromatography, and characterized by matrix-assisted laser desorption ionizationCtime-of-flight mass spectrometry (purity 95%). Open in a separate window Figure 1 Designation of hydrophobic-at-interface HIV-1 peptides and their antiviral and membrane-restructuring activities. (is the intensity in the blue channel, and is the intensity in the green channel. The factor accounts for the relative sensitivity of the two channels, calibrated with a 5?displays the distribution of Wimley-White interfacial hydrophobicity (33) along the MPER-TMD region of HIV-1 gp41. At the N-terminus, an amphipathic-at-interface helix (residues 656C671) is followed by a fully hydrophobic-at-interface stretch (residues 672C683). Supporting the biological relevance of these MPER segments,.