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• W1986824098 abstract "Double-stranded RNA (dsRNA) virions constitute transcriptionally competent machines that must translocate across cell membranes to function within the cytoplasm. The entry mechanism of such non-enveloped viruses is not well described. Birnaviruses are unique among dsRNA viruses because they possess a single shell competent for entry. We hereby report how infectious bursal disease virus, an avian birnavirus, can disrupt cell membranes and enter into its target cells. One of its four structural peptides, pep46 (a 46-amino acid amphiphilic peptide) deforms synthetic membranes and induces pores visualized by electron cryomicroscopy, having a diameter of less than 10 nm. Using both biological and synthetic membranes, the pore-forming domain of pep46 was identified as its N terminus moiety (pep22). The N and C termini of pep22 are shown to be accessible during membrane destabilization and pore formation. NMR studies show that pep46 inserted into micelles displays a cis-trans proline isomerization at position 16 that we propose to be associated to the pore formation process. Reverse genetic experiments confirm that the amphiphilicity and proline isomerization of pep46 are both essential to the viral cycle. Furthermore, we show that virus infectivity and its membrane activity (probably because of the release of pep46 from virions) are controlled differently by calcium concentration, suggesting that entry is performed in two steps, endocytosis followed by endosome permeabilization. Our findings reveal a possible entry pathway of infectious bursal disease virus: in endosomes containing viruses, the lowering of the calcium concentration promotes the release of pep46 that induces the formation of pores in the endosomal membrane. Double-stranded RNA (dsRNA) virions constitute transcriptionally competent machines that must translocate across cell membranes to function within the cytoplasm. The entry mechanism of such non-enveloped viruses is not well described. Birnaviruses are unique among dsRNA viruses because they possess a single shell competent for entry. We hereby report how infectious bursal disease virus, an avian birnavirus, can disrupt cell membranes and enter into its target cells. One of its four structural peptides, pep46 (a 46-amino acid amphiphilic peptide) deforms synthetic membranes and induces pores visualized by electron cryomicroscopy, having a diameter of less than 10 nm. Using both biological and synthetic membranes, the pore-forming domain of pep46 was identified as its N terminus moiety (pep22). The N and C termini of pep22 are shown to be accessible during membrane destabilization and pore formation. NMR studies show that pep46 inserted into micelles displays a cis-trans proline isomerization at position 16 that we propose to be associated to the pore formation process. Reverse genetic experiments confirm that the amphiphilicity and proline isomerization of pep46 are both essential to the viral cycle. Furthermore, we show that virus infectivity and its membrane activity (probably because of the release of pep46 from virions) are controlled differently by calcium concentration, suggesting that entry is performed in two steps, endocytosis followed by endosome permeabilization. Our findings reveal a possible entry pathway of infectious bursal disease virus: in endosomes containing viruses, the lowering of the calcium concentration promotes the release of pep46 that induces the formation of pores in the endosomal membrane. Biological membranes represent a physical barrier that isolates cellular components from the external environment. Exchanges between cells and their surroundings are brought about by structural changes of the lipid bilayer that lead either to fusion events (vesicular trafficking) or to the formation of pores (exchange through channels). Membrane-enveloped and non-enveloped viruses represent a paradigm for the study of membrane deformations (1Smith A.E. Helenius A. Science. 2004; 304: 237-242Crossref PubMed Scopus (615) Google Scholar). Enveloped viruses carry membrane-anchored glycoproteins that mediate fusion between viral and cellular membranes. The considerable number of studies carried out on viral glycoproteins, in particular the large number of x-ray crystal structure determinations (2Wilson I.A. Skehel J.J. Wiley D.C. Nature. 1981; 289: 366-373Crossref PubMed Scopus (1967) Google Scholar, 3Bullough P.A. Hughson F.M. Skehel J.J. Wiley D.C. Nature. 1994; 371: 37-43Crossref PubMed Scopus (1368) Google Scholar, 4Roche S. Bressanelli S. Rey F.A. Gaudin Y. Science. 2006; 313: 187-191Crossref PubMed Scopus (351) Google Scholar, 5Heldwein E.E. Lou H. Bender F.C. Cohen G.H. Eisenberg R.J. Harrison S.C. Science. 2006; 313: 217-220Crossref PubMed Scopus (465) Google Scholar) have shed light on the membrane fusion mechanism. In contrast, the entry pathway of non-enveloped viruses is not as well understood. For positive-strand RNA (+sRNA) viruses such as polio-virus, binding of the virus to its receptor results in large capsid rearrangements. The hydrophobic N terminus of the VP1 protein moves to the particle surface while the myristoylated internal protein VP4 inserts into the target membrane (6Hogle J.M. Annu. Rev. Microbiol. 2002; 56: 677-702Crossref PubMed Scopus (261) Google Scholar, 7Bubeck D. Filman D.J. Hogle J.M. Nat. Struct. Mol. Biol. 2005; 12: 615-618Crossref PubMed Scopus (71) Google Scholar). Noda-viruses, others +sRNA viruses, have a unique capsid protein (8Wery J.P. Reddy V.S. Hosur M.V. Johnson J.E. J. Mol. Biol. 1994; 235: 565-586Crossref PubMed Scopus (74) Google Scholar) associated to a peptide, the γ peptide (44 residues). Both capsid protein and peptide result from the self-cleavage of a capsid protein precursor. The γ peptide has the capacity to permeabilize biological membranes allowing genome translocation through the membrane (9Bong D.T. Steinem C. Janshoff A. Johnson J.E. Reza Ghadiri M. Chem. Biol. 1999; 6: 473-481Abstract Full Text PDF PubMed Scopus (59) Google Scholar, 10Janshoff A. Bong D.T. Steinem C. Johnson J.E. Ghadiri M.R. Biochemistry. 1999; 38: 5328-5336Crossref PubMed Scopus (66) Google Scholar). The recent determination of the atomic structure of the γ peptide membrane-active domain demonstrates similarities with the fusion peptides of glycoproteins of enveloped viruses: both are formed by a kinked helix (11Maia L.F. Soares M.R. Valente A.P. Almeida F.C. Oliveira A.C. Gomes A.M. Freitas M.S. Schneemann A. Johnson J.E. Silva J.L. J. Biol. Chem. 2006; 238: 29278-29286Abstract Full Text Full Text PDF Scopus (23) Google Scholar). The γ peptide is located inside the capsid and is brought toward the membrane during entry. For these viruses, all these rearrangements are believed to result in the formation of a narrow channel that allows the genomic RNA to reach the cytoplasm. In contrast to +sRNA viruses, dsRNA viruses have to maintain their genome hidden from the cellular defense mechanisms at all steps of the viral cycle. Consequently, a large object, the capsid-protected genome, needs to cross the cell membrane. Most dsRNA viruses possess several concentric shells. During entry, the external capsid layer is generally lost and its constitutive proteins, or their cleavage products, are thought to induce a local destabilization of the cellular membrane allowing virus translocation (12Chandran K. Nibert M.L. Trends Microbiol. 2003; 11: 374-382Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Recently, it has been shown that mammalian reovirus produce a myristoylated peptide (μ1N) that can induce size-selective pores in membranes (13Agosto M.A. Ivanovic T. Nibert M.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16496-16501Crossref PubMed Scopus (87) Google Scholar). The release of this peptide associated to pore formation (13Agosto M.A. Ivanovic T. Nibert M.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16496-16501Crossref PubMed Scopus (87) Google Scholar) and structural rearrangements of the remaining domain of μ1 (μ1 C) (14Zhang L. Chandran K. Nibert M.L. Harrison S.C. J. Virol. 2006; 80: 12367-12376Crossref PubMed Scopus (39) Google Scholar) are thought to lead to virus entry into the cell. Birnaviruses only possess a single-layered capsid (15Coulibaly F. Chevalier C. Gutsche I. Pous J. Navaza J. Bressanelli S. Delmas B. Rey F.A. Cell. 2005; 120: 761-772Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) that is assumed to be competent for both virus translocation and genome replication-transcription. For all dsRNA 5The abbreviations used are: dsRNA, double-stranded RNA; LDH, lactate dehydrogenase; CF, carbofluoresceine; IBDV, infectious bursal disease virus; DPC, dodecylphosphocholin; VLP, virus-like particles; r.m.s.d., root mean-squared deviation. 5The abbreviations used are: dsRNA, double-stranded RNA; LDH, lactate dehydrogenase; CF, carbofluoresceine; IBDV, infectious bursal disease virus; DPC, dodecylphosphocholin; VLP, virus-like particles; r.m.s.d., root mean-squared deviation. viruses, the mechanisms of membrane destabilization associated to entry remain hypothetical. Totiviruses, another group of dsRNA viruses that lack additional shells constitute a special case because they are not able to enter into a cell and are therefore only transmitted during cell division.The capsid of birnaviruses is formed by 260 VP2 protein trimers (15Coulibaly F. Chevalier C. Gutsche I. Pous J. Navaza J. Bressanelli S. Delmas B. Rey F.A. Cell. 2005; 120: 761-772Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 16Saugar I. Luque D. Ona A. Rodriguez J.F. Carrascosa J.L. Trus B.L. Caston J.R. Structure. 2005; 13: 1007-1017Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) which obey T = 13laevo icosahedral symmetry (17Bottcher B. Kiselev N.A. Stel'Mashchuk V.Y. Perevozchikova N.A. Borisov A.V. Crowther R.A. J. Virol. 1997; 71: 325-330Crossref PubMed Google Scholar, 18Pous J. Chevalier C. Ouldali M. Navaza J. Delmas B. Lepault J. J. Gen. Virol. 2005; 86: 2339-2346Crossref PubMed Scopus (28) Google Scholar). VP3, the second major viral protein, binds the RNA-dependent RNA polymerase VP1 and the genomic dsRNA (19Tacken M.G. Peeters B.P. Thomas A.A. Rottier P.J. Boot H.J. J. Virol. 2002; 76: 11301-11311Crossref PubMed Scopus (77) Google Scholar, 20Lombardo E. Maraver A. Cast n J.R. Rivera J. Fernandez-Arias A. Serrano A. Carrascosa J.L. Rodriguez J.F. J. Virol. 1999; 73: 6973-6983Crossref PubMed Google Scholar). Birnaviruses present characteristic peptides associated to the virus particle (21Delmas B. Kibenge F.S.B. Leong J.C. Mundt E. Vakharia V.N. Wu J.L. Birnaviridae. Academic Press, London2005Google Scholar). Four peptides (pep46, pep7a, pep7b, and pep11) being 46, 7, 7, or 11 amino acids in length, respectively, are found in the case of infectious bursal disease virus (IBDV) (22Da Costa B. Chevalier C. Henry C. Huet J.C. Petit S. Lepault J. Boot H. Delmas B. J. Virol. 2002; 76: 2393-2402Crossref PubMed Scopus (108) Google Scholar). These peptides are generated through the processing of the viral polyprotein pVP2-VP4-VP3 (Fig. 1). VP4 is a protease that cleaves its own N and C termini, thus releasing pVP2 and VP3 within the infected cell (23Birghan C. Mundt E. Gorbalenya A.E. EMBO J. 2000; 19: 114-123Crossref PubMed Scopus (166) Google Scholar). Subsequent serial cleavages at the C terminus of pVP2 yield the mature VP2 capsid protein and the four peptides.In this study, we show that pep46 deforms biological membranes, leading to the formation of pores. We determined by NMR the atomic structure of this peptide. The membrane-active domain is formed by two kinked α-helices linked by a proline displaying cis-trans isomerization that controls peptide hydrophobicity. These results combined with reverse genetic studies permit the proposition of a general model describing the cell entry pathway of a non-enveloped virus.EXPERIMENTAL PROCEDURESPeptide Synthesis—The peptides were obtained by automated solid-phase synthesis using the 9-fluorenylmethoxy carbonyl strategy and purified by reversed-phase high-performance liquid chromatography using standard procedures. The peptides were analyzed by mass spectrometry and confirmed to have purity higher than 98%.Peptide Activity—The hepatocarcinoma epithelial chicken LMH cell line was grown in RPMI medium supplemented with 10% fetal calf serum, 2 mm glutamine, and 1 mm sodium pyruvate. The activity of the peptides was determined by studying their effects on live cells. After incubation at 37 °C, LDH release was measured using the cytotox 96 nonradioactive kit (Promega), as described by the manufacturer. The percentage of LDH release was determined taking in account the OD values found on untreated and Triton X-100-treated cells. All experiments were carried out five times on duplicated samples; all measurements gave similar results. Liposomes containing carbofluoresceine (CF) were prepared as described by Nandi et al. (24Nandi P. Charpilienne A. Cohen J. J. Virol. 1992; 66: 3363-3367Crossref PubMed Google Scholar). The release of CF after addition of peptides or virus was monitored by the increase of fluorescence at 520 nm, using a 492-nm excitation band in a thermostatted Perkin-Elmer LS50B spectrofluorimeter. The value for 100% release was obtained by addition of Triton X-100 at the end of the reaction. All fluorescent experiments were carried out at least three times and gave similar results.Electron Cryomicroscopy—Microscopy was performed as previously described (25Adrian M. Dubochet J. Lepault J. McDowall A.W. Nature. 1984; 308: 32-36Crossref PubMed Scopus (947) Google Scholar). A drop of the studied sample was adsorbed to air glow-discharged holey carbon films. The excess of liquid was removed and the grid frozen in liquid nitrogen cooled ethane. The grid was transferred into a Gatan 6226 cryo-holder and observed with a Philips CM12 electron microscope operated at 80 kV. The micrographs were recorded at a magnification of 35,000 under standard low dose exposition conditions.Sample Preparation and NMR Spectroscopy—Pep46 was dissolved at pH 3.5 in an aqueous 40 mm dodecylphosphocholin (DPC) solution at a final concentration of 1 mm. Two-dimensional phase-sensitive 1H Clean-TOCSY with 70 ms spin lock, and 100 ms and 200 ms mixing times NOESY experiments (26Kumar A. Ernst R.R. Wuthrich K. Biochem. Biophys. Res. Commun. 1980; 95: 1-6Crossref PubMed Scopus (2014) Google Scholar) were recorded at 313 K and 333 K on an AVANCE Bruker spectrometer operating at 600.14 MHz without sample spinning with 2K real points in t2, with a spectral width of 6000 Hz and 512 t1-increments. Pulsed-field gradients (27Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3500) Google Scholar) were used for water suppression. The data were processed using XWIN-NMR software (Bruker). A π/6 phase-shifted sine bell window function was applied prior to Fourier transformation in both dimensions (t1 and t2). The temperature was externally controlled using a special temperature control system (BCU 05 Bruker).NMR Structure Determination—NOE cross-peak volumes measured on NOESY spectrum (NOESY, 200 ms mixing time, 333 K) were converted into distances, semiquantitatively, by counting contour levels. Using the Tyr-2,6H geminal, and Asp-Hβ protons as calibration peak, NOE signals were classified into 5 categories with upper distance limits ranging from 2.5 to 5 Å. Pseudo-atom corrections were added when necessary. Calculations were performed using the standard procedures in X-PLOR version 3.84 (28Nilges M. Clore G.M. Gronenborn A.M. FEBS Lett. 1988; 239: 129-136Crossref PubMed Scopus (524) Google Scholar, 29Brünger A.T. Adams P.D. Clore J.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Acta. Crystallogr. 1998; D54: 905-921Crossref Scopus (16929) Google Scholar). Quality of structures was evaluated with PROCHECK (30Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4329) Google Scholar).Virus Rescue Experiments—To generate infectious IBDV mutants from cDNA clones, a previously described strategy was used (22Da Costa B. Chevalier C. Henry C. Huet J.C. Petit S. Lepault J. Boot H. Delmas B. J. Virol. 2002; 76: 2393-2402Crossref PubMed Scopus (108) Google Scholar). Cells at 90% confluence in P12 wells were infected with the vaccinia virus MVA-T7 at a multiplicity of infection of 1. After a 1-h adsorption, the cells were rinsed with the RPMI medium. In the mean time, pT7-A-HDR derivates and pT7-B-HDR plasmids allowing the transcription of genome segments A and B, respectively, were mixed (0.5 μg of each plasmid) with 3.5 μl of Lipofectamine 2000 (Invitrogen) in 350 μl of Opti-MEM and were kept at room temperature for 30 min. The cells were again rinsed in OptiMEM and incubated with the DNA-Lipofectamine 2000 mixture at 37 °C for 5 h. Next, 1.5 ml of complemented RPMI medium was added to each well. Recombinant mutant viruses were recovered 48-h post-transfection after filtration through a 0.22-μm-pore-size filter. At least three independent transfection experiments were carried out to analyze each pT7-A-HDR derivative. The viruses were amplified on LSCC-BK3 cells. IBDV-infected LSCC-BK3 cells were analyzed 3 or 5 days postinfection. Briefly, the cells were fixed with 2.5% PFA in phosphate-buffered saline for 30 min at room temperature and permeabilized by incubation for 10 min in 0.1% Triton X-100. Fixed cells were incubated with a 1:250 dilution of an anti-VP3 monoclonal antibody. Next, the cells were rinsed and incubated with an anti-immunoglobin mouse fluorescein isothiocyanate conjugate in phosphate-buffered saline-0.05% Tween. The cells were rinsed three times and subjected to FACScalibur (Becton Dickinson) analysis using the Cell Quest Pro software.VLP-GFP Cell Binding Assay—Fluorescent VLP were produced as described by Chevalier et al. (31Chevalier C. Lepault J. Erk I. Da Costa B. Delmas B. J. Virol. 2002; 76: 2384-2392Crossref PubMed Scopus (67) Google Scholar). The VLP were incubated on permissive and non-permissive cells at 4 °C for 30 min in the presence and absence of calcium. The cells were rinsed twice with OptiMEM (Invitrogen) containing the adequate concentration of calcium and analyzed with the FACScalibur.RESULTS AND DISCUSSIONThe Membrane-active Domain of pep46—Fig. 1A shows the schematic representation of the IBDV polyprotein maturation process. A sequence alignment between different birnavirus pep46 homologues reveals only six conserved residues, three of them proline. All sequences display identical clusters of similar amino acids allowing the definition of three domains: the central hydrophobic domain containing most of the conserved residues (three prolines at positions 16, 23, and 27, a threonine at position 20 and the following leucine) and the two flanking domains mainly constituted by both hydrophobic and charged residues. Whereas the N-terminal domain is positively charged, the C-terminal is some-what negatively charged. The N-terminal domain is predicted as forming an amphipathic α-helix with positively charged residues on one side of the helix and hydrophobic residues on the other side (22Da Costa B. Chevalier C. Henry C. Huet J.C. Petit S. Lepault J. Boot H. Delmas B. J. Virol. 2002; 76: 2393-2402Crossref PubMed Scopus (108) Google Scholar). As far as the positions of charged and hydrophobic residues are concerned, the sequence similarity between pep46 of different IBDV strains (data not shown) and different birnaviruses (Fig. 1A) is higher in the N-terminal than in the C-terminal domain, suggesting a critical role of this α-helix in the viral cycle. The overall hydrophobicity of pep46 strongly suggests potential interactions of this peptide with membranes. Fig. 1B shows that cells incubated with pep46 undergo spectacular morphological modifications characterized by significant shrinkage and cytoplasm vacuolization. Video light microscopy shows that pep46-treated cells first swell and then undergo important membrane deformations that are associated to massive losses of cellular material (not shown). All these changes result in round ghost cells that have lost their initial morphology (Fig. 1B). As already shown for mammalian reoviruses (Agosto et al., 13Agosto M.A. Ivanovic T. Nibert M.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16496-16501Crossref PubMed Scopus (87) Google Scholar), these observations can be interpreted as arising from partial permeabilization of the cellular membrane that leads to the establishment of a high osmotic pressure, resulting in severe cell damage. To identify the membrane-active domain of pep46, several peptides derived from its primary sequence, were synthesized and their effects analyzed on cells. Peptides made of the first 15 (pep15), of the 22 N-terminal amino acids (pep22) and of the last 24 C-terminal residues (pep24) were synthesized (Fig. 1B). Although attenuated, the effects of both pep22 and pep15 were similar to that of pep46 with a rounding of the cells and a loss of cellular material. While pep22 was found to be more efficient than pep15, pep24 had no effect on cells. All these observations demonstrate that pep46 and in particular its N-terminal moiety, pep22, destabilize cell membranes.The Permeabilizing Activity of pep46—The permeabilizing activity of pep46 and its shorter derivates, pep22, pep15, and pep24 was quantified by measuring the release of a cytoplasmic enzyme (LDH) induced by each peptide. Although the results are only illustrated for an IBDV-permissive cell line (LMH cells; Fig. 2), similar data were obtained for both permissive and non-permissive cells. The effects of the peptides were analyzed for time (Fig. 2A) and concentration (Fig. 2B) dependence. Fig. 2A shows that the peptides permeabilize membranes with characteristic kinetic constants and lag periods. While a lag period cannot be evaluated in the case of pep46, its existence is clearly visible for pep22 and pep15. Pep15 displays a longer lag period than pep22. The existence of a lag period indicates that the membrane permeabilization is a multi-step event. No activity could be detected for pep24. Fig. 2B shows that all peptides are characterized by a critical concentration below which the peptides have no effect on the LDH release. The critical concentration increases evenly following the order of the series: pep46, pep22, and pep15. No effect could be detected for pep24 up to a concentration of 20 μm. All these experiments demonstrate that pep46 and particularly its N-terminal moiety permeabilize cell membranes. To assess the efficiency of pep46, we compared its activity to the one of the membrane-active domain (γ1) of the nodavirus γ peptide. We found that γ1 is 10-20-fold less efficient than pep22. While it can be seen on Fig. 2B that the critical concentration of pep22 is about 3 μm, the one of γ1 peptide is equal to 30-50 μm (data not shown).FIGURE 2Membrane permeabilization activity of pep46 and derivates. A and B show the peptide activity on cells quantified by LDH release and expressed as a percentage of the total cellular LDH. A, effect of time on LDH release. Peptides were incubated with cells at a concentration of 10 μm. The amino acid sequence of pep22P16A is identical to the one of pep22 except proline in position 16 that is substituted by an alanine. No release was observed with pep24. B, effect of concentration on LDH release. The activities of pep15 and pep22P16A display the same concentration dependence. Again, no activity could be observed with pep24. C and D show the release of the fluorescent probe from synthetic liposomes. The peptide activity was quantified by the fluorescent probe leakage and expressed as a percentage of the total unquenched fluorescence (when liposomes were treated with Triton X-100). In A and B, results are mean ± S.D. of five distinct experiments. C, activities of pep22 and pep24 at a concentration of 1 μm were compared. Triton X-100 indicates the time at which Triton X-100 has been added to the samples to validate the quality of the liposomes containing the fluorescent probe. Again, no activity of pep24 was detected. D, effect of peptide concentration and time. The activity of pep46 was measured and found concentration dependent in the nanomolar to micromolar range. E, comparison of the effects of pep46 and pep22 on liposomes. F, effect of avidin binding at the N and C terminus of pep22.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The membrane permeabilization induced by pep46 could depend on a perturbation of the metabolism that controls cellular entry processes such as endocytosis. To test this hypothesis, we analyzed the influence of a metabolic inhibitor. The depletion of the cellular ATP pool did not influence the activity of pep46 and its shorter derivates (not shown). Therefore, pep46 and in particular, its N-terminal moiety (pep22), display a membrane activity that is energy-independent. All these features were observed on permissive and non-permissive cells, demonstrating that pep46 or derivates interact with the lipid bilayer, resulting in the formation of a pore, even in the absence of a specific receptor.To further characterize the pore formation mechanism, we studied the activity of these peptides on synthetic membranes. The peptides were incubated with liposomes containing a fluorescent probe, which due to quenching effects has a fluorescence signal strongly depending upon concentration. The release of the probe from the liposomes leads to a lowering of the probe concentration and thus to an increase of the fluorescence signal. With pep22, at a concentration of 1 μm, the release of the probe from liposomes is achieved within a few seconds (Fig. 2C). Up to a concentration of 2 mm, the release is independent of the presence of calcium or a chelating molecule (EDTA, data not shown). With this assay as with others, pep24 is found to be inactive on membranes. Fig. 2, D and E panels illustrate the results obtained with both pep46 and pep22. The release of the probe from the liposomes has kinetic characteristics similar to those previously described on cultured cells: the existence of a critical concentration and short lag period particularly visible at low concentrations. Because of the membrane composition and cell organization, the fluorescence assay on liposomes allows the detection of pores at concentrations that are three orders of magnitude lower than the LDH release assay.Polarity in the Interaction of pep22 with Membranes—To analyze the importance of the N and C termini of pep22 in membrane destabilization, a reporter protein, fluorescent streptavidin, was attached either at its N or C terminus. The labeled pep22 were added to liposomes containing a fluorescent probe. Fig. 2F shows that the binding of avidin to either the N or the C termini of pep22 inhibits, but does not block the release of the probe from the liposomes treated with the peptide. These results demonstrate that the addition of an exogenous charged polypeptide at both termini does not prevent the formation of pores in synthetic membranes. Both peptide termini are thus accessible during the pore formation process and remain in the vicinity of the polar heads of the membrane.Visualization of the Pores by Electron Cryomicroscopy—The deformations of synthetic membranes induced by pep46 and shorter derivates were analyzed by electron cryomicroscopy. Fig. 3a shows liposomes visualized in the absence (left) and presence (right) of pep22. Pep46 has similar effects on liposomes than pep22. At low magnification, both samples appear similar. Although multi-lamellar vesicles are visible, most of the liposomes are uni-lamellar and characterized by heterogeneous diameter values. Polar head and aliphatic chain domains of the lipid constituting the liposome bilayer are easily discerned. At higher magnification, a significant effect of pep22 on liposomes is observed (Fig. 3b). Whereas in the absence of pep22, the polar heads constituting the liposome bilayers form lines that are parallel over long distances (left), in the presence of pep22, the lines are parallel over much smaller distances (right). In fact, in this latter case, the bilayer thickness varies giving rise to areas characterized by the presence of parallel lines that rapidly alternate with fuzzy lines. In accordance with this observation, the contrast of the membrane is lowered when pep22 is added to the liposomes. The fuzzy domains often show fusion of the external and internal leaflets of the bilayers (Fig. 3c) demonstrating the formation of an aqueous channel within the membrane that will be defined as a pore. The diameter of the pore is not constant; while it is smaller than the picture resolution (about 2 nm) in the left and middle panels, it is larger in the right panel, but the diameter of the pores were always found less than 10 nm. In all cases, the diameter of the pore is smaller than the diameter of the IBDV virion (60-70 nm). No significant effect of pep24 on liposomes could be observed (not shown).FIGURE 3Visualization by cryomicroscopy of the structural effects induced by pep22. a, panels show the liposomes in the absence (left) and the presence (right) of pep22. In both cases, liposomes having various diameters are observed. In general, they are unilamellar. The bilayered structure of the membrane is clearly visible. b, when the membranes are observed at higher magnification, significant differences are observed in the presence of pep22. While the position of the polar heads of the lipids gives rise to parallel lines over long distances when" @default.
• W1986824098 created "2016-06-24" @default.
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• W1986824098 date "2007-07-01" @default.
• W1986824098 modified "2023-10-14" @default.
• W1986824098 title "Infectious Bursal Disease Virus, a Non-enveloped Virus, Possesses a Capsid-associated Peptide That Deforms and Perforates Biological Membranes" @default.
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