1

Infectious bursal disease virus, a non-enveloped virus, possesses a capsid-

associated peptide that deforms and perforates biological membranes

Marie Galloux, Sonia Libersou1, Nelly Morellet

2, Serge Bouaziz

2, Bruno Da

Costa, Malika Ouldali, Jean Lepault1*

and Bernard Delmas*

Unité de Virologie et Immunologie moléculaires, UR892, Batiment de Biotechnologies,

INRA, Domaine de Vilvert, 78350 Jouy-en-Josas, 1 Laboratoire de Virologie Moléculaire

et Structurale, CNRS, UMR 2472, IFR 115, INRA, UMR 1157, 91198 Gif-sur-Yvette, 2

Unité de Pharmacologie chimique et génétique, INSERM, U640, CNRS, UMR 8151,

75006 Paris, France.

Running title: A viral peptide deforms and perforates membrane

Address correspondence: bernard.delmas@jouy.inra.fr Tel: 33 1 3465 2627 Fax: 33 1 3465

2621, or jean.lepault@vms.cnrs-gif.fr Tel: 33 1 6982 3855

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 since 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 cryo-

microscopy, having a diameter 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 due to 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 aphysical barrier that isolates cellularcomponents from the external environment.Exchanges between cells and theirsurroundings are brought about by structural

http://www.jbc.org/cgi/doi/10.1074/jbc.M701048200The latest version is at JBC Papers in Press. Published on May 8, 2007 as Manuscript M701048200

Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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changes of the lipid bilayer that lead either tofusion events (vesicular trafficking) or to theformation of pores (exchange throughchannels). Membrane-enveloped and non-enveloped viruses represent a paradigm forthe study of membrane deformations (1).Enveloped viruses carry membrane-anchoredglycoproteins that mediate fusion betweenviral and cellular membranes. Theconsiderable number of studies carried outon viral glycoproteins, in particular the largenumber of X-ray crystal structuredeterminations (2-5), have shed light on themembrane fusion mechanism. In contrast,the entry pathway of non-enveloped virusesis not as well understood. For positive-strandRNA (+sRNA) viruses such as poliovirus,binding of the virus to its receptor results inlarge capsid rearrangements. Thehydrophobic N-terminus of the VP1 proteinmoves to the particle surface while themyristoylated internal protein VP4 insertsinto the target membrane (6,7). Nodaviruses,others +sRNA viruses, have a unique capsidprotein (8) associated to a peptide, the peptide (44 residues). Both capsid proteinand peptide result from the self-cleavage of acapsid protein precursor. The peptide hasthe capacity to permeabilize biologicalmembranes allowing genome translocationthrough the membrane (9,10). The recentdetermination of the atomic structure of the

peptide membrane-active domaindemonstrates similarities with the fusionpeptides of glycoproteins of envelopedviruses: both are formed by a kinked helix(11). The peptide is located inside thecapsid and is brought towards the membraneduring entry. For these viruses, all theserearrangements are believed to result in theformation of a narrow channel that allowsthe genomic RNA to reach the cytoplasm. Incontrast to +sRNA viruses, dsRNA viruseshave to maintain their genome hidden fromthe cellular defense mechanisms at all stepsof the viral cycle. Consequently, a largeobject, the capsid-protected genome, needsto cross the cell membrane. Most dsRNAviruses possess several concentric shells.During entry, the external capsid layer is

generally lost and its constitutive proteins, ortheir cleavage products, are thought toinduce a local destabilization of the cellularmembrane allowing virus translocation (12).Recently, it has been shown that mammalianreovirus produce a myristoylated peptide(µ1N) that can induce size-selective pores inmembranes (13). The release of this peptideassociated to pore formation (13) andstructural rearrangements of the remainingdomain of µ1 (µ1 C) (14) are thought to leadto virus entry into the cell. Birnaviruses onlypossess a single-layered capsid (15) that isassumed to be competent for both virustranslocation and genome replication-transcription. For all dsRNA viruses, themechanisms of membrane destabilizationassociated to entry remain hypothetical.Totiviruses, another group of dsRNA virusesthat lack additional shells constitute a specialcase because they are not able to enter into acell and are therefore only transmitted duringcell division.

The capsid of birnaviruses is formedby 260 VP2 protein trimers (15,16) whichobey T=13laevo icosahedral symmetry(17,18). VP3, the second major viral protein,binds the RNA-dependent RNA polymeraseVP1 and the genomic dsRNA (19,20).Birnaviruses present characteristic peptidesassociated to the virus particle (21). Fourpeptides (pep46, pep7a, pep7b and pep11)being 46, 7, 7 or 11 amino acids in length,respectively, are found in the case ofinfectious bursal disease virus (IBDV) (22).These peptides are generated through theprocessing of the viral polyprotein pVP2-VP4-VP3 (Fig. 1). VP4 is a protease thatcleaves its own N- and C-termini, thusreleasing pVP2 and VP3 within the infectedcell (23). Subsequent serial cleavages at theC terminus of pVP2 yield the mature VP2capsid protein and the four peptides.

In this study, we show that pep46deforms biological membranes, leading tothe formation of pores. We determined byNMR the atomic structure of this peptide.The membrane-active domain is formed bytwo kinked helices linked by a prolinedisplaying cis-trans isomerization that

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controls peptide hydrophobicity. Theseresults combined with reverse genetic studiespermit the proposition of a general modeldescribing the cell entry pathway of a non-enveloped virus.

Experimental ProceduresPeptide synthesis - The peptides wereobtained by automated solid-phase synthesisusing the 9-fluorenylmethoxy carbonylstrategy and purified by reversed-phase high-performance liquid chromatography usingstandard procedures. The peptides wereanalyzed by mass spectrometry andconfirmed to have purity higher than 98%.Peptide activity - The hepatocarcinomaepithelial chicken LMH cell line was grownin RPMI medium supplemented with 10%fetal calf serum, 2 mM glutamine and 1 mMsodium pyruvate. The activity of the peptideswas determined by studying their effects onlive cells. After incubation at 37°C, LDHrelease was measured using the cytotox 96nonradioactive kit (Promega), as describedby the manufacturer. The percentage of LDHrelease was determined taking in account theODs found on untreated and Triton X-100-treated cells. All experiments were carriedout five times on duplicated samples; allmeasurements gave similar results.Liposomes containing carbofluoresceine(CF) were prepared as described by Nandi etal. (24). The release of CF after addition ofpeptides or virus was monitored by theincrease of fluorescence at 520nm, using a492nm exciting band in a thermostatedPerkin-Elmer LS50B spectrofluorimeter. Thevalue for 100% release was obtained byaddition of Triton X-100 at the end of thereaction. All fluorescent experiments werecarried out at least three times and gavesimilar results.Electron cryomicroscopy – Microscopy wasperformed as previously described (25). Adrop of the studied sample was adsorbed toair glow-discharged holey carbon films. Theexcess of liquid was removed and the gridfrozen in liquid nitrogen cooled ethane. The

grid was transferred into a Gatan 6226 cryo-holder and observed with a Philips CM12electron microscope operated at 80 kV. Themicrographs were recorded at amagnification of 35,000 under standard lowdose exposition conditions.Sample preparation and NMR spectroscopy -Pep46 was dissolved at pH 3.5 in an aqueous40mM dodecylphosphocholin (DPC)solution at a final concentration of 1mM.Two-dimensional phase-sensitive 1H Clean-TOCSY with 70 ms spin lock, and 100msand 200ms mixing times NOESYexperiments (26) were recorded at 313K and333K on an AVANCE Bruker spectrometeroperating at 600.14 MHz without samplespinning with 2K real points in t2, with aspectral width of 6000 Hz and 512 t1-increments. Pulsed-field gradients (27) wereused for water suppression. The data wereprocessed using XWINNMR software(Bruker). A /6 phase-shifted sine bellwindow function was applied prior toFourier transformation in both dimensions(t1 and t2). The temperature was externallycontrolled using a special temperaturecontrol system (BCU 05 Bruker).

NMR structure determination - NOE cross-peak volumes measured on NOESYspectrum (NOESY, 200 ms mixing time, 333K) were converted into distances, semi-quantitatively, by counting contour levels.Using the Tyr-2,6H geminal, and Asp-Hprotons as calibration peak, NOE signalswere classified into 5 categories with upperdistance limits ranging from 2.5 to 5 Å.Pseudo-atom corrections were added whennecessary. Calculations were performedusing the standard procedures in X-PLORversion 3.84 (28,29). Quality of structureswas evaluated with PROCHECK (30).Virus rescue experiments - To generateinfectious IBDV mutants from cDNA clones,a previously described strategy was used(22). Cells at 90% confluence in P12 wellswere infected with the vaccinia virus MVA-T7 at a multiplicity of infection of 1. After a1-h adsorption, the cells were rinsed with theRPMI medium. In the mean time, pT7-A-HDR derivates and pT7-B-HDR plasmids

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allowing the transcription of genomesegments A and B, respectively, were mixed(0.5 g of each plasmid) with 3.5 L ofLipofectAMINE 2000 (Gibco-BRL) in 350

L of OptiMEM and were kept at roomtemperature for 30 min. The cells were againrinsed in OptiMEM and incubated with theDNA- LipofectAMINE 2000 mixture at37°C for 5h. Next, 1.5 mL of complementedRPMI medium was added to each well.Recombinant mutant viruses were recovered48 h post-transfection after filtration througha 0.22- m-pore-size filter. At least threeindependent transfection experiments werecarried out to analyze each pT7-A-HDRderivative. The viruses were amplified onLSCC-BK3 cells. IBDV-infected LSCC-BK3 cells were analyzed 3 or 5 dayspostinfection. Briefly, the cells were fixedwith 2.5% PFA in PBS for 30 min at roomtemperature and permeabilized by incubationfor 10 min in 0.1% Triton X-100. Fixed cellswere incubated with a 1:250 dilution of ananti-VP3 monoclonal antibody. Next, thecells were rinsed and incubated with an anti-immunoglobin mouse f luoresceinisothiocyanate conjugate in PBS-0.05%Tween. The cells were rinsed three times andsubjected to FACScalibur (BectonDickinson) analysis using the Cell Quest Prosoftware.VLP-GFP cell binding assay. FluorescentVLP were produced as described byChevalier et al. (31). The VLP wereincubated on permissive and non-permissivecells at 4°C for 30 minutes in the presenceand absence of calcium. The cells wererinsed twice with OptiMEM (GIBCO)containing the adequate concentration ofcalcium and analyzed with the FACScalibur.

RESULTS AND DISCUSSION

The membrane-active domain of

pep46. Figure 1A shows the schematicrepresentation of the IBDV polyproteinmaturation process. A sequence alignmentbetween different birnavirus pep46homologues reveals only six conservedresidues, three of them proline. Allsequences display identical clusters of

similar amino acids allowing the definitionof three domains: the central hydrophobicdomain containing most of the conservedresidues (three prolines at positions 16, 23and 27, a threonine at position 20 and thefollowing leucine) and the two flankingdomains mainly constituted by bothhydrophobic and charged residues. Whereasthe N-terminal domain is positively charged,the C-terminal is somewhat negativelycharged. The N-terminal domain is predictedas forming an amphipathic -helix withpositively charged residues on one side ofthe helix and hydrophobic residues on theother side (22). As far as the positions ofcharged and hydrophobic residues areconcerned, the sequence similarity betweenpep46 of different IBDV strains (data notshown) and different birnaviruses (Fig. 1A)is higher in the N-terminal than in the C-terminal domain, suggesting a critical role ofthis -helix in the viral cycle. The overallhydrophobicity of pep46 strongly suggestspotential interactions of this peptide withmembranes. Figure 1B shows that cellsincubated with pep46 undergo spectacularmorphological modifications characterizedby significant shrinkage and cytoplasmvacuolization. Video light microscopy showsthat pep46-treated cells first swell and thenundergo important membrane deformationsthat are associated to massive losses ofcellular material (not shown). All thesechanges result in round ghost cells that havelost their initial morphology (Fig. 1B). Asalready shown for mammalian reoviruses(Agosto et al., 2006), these observations canbe interpreted as arising from partialpermeabilization of the cellular membranethat leads to the establishment of a highosmotic pressure, resulting in severe celldamage. To identify the membrane-activedomain of pep46, several peptides derivedfrom its primary sequence, were synthesizedand their effects analyzed on cells. Peptidesmade of the first 15 (pep15), of the 22 N-terminal amino acids (pep22) and of the last24 C-terminal residues (pep24) weresynthesized (Fig. 1B). Although attenuated,the effects of both pep22 and pep15 were

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similar to that of pep46 with a rounding ofthe cells and a loss of cellular material.While pep22 was found to be more efficientthan pep15, pep24 had no effect on cells. Allthese observations demonstrate that pep46and in particular its N-terminal moiety,pep22, destabilize cell membranes.

The permeabilizing activity of

pep46. The permeabilizing activity of pep46and its shorter derivates, pep22, pep15 andpep24 was quantified by measuring therelease of a cytoplasmic enzyme (lactatedehydrogenase [LDH]) induced by eachpeptide. Although the results are onlyillustrated for an IBDV-permissive cell line(LMH cells; Fig.2), similar data wereobtained for both permissive and non-permissive cells. The effects of the peptideswere analyzed for time (Fig. 2A) andconcentration (Fig. 2B) dependence. Figure2A shows that the peptides permeabilizemembranes with characteristic kineticconstants and lag periods. While a lag periodcannot be evaluated in the case of pep46, itsexistence is clearly visible for pep22 andpep15. Pep15 displays a longer lag periodthan pep22. The existence of a lag periodind i ca t e s t ha t t he membranepermeabilization is a multi-step event. Noactivity could be detected for pep24. Figure2B shows that all peptides are characterizedby a critical concentration below which thepeptides have no effect on the LDH release.The critical concentration increases evenlyfollowing the order of the series: pep46,pep22 and pep15. No effect could bedetected for pep24 up to a concentration of20µM. All these experiments demonstratethat pep46 and particularly its N-terminalmoiety permeabilize cell membranes. Toassess the efficiency of pep46, we comparedits activity to the one of the membrane-activedomain ( 1) of the nodavirus peptide. Wefound that 1 is 10 to 20-fold less efficientthan pep22. While it can be seen on figure2B that the critical concentration of pep22 isabout 3µ , the one of 1 peptide is equal to30 -50µM (data not shown).

The membrane permeabilizationinduced by pep46 could depend on a

perturbation of the metabolism that controlscellular entry processes such as endocytosis.To test this hypothesis, we analyzed theinfluence of a metabolic inhibitor. Thedepletion of the cellular ATP pool did notinfluence the activity of pep46 and its shorterderivates (not shown). Therefore, pep46 andin particular, its N-terminal moiety (pep22),display a membrane activity that is energy-independent. All these features wereobserved on permissive and non-permissivecells, demonstrating that pep46 or derivatesinteract with the lipid bilayer, resulting in theformation of a pore, even in the absence of aspecific receptor.

To further characterize the poreformation mechanism, we studied theactivity of these peptides on syntheticmembranes. The peptides were incubatedwith liposomes containing a fluorescentprobe, which due to quenching effects has afluorescence signal strongly depending uponconcentration. The release of the probe fromthe liposomes leads to a lowering of theprobe concentration and thus to an increaseof the fluorescence signal. With pep22, at aconcentration of 1 µM, the release of theprobe from liposomes is achieved within afew seconds (Fig. 2C). Up to a concentrationof 2 mM, the release is independent of thepresence of calcium or a chelating molecule(EDTA, data not shown). With this assay aswith others, pep24 is found to be inactive onmembranes. Figure 2D and 2E panelsillustrate the results obtained with bothpep46 and pep22. The release of the probefrom the liposomes has kineticcharacteristics similar to those previouslydescribed on cultured cells: the existence of acritical concentration and short lag periodparticularly visible at low concentrations.Due to the membrane composition and cellorganization, the fluorescence assay onliposomes allows the detection of pores atconcentrations that are three orders ofmagnitude lower than the LDH release assay.

Polarity in the interaction of pep22

with membranes. To analyze theimportance of the N- and C-termini of pep22in membrane destabilization, a reporter

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protein, fluorescent streptavidin, wasattached either at its N- or C-terminus. Thelabeled pep22 were added to liposomescontaining a fluorescent probe. Figure 2Fshows that the binding of avidin to either theN- or the C-termini of pep22 inhibits, butdoes not block the release of the probe fromthe liposomes treated with the peptide. Theseresults demonstrate that the addition of anexogenous charged polypeptide at bothtermini does not prevent the formation ofpores in synthetic membranes. Both peptidetermini are thus accessible during the poreformation process and remain in the vicinityof the polar heads of the membrane.

Visualization of the pores by

electron cryomicroscopy. The deformationsof synthetic membranes induced by pep46and shorter derivates were analyzed byelectron cryo-microscopy. Figure 3a showsliposomes visualized in the absence (left)and presence (right) of pep22. Pep46 hassimilar effects on liposomes than pep22. Atlow magnification, both samples appearsimilar. Although multi-lamellar vesicles arevisible, most of the liposomes are uni-lamellar and characterized by heterogeneousdiameter values. Polar head and aliphaticchain domains of the lipid constituting theliposome bilayer are easily discerned. Athigher magnification, significant effect ofpep22 on liposomes is observed (Figure 3b).Whereas in the absence of pep22, the polarheads constituting the liposome bilayersform lines that are parallel over longdistances (left), in the presence of pep22, thelines are parallel over much smallerdistances (right). In fact, in this latter case,the bilayer thickness varies giving rise toareas characterized by the presence ofparallel lines that rapidly alternate with fuzzylines. In accordance with this observation,the contrast of the membrane is loweredwhen pep22 is added to the liposomes. Thefuzzy domains often show fusion of theexternal and internal leaflets of the bilayers(Fig. 3c) demonstrating the formation of anaqueous channel within the membrane thatwill be defined as a pore. The diameter of thepore is not constant; while it is smaller than

the picture resolution (about 2nm) in the leftand middle panels, it is larger in the rightpanel, but the diameter of the pores werealways found less than 10 nm. In all cases,the diameter of the pore is smaller than thediameter of the IBDV virion (60-70 nm). Nosignificant effect of pep24 on liposomescould be observed (not shown).

NMR structure of pep46 in

dodecylphosphocholine (DPC) micelles. Inorder to further study the ability of thepeptides to form pores, the structure ofpep46 in a detergent mimicking the lipidbilayer environment was determined byNMR spectroscopy. Medium range NOEdemonstrate that in DPC, pep46 is organizedas four -helices (amino acids 3 to 15 or (3-15)), (16-22), (27-33) and (35-40) (Figure4A and 4B; Table 1). The proline-richdomain is thus constituted by the second -helix and the following loop. Long rangeNOE are found between Val18 and Ala25and between Ser19 and Ala26 showing thatthis domain has a tertiary folding mainlymaintained by hydrophobic interactions.Importantly, characteristic sequential NOEcross-peaks reveal that Pro16 and Pro23 arepresent in both cis and trans conformations.The doubling of the resonances of Ala14,Val15, Val17, Val18, Phe22, Ala25, Ala26,His30 confirms the cis/trans isomerizationof several peptidyl-prolyl bonds. The all-trans conformation of pep46 was initiallycalculated. The N-terminal moiety displaystwo -helices with well-defined orientations(Figure 4A). This structure is similar to thatof the nodavirus membrane-active domain ofthe peptide (11). In the case of IBDV, theelbow between the two helices is generatedby the presence of a proline, Pro16. The C-terminal moiety also shows two helices buthaving various possible orientations. Figure4B shows the best structure calculated forpep46. The (3-15) helix displays thecharacteristic features of an amphipathic

helix with the polar amino acids (Lys4,Asp5, Arg8, Arg11 and Arg12) located atone side and the hydrophobic residues (Ile6,Ile7, Ile10 and Ile13) at the opposite side.The second helix (16-22) is mainly

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hydrophobic and is followed by a proline-rich loop (23-27). The C-terminal moiety isconstituted by two short helices (27-33) and(35-40) is separated by a flexible segmentcontaining two glycines, Gly33 and Gly35.The (35-40) helix is also amphipathic, withthe hydrophilic face constituted by the aminoacid side chain of Glu34, Asp37, and Tyr38,and the hydrophobic one by the side chainsof Val36, Leu39 and Leu40.

To emphasize the influence of thePro16 cis conformation on pep46 structure,we generated a model by simulatedannealing. Figure 4C and 4D show that thecis-isomerization of Pro16 induces areorientation of the second helix. In fact, thehydrophobic side of the second helix (16-22)turns towards the hydrophobic side of thefirst helix (3-15) and the hydrophobicresidues Phe1 and Phe3. The trans to cisisomerization of Pro16 results in the spatialclustering of hydrophobic residues of pep46and thus may play an important role inmembrane permeabilization.

To confirm the importance of Pro16in the membrane destabilization process, apeptide corresponding to the 22 first N-terminal residues of pep46 with the Pro16substituted to an alanine (namedpep22P16A) was synthesized and assayedfor its ability to form pores in liposomes.Figure 2A and B shows that this peptide hasa low permeabilization activity incomparison to pep22. It has in fact anactivity similar to that of the 15 amino acidlong peptide, pep15. These data show thatthe first 15 N-terminal amino acids aresufficient to destabilize membranes;however, the activity is increased when aproline, and its isomerization property, ispresent at position 16. All these observationssuggest that pep46 plays a crucial role in cellentry. To be active, pep46 in the virus needsto be accessible to membranes during entry.

The activity of pep46 present in the

virus particle is controlled by the divalent

ion concentration. Using the fluorescentprobe release assay, we found that purifiedviruses induce a weak leakage of theliposomes (see Fig. 5A). The release of the

probe is greatly increased when calcium ischelated by addition of EDTA and reducedwhen calcium is added. Subviral particlesonly constituted by the capsid protein VP2(particles that do not contain pep46) do notinduce liposome leakage. Neither pHvariations nor protease treatments arecapable of promoting pore formation by thevirus particles (not shown). These resultsdemonstrate that the main effector of theactivity of the virus on membranes is thecalcium concentration. It is reasonable toassume that the membrane activity of thevirus is due to the release of pep46 from thevirions. We attribute the membrane activityof purified IBDV preparations to thepresence of partially disrupted virions.Interestingly, it was previously demonstratedfor rotavirus, another dsRNA virus, thatrelease of the external capsid proteins (VP4and VP7) that leads to the generation ofmembrane-active peptides is also controlledby calcium concentration (24). It has beenshown that the concentration of calcium inendosomes is as low as 3-30 µM (32,33),suggesting that the peptide is released in theendosomes. All these observations suggestthat birna- and rotaviruses use similar entrypathways.

To further investigate the role ofcalcium in cell entry, infections were carriedout at various calcium concentrations. Figure5B shows indeed that infection is highlydependent on calcium concentration.Surprisingly, no infection occurs when thecalcium concentration in the cell culturemedium is less than 200 µM. Fluorescence-activated cell sorter analysis carried out withpermissive and non-permissive cell linesdemonstrates that virus binding requirescalcium. For non-permissive cells,fluorescent virus-like particles (VLP) do notbind independently of the calcium conditions(Fig. 5C, left panel). For permissive cells,VLP only bind to the cells in the presence ofcalcium (Fig. 5C, middle panel). At thetemperature used for the experiments (4°C)and in the presence of calcium, VLP mainlybind the cells and do not enter because most

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bound VLP can be removed by EDTAtreatment (Fig. 5C, right panel).This data show that calcium is involved intwo different steps of the entry: cell bindingrequires calcium while pore formation needsa calcium concentration decrease. Theopposite effects of calcium on binding andperforation show that these two events occurin different compartments. We thus proposethat virus entry is performed in four steps: i)binding requiring a high calciumconcentration, ii) endocytosis, iii) release ofpep46 from the virus particle due to acalcium concentration decrease and iv) poreformation in the endosomal membrane bypep46 that establishes a continuity betweenthe endosomal lumen and the cytoplasm.

Effect of pep46 mutations in virus

rescue. To identify critical residues of pep46in the viral cycle, we carried out a deletion-substitution mutagenesis analysis using areverse genetic system previously described(22). Firstly, eight deletion mutants with fiveconsecutive deleted residues wereconstructed (Fig. 6). Secondly, all residueswere substituted by an alanine (or anarginine when an alanine was present in thewild-type sequence) to generate 46 singlesubstitution mutants. With the first group ofmutants for which the pep46 secondarystructure is disrupted, no virus could berescued, demonstrating that pep46 isessential for virus viability. In agreementwith this conclusion, as much as 17 singleamino acid substitutions block virus recoveryfor single amino acid mutants. These criticalsubstitutions are mainly present in the N- andC-terminal domains of pep46. Two of thefour positively charged residues of the N-terminal domain (Lys4 and Arg11) cannot besubstituted. Conversely, single substitutionsof the negatively charged residues of the C-terminal domain allow virus amplification.No virus was recovered with mostsubstitutions of the hydrophobic residues ofthe N- and C- terminal domains. In thecentral proline-rich domain, only tworesidues are critical for virus rescue: Pro16and Ala25. Most of the substitutions ofalanine by arginine are critical to virus

rescue, suggesting that such mutationsinduce important structural destabilization.Interestingly, the substitution of a hydrogenatom by a methyl group (glycine to alanineat position 35) is deleterious for virus rescue,showing that the flexibility between the twolast helices is essential for pep46 function.

In conclusion, the reverse geneticexperiments indicate that the two terminaldomains of pep46 are critical to the viralcycle. Pep46 may be involved in numerousvirus functions such as assembly (34),genome packaging… However, as expectedfor a membrane-interacting peptide, theamphipathicity of pep46 has to be preservedfor virus viability. Pro16, the prolinebelonging to the membrane-active domain ofpep46, cannot be substituted, suggesting thatthe bending induced by this residue betweenthe two first helices and its isomerizationproperties are critical to the virus survey.Reverse genetics also shows that the C-terminal domain plays a role in the viralcycle. Because the N-terminus of pep46 issufficient to induce pores in liposomes, theC-terminus appears involved in otherfunction such as the association of pep46 tothe virions.

Conclusions: a model for the entry

of birnaviruses. Altogether, theseobservations suggest that pep46 is involvedin a multi-step entry mechanism. In thepresence of calcium, the virus recognizes itstarget cell and is endocytosed. Next, wepropose that the low calcium concentrationof the endosome allows the release of pep46from the virus particle and the deformationof the membranes. The final step of themembrane destabilization is the formation ofpores. The model shown in Fig. 7 is atentative explanation of the pore formationprocess that takes into account thebiophysical properties of the peptide and theobserved structure of the membranes. Theamphipathic nature of the N-terminal domainof pep46 at first permits interactions with thepolar heads of the lipids constituting themembrane and exposing hydrophobicresidues to the solvent (Fig. 7A). We proposethat isomerization of Pro16 increases the

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exposed hydrophobicity and leads to arotation of the peptide at the membranesurface. The peptide inserts into the lipidsand segregates the aliphatic chains creatinginstability (Fig. 7B). The lipid segregationwithin a leaflet and the resulting aliphaticchain interdigitation between leaflets lead tomembrane thickness variation as observed byelectron cryomicroscopy (Fig. 7C). Thepeptide insertion orders the aliphatic chainsof the target leaflet while it disorders theones of the opposite leaflet. In other words,according to the Luzzati nomenclature (35),the peptide insertion has a tendency to favora lipid conformation in the insertion leafletand an conformation in the opposite one.In others words, the peptide “alpha-beta-zes”the membrane around its insertion position.We do not know how many peptides arerequired for the formation of one pore butthe existence of a lag time in this processindeed suggests that several peptides areinvolved. When two or more “alpha-beta-zed” membrane domains come close together(Fig. 7D), the instability of the membraneleads to the formation of a pore (Fig. 7E).The fact that both termini of pep22 areaccessible suggests that the peptide remainsclose to the polar heads of the lipids. Thediameter of the pore (less than 10 nm) issmaller than that of the virus (70 nm),suggesting that exchange of small moleculesbetween endosomal ghosts and thecytoplasm is sufficient to allow the initialtranscription of the genome and then

translation. At this moment, the becoming ofthe destabilized membrane is not understood.It may stay around the viral particle or befurther degraded through physical (osmoticpressure) or biochemical (degradationenzymes) processes.

Finally, this entry model elaboratedon a double-stranded RNA virus, abirnavirus, applies to single-strand RNAviruses such as noda- and tetra-viruses (9).All these viruses possess at least onestructural peptide of about 40 residues ableto destabilize membranes. In agreement withthe structural homologies observed for theircapsid proteins, single and double-strandedRNA viruses display almost identicalmembrane destabilization properties. Thisobservation suggests, as already proposed(15) that single-strand RNA viruses andbirnaviruses have a common evolutionlineage. Because the replication of doublestranded RNA viruses require thetranslocation of a large particle throughmembranes, birnaviruses appeared to havedeveloped high pore formation efficiency byencoding a membrane active peptidecontaining a proline and its associated cis-trans isomerization.

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REFERENCES

1. Smith, A. E., and Helenius, A. (2004) Science 304(5668), 237-2422. Wilson, I. A., Skehel, J. J., and Wiley, D. C. (1981) Nature 289(5796), 366-3733. Bullough, P. A., Hughson, F. M., Skehel, J. J., and Wiley, D. C. (1994) Nature

371(6492), 37-434. Roche, S., Bressanelli, S., Rey, F. A., and Gaudin, Y. (2006) Science 313(5784), 187-

1915. Heldwein, E. E., Lou, H., Bender, F. C., Cohen, G. H., Eisenberg, R. J., and Harrison,

S. C. (2006) Science 313(5784), 217-2206. Hogle, J. M. (2002) Annu Rev Microbiol 56, 677-7027. Bubeck, D., Filman, D. J., and Hogle, J. M. (2005) Nat Struct Mol Biol 12(7), 615-6188. Wery, J. P., Reddy, V. S., Hosur, M. V., and Johnson, J. E. (1994) J Mol Biol 235(2),

565-5869. Bong, D. T., Steinem, C., Janshoff, A., Johnson, J. E., and Reza Ghadiri, M. (1999)

Chem Biol 6(7), 473-48110. Janshoff, A., Bong, D. T., Steinem, C., Johnson, J. E., and Ghadiri, M. R. (1999)

Biochemistry 38(17), 5328-533611. Maia, 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., and Silva, J. L. (2006) J Biol Chem238(39), 29278-29286

12. Chandran, K., and Nibert, M. L. (2003) Trends Microbiol 11(8), 374-38213. Agosto, M. A., Ivanovic, T., and Nibert, M. L. (2006) Proc. Natl. Acad. Sci. U.S.A.

103(44), 16496-1650114. Zhang, L., Chandran, K., Nibert, M. L., and Harrison, S. C. (2006) J Virol 80(24),

12367-1237615. Coulibaly, F., Chevalier, C., Gutsche, I., Pous, J., Navaza, J., Bressanelli, S., Delmas,

B., and Rey, F. A. (2005) Cell 120(6), 761-77216. Saugar, I., Luque, D., Ona, A., Rodriguez, J. F., Carrascosa, J. L., Trus, B. L., and

Caston, J. R. (2005) Structure 13(7), 1007-101717. Bottcher, B., Kiselev, N. A., Stel'Mashchuk, V. Y., Perevozchikova, N. A., Borisov,

A. V., and Crowther, R. A. (1997) J Virol 71(1), 325-33018. Pous, J., Chevalier, C., Ouldali, M., Navaza, J., Delmas, B., and Lepault, J. (2005) J

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Virol 76(22), 11301-1131120. Lombardo, E., Maraver, A., Cast n, J. R., Rivera, J., Fernandez-Arias, A., Serrano, A.,

Carrascosa, J. L., and Rodriguez, J. F. (1999) J Virol 73(8), 6973-698321. Delmas, B., F.S.B., K., Leong, J. C., Mundt, E., V.N., V., and WU, J. L. (2005)

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Delmas, B. (2002) J Virol 76(5), 2393-240223. Birghan, C., Mundt, E., and Gorbalenya, A. E. (2000) Embo J 19(1), 114-12324. Nandi, P., Charpilienne, A., and Cohen, J. (1992) J Virol 66(6), 3363-336725. Adrian, M., Dubochet, J., Lepault, J., and McDowall, A. W. (1984) Nature 308(5954),

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33. Gerasimenko, O. V., and Tepikin, A. V. (2005) Cell calcium 38, 201-21134. Chevalier, C., Galloux, M., Pous, J., Henry, C., Denis, J., Da Costa, B., Navaza, J.,

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1030

FOOTNOTES

We thank Yves Gaudin, Jean-Michel Peyrin, Félix Rey, Human Rezaei and Stéphane

Roche for helpful discussions. This work was supported by a grant of the ACI

“Microbiologie” from the French MRT, by the EU COST action 892, and the Agence

Nationale de la Recherche “Projets blancs” programs. M.G. was funded by an INRA/Région

Ile-de-France fellowship. S.L. was supported by an INRA fellowship.

Coordinates of pep46 have been deposited in the Protein Data Bank under accession

number 2IMU.

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FIGURE LEGENDS

Figure 1: Schematic representation of the IBDV polyprotein processing and effects of pep46and derivates on cell morphology. (A) The IBDV polyprotein and the four structural peptidespep46, pep7a, pep7b and pep11 deriving from the C-terminal domain of pVP2, arerepresented. Numbering refers to the amino acid position in the polyprotein. Hydrophobicresidues are red and charged residues are blue. Strictly conserved residues are indicated in theconsensus line. The main characteristics of the pep46 domains are indicated. (B) Effect ofpep46 and derivates on the morphology of LMH cells. After 10 min, drastic effects on themorphology and the size of the cells are observed with pep46 and pep22 and to a lesser extentwith pep15. Pep24 has no effect on cells. Major morphological effects are indicated by whitearrowheads.

Figure 2: Membrane permeabilization activity of pep46 and derivates. A and B show thepeptide activity on cells quantified by LDH release and expressed as a percentage of the totalcellular LDH. (A) Effect of time on LDH release. Peptides were incubated with cells at aconcentration of 10 µM. The amino acid sequence of pep22P16A is identical to the one ofpep22 except proline in position 16 that is substituted by an alanine. No release was observedwith pep24. (B) Effect of concentration on LDH release. The activities of pep15 andpep22P16A display the same concentration dependence. Again, no activity could be observedwith pep24. C and D show the release of the fluorescent probe from synthetic liposomes. Thepeptide activity was quantified by the fluorescent probe leakage and expressed as a percentageof the total unquenched fluorescence (when liposomes were treated with Triton X-100). In(A) and (B), results are mean +/- SD of five distinct experiments. (C) The activities of pep22and pep24 at a concentration of 1 µM were compared. Triton X-100 indicates the time atwhich Triton X-100 has been added to the samples to validate the quality of the liposomescontaining the fluorescent probe. Again, no activity of pep24 was detected. (D). Effect ofpeptide concentration and time. The activity of pep46 was measured and found concentrationdependent in the nanomolar to micromolar range. (E) Comparison of the effects of pep46 andpep22 on liposomes. (F). Effect of avidin binding at the N- and C-terminus of pep22.

Figure 3: Visualization by cryomicroscopy of the structural effects induced by pep22. (a) Thepanels show the liposomes in the absence (left) and the presence (right) of pep22. In bothcases, liposomes having various diameters are observed. In general, they are unilamellar. Thebilayered structure of the membrane is clearly visible. (b) When the membranes are observedat higher magnification, significant differences are observed in the presence of pep22. Whilethe position of the polar heads of the lipids gives rise to parallel lines over long distanceswhen pep22 is absent (left), the positions of the polar heads differ in the presence of pep22(right). For this last case, the raw micrograph is presented (upper panel) as well as a filteredimage for the low (1/100nm) and high (1/2nm) spatial frequencies (lower panel). In this lastcase, the thickness of the membrane seems to vary and parallel lines alternate with fuzzy area.The arrows indicate a thinner membrane area (t). (c) Fusion events between the external andinternal leaflets are observed giving rise to an aqueous channel or pore. The membranedeformations (arrows 1) and the pores (2 and 3) are better observed on images that have beenfiltered (lower panel). The diameter of the pores are less than 10nm.

Figure 4: NMR structure of pep46 in DPC micelles. (A) Stereoview of the twelve pep46accepted refined structures with the peptide bonds of Pro16, 23, 24 and 27 in the transconformation. The superposition is performed on the (3-22) backbone atoms of pep46. Theribbons corresponding to the (1-22) and (23-46) domains are respectively colored in blue and

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dark green. The four -helices are represented as orange cylinders. Proline 16 is colored inpurple. The hydrophobic and aromatic residues, Phe1 (red) and Phe3 (yellow) of pep46, showa very great flexibility, which could be important for the membrane interaction. (B) Structureof one of the pep46 structures with all the prolines in the trans conformation, showing thedistribution of the hydrophobic and hydrophilic residues along the four helices (red cylinders).(C) Influence of the cis-trans isomerism of the peptide bond between Val15 and Pro16 on therelative orientation of the (3-15) and (16-22) helices (blue cylinders). The structure should becompared with that represented in (B). The superposition between the two structures wasdone on the (3-15) backbone atoms. The second helix (16-22), entirely hydrophobic, tends tobe directed to the same side as the hydrophobic face of the first amphipathic helix, and, morespecifically toward the residues Phe1 and Phe3 located at the N-extremity of Pep46, whenPro16 adopts a cis conformation. (D) Superposition on the (3-15) backbone atoms of the bestpep46 structures calculated with the proline 16 in trans (red cylinders) and cis (blue cylinders)conformation, showing the orientation distribution of the (16-22) helix toward the (3-15)helix.

Figure 5: Effect of calcium on pore formation and virus infection. (A) Release of a fluorescentprobe from synthetic liposomes induced by a virus preparation. Standard virus preparation(IBDV) induces a small leak of the liposomes. When EDTA is added at a 1 mMconcentration, the leakage of the fluorescent compound is increased (IBDV+EDTA). Whencalcium at a concentration of 5mM is present, the leakage of the fluorescent compound isinhibited. Subviral particles only containing the capsid protein VP2 (SVP/VP2) do not haveany effect on liposomes. EDTA does not severely damage the virus as observed by electronmicroscopy and by the fact that EDTA treated virions recovered partial infectivity in thepresence of calcium. (B) Effect of calcium concentration on virus infectivity. Numbers ofinfected cells, expressed as a percentage of cells infected in a Tris-buffered isotonic solution(NaCl 0.9%), are presented for different calcium concentrations. To efficiently infect a cellculture, the inoculum needs to contain 0.2mM calcium. Altogether, this figure shows thatcalcium plays a critical role in infection; it is required for an early step of the infection andneeds to be depleted to promote pep46 release from the particles and destabilization of theendosome membrane. (C) Fluorescence-activated cell sorter analysis of the binding offluorescent virus-like particles (VLP) on permissive (LSCC-BK3) and nonpermissive (DT40)avian lymphoid cell lines. On the left and middle panels, binding was measured in thepresence (red lines) or in the absence (blues lines) of calcium. The left panel shows thatnonpermissive cells do not allow the binding of VLP independently of the calciumconcentration. The middle panel shows that permissive cells bind VLP in the presence ofcalcium. The right panel shows that VLP bound to permissive cells in the presence of calcium(red line) are partially released by EDTA treatment (blue lines). The green lines in all panelsindicate the fluorescence signal detected on cells that are not incubated with fluorescent VLP.

Figure 6: Rescue of viruses engineered with pep46 mutations. The amino acid sequence ofpep46 is indicated in the single letter-code at the top of the figure. The hydrophobic residuesare red and charged residues in blue. Deleted amino acids are indicated by asterisks, andsubstitutions are indicated for each plasmid in the single-letter code. Depending on the virusrecovery results, the plasmids are indicated by + (recovery) or – (nonrecovery).

Figure 7: Schematic model for membrane deformation and pore formation induced by a viralamphipathic peptide. (A) The peptide adsorbed to the lipid polar head surface (colored inblue) by its hydrophilic side (blue), presenting its hydrophobic side (colored in red) to theaqueous solvent. Prolines in the trans conformation are indicated by black filled circles. The

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lipid aliphatic chains are colored in red. The circle and the kinked arrow indicate the domainthat can change orientation with the proline isomerisations when adsorbed to the lipid polarheads. (B) The prolines change conformation from trans to cis (black squares) and the peptiderotates and inserts its hydrophobic side into the bilayer, excluding lipid molecules. The redstar indicates instability in the membrane introduced by the peptide insertion. (C) The bilayerrearranges leading to a thinning of the membrane as observed by microscopy. Peptideinsertion results in an ordering of the lipid aliphatic chains; the peptide forces the aliphaticchains of the target leaflet to adopt a conformation. To compensate the membrane surfaceincrease resulting from the peptide insertion, the aliphatic chains of the opposite layer aredisorganized and adopt an conformation. The peptide “alpha-beta-zes” the membrane. (D)When several alpha-beta-zed domains become adjacent, instability is created that leads to theformation of a pore (E).

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4612061341165

0

(479*) 0.020 ± 0.003

(715) 0.0041 ± 0.0001 (1299) 0.69 ± 0.02 (45) 0.47 ± 0.02

10.71 ± 3.1 -167.8 ± 12.1 -1665.3 ± 24.2

86.3 12.8 0.4 0.4

Backbone atoms Heavy atoms

0.41 ± 0.18 1.49 ± 0.44 0.36 ± 0.17 0.92 ± 0.30 0.77 ± 0.33 1.63 ± 0.48 0.87 ± 0.39 1.14 ± 0.40

0.48 ± 0.18 1.09 ± 0.36 0.32 ± 0.14 1.17 ± 0.42

NMR derived distance restraints Total Intraresidue Sequential (|i-j| =1) Medium-range (1< |i-j| 4) Long-range (|i-j| >5)

Distance violations > 0.2r.m.s.d. (Å) from experimental distance restraints

r.m.s.d. from idealized geometry bonds (Å) angles (deg) improper angles (deg)a

Xplor potential energiesb

ENOE (Kcal/mol) Evdw (Kcal/mol) Etotal (Kcal/mol)

Ramachandran analysis of residues favored region (%) additional allowed regions (%) generously allowed regions (%) disallowed regions (%)

Pairwise rms deviation (Å)

(3-15) (17-22) (3-22) (18-27)

(27-33) (35-40)

Table 1 : NMR restraints and structural statistics

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VP2 VP4 VP3

pep46 pep7a pep7b pep11

pVP2

442 512

Figure 1: Galloux et al.

Positively chargeddomain predicted

forming anamphipathic helix

Negativelycharged domain

Proline richdomain

IBDV FGFKDIIRAIRRIAVPVVSTLFPPAAPLAHAIGEGVDYLLGDEAQAIPNV WGWRDIVRGIRKVAAPVLSTLFPMAAPLIGMADQFIGDLTK..TNABSNV FGWGDLLSGIRKWVFPVVDTLLPAARPLTDLASGWIKNKY...PEADrXV FDFWQLLKQIKNVAVPLAATLAPQFAPIIGAADGLANAIL...GDSConsensus I P TL P P

A.B.

t = 1

0min

pep46 pep24pep22 pep15

t = 0

min

pep46pep22

pep24pep15

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Time (s)

% F

luo

resc

ence

1 M

100 nM75 nM50 nM

10 nM

7,5 nM

5 nM

1 nM0,6

0,7

0,8

0,9

1

1,1

0 50 100 150 200 250 300 350 40050 100 150 200 250 300 350 4000

D.

A.

Time (min)

pep46

pep22

pep15

pep24

pep22P16A

0

20

40

60

80

100

0 30 60 90 120

100

80

60

40

20

030 60 1200

% L

DH

rel

ease

90

B.

pep15

0

20

40

60

80

100

0 5 10 15 20

% L

DH

rel

ease

100

80

60

40

20

0201050

Concentration ( M)

pep46

pep22

pep22P16A

pep24

15

Figure 2: Galloux et al.

C.

0

20

40

60

80

100

0 50 100 150 200 250 300

Time (s)

50 100 150 200 250 3000

% F

luo

resc

ence

100

80

60

40

20

0

pep22

pep24

Triton X-100peptides

100

80

60

40

20

0

E.

0

20

40

60

80

100

0 100 200 300 400 500

% F

luo

resc

ence

Concentration (nM)

F.

0

20

40

60

80

100

00

% F

luo

resc

ence

100

80

60

40

20

0250 500125

Concentration (nM)

BiotNpep22

pep22CBiot

pep22CBiot-Avidin

Avidin-BiotNpep22

pep46

pep22

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A.

B.C.

D.

Figure 4: Galloux et al.

N-ter

C-ter

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20

40

60

80

100

0 500 1000 1500 2000 2500 3000

IBDV+EDTA

IBDV

IBDV+Ca++

SVP/VP2

Time (s)

% F

luor

esce

nce

100

75

50

25

0500 1000 1500 2000 2500 30000

A.

Figure 5: Galloux et al.

100

50

RP

M1

5 m

M

3 m

M

2 m

M

1 m

M

0.4

mM

0.2

mM

0.1

mM

50 µ

M

40 µ

M

4 µM

0.4 µM

0.1 µM

EG

TA

-100

nM

0

0 µM

Num

ber

of in

fect

ed c

ells

(% o

f co

ntro

l)

Ca++ concentration

B.

C.

DT40 LSCC-BK3 LSCC-BK3

Figure 5: Galloux et al.

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FGFKDIIRAIRRIAVPVVSTLFPPAAPLAHAIGEGVDYLLGDEAQA ---*****-------------------------------------- --------*****--------------------------------- -------------*****---------------------------- ------------------*****----------------------- -----------------------*****------------------ ----------------------------*****------------- ---------------------------------*****-------- --------------------------------------*****--- A--------------------------------------------- -A-------------------------------------------- --A------------------------------------------- ---A------------------------------------------ ----A----------------------------------------- -----A---------------------------------------- ------A--------------------------------------- -------A-------------------------------------- --------R------------------------------------- ---------A------------------------------------ ----------A----------------------------------- -----------A---------------------------------- ------------A--------------------------------- -------------R-------------------------------- --------------A------------------------------- ---------------A------------------------------ ----------------A----------------------------- -----------------A---------------------------- ------------------A--------------------------- -------------------A-------------------------- --------------------A------------------------- ---------------------A------------------------ ----------------------A----------------------- -----------------------A---------------------- ------------------------R--------------------- -------------------------R-------------------- --------------------------A------------------- ---------------------------A------------------ ----------------------------R----------------- -----------------------------A---------------- ------------------------------R--------------- -------------------------------A-------------- --------------------------------A------------- ---------------------------------A------------ ----------------------------------A----------- -----------------------------------A---------- ------------------------------------A--------- -------------------------------------A-------- --------------------------------------A------- ---------------------------------------A------ ----------------------------------------A----- -----------------------------------------A---- ------------------------------------------A--- -------------------------------------------R-- --------------------------------------------A- ---------------------------------------------R FG K I IR A P A LA I G LL QA

plasmids

WtΔ4-8Δ9-13Δ14-18Δ19-23Δ24-28Δ29-33Δ34-38Δ39-43

F1AG2AF3AK4AD5AI6AI7AR8AA9R

I10AR11AR12AI13AA14RV15AP16AV17AV18AS19AT20AL21AF22AP23AP24AA25RA26RP27AL28AA29RH30AA31RI32AG33AE34AG35AV36AD37AY38AL39AL40AG41AD42AE43AA44RQ45AA46R

Crucial residues

Virusrecovery

+----------+-++-++--++-+-++++++++-++--++-++-+++--++++--

Positively-chargeddomain

Negatively-chargeddomain

Proline-richdomain

Figure 6: Galloux et al.

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beta

NC

A. Adsorption B. Isomerisation-Insertion

C. « alpha-beta-sation »

NC

Figure 7: Galloux et al.

alpha

E. Pore formation

Instability

D. Association of destabilized domains

Instability

2 to 10 nm

pore

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Ouldali, Jean Lepault and Bernard DelmasMarie Galloux, Sonia Libersou, Nelly Morellet, Serge Bouaziz, Bruno Da Costa, Malika

peptide that deforms and perforates biological membranesInfectious bursal disease virus, a non-enveloped virus, possesses a capsid-associated

published online May 8, 2007J. Biol. Chem. 

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