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Dissecting Quasi-Equivalence in Nonenveloped Viruses: Membrane Disruption Is Promoted by Lytic Peptides Released from Subunit Pentamers, Not Hexamers

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Disruption Is Promoted by Lytic Peptides Released from Subunit

Pentamers, Not Hexamers

Tatiana Domitrovic,aTsutomu Matsui,band John E. Johnsona

Department of Molecular Biology, The Scripps Research Institute, La Jolla California, USA,a

and Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, California, USAb

Nonenveloped viruses often invade membranes by exposing hydrophobic or amphipathic peptides generated by a proteolytic maturation step that leaves a lytic peptide noncovalently associated with the viral capsid. Since multiple copies of the same pro-tein form many nonenveloped virus capsids, it is unclear if lytic peptides derived from subunits occupying different positions in

a quasi-equivalent icosahedral capsid play different roles in host infection. We addressed this question withNudaurelia capensis

omega virus(NV), an insect RNA virus with an icosahedral capsid formed by protein, which undergoes autocleavage during

maturation, producing the lyticpeptide. NV is a unique model because autocatalysis can be precisely initiatedin vitroand is

sufficiently slow to correlate lytic activity withpeptide production. Using liposome-based assays, we observed that

autocataly-sis is essential for the potent membrane disruption caused by NV. We observed that lytic activity is acquired rapidly during the

maturation program, reaching 100% activity with less than 50% of the subunits cleaved. Previous time-resolved structural

stud-ies of partially mature NV particles showed that, during this time frame,peptides derived from the pentamer subunits are

produced and are organized in a vertical helical bundle that is projected toward the particle surface, while identical polypeptides in quasi-equivalent subunits are produced later or are in positions inappropriate for release. Our functional data provide experi-mental support for the hypothesis that pentamers containing a central helical bundle, observed in different nonenveloped virus families, are a specialized lytic motif.

A

nimal virus infection generally involves an interaction of the particle with a cell surface receptor, followed by any of a num-ber of cell entry processes that depend on the virus. Enveloped viruses frequently fuse with either the plasma membrane or the endocytic membrane and deliver their genomic contents by this well-understood process. Following receptor attachment, nonen-veloped viruses also enter through a variety of pathways but must cope with membrane translocation of their genomes without the aid of fusion. A common feature of many nonen-veloped animal viruses is a capsid-associated lytic peptide that is activated by a chemical cue and assumed to facilitate membrane rupture for genome or particle delivery to the cyto-plasm (1, 20,31). This activity is commonly studiedin vitro

with artificial liposomes filled with a fluorescent dye that is quenched within the liposome but fluoresces strongly when the liposome is breached and the dye is released (2,16). The role of the lytic peptide, and associated infectivity, in Flock House virus (FHV), a nonenveloped, T⫽3 quasi-equivalent, small-subunit RNA (ssRNA) insect virus, was investigated with extensive mu-tagenesisin vivoemploying a novel protocol in which the lytic peptide was provided intranswith a virus-like particle (VLP) (32). There was a striking correlation between mutants that show re-duced dye releasein vitro and reduced infectivityin vivo, thus confirming the biological relevance of the liposome assay (2).

There are generally two steps in activating the lytic peptide. The first is part of the virus’s maturation and is often an autocatalytic cleavage of a subunit polypeptide rendering the lytic peptide co-valently independent but still associated with the particle. The second step occurs during infection, where the peptide activity is triggered by a chemical cue (usually a low pH) in a particular

cellular compartment appropriate for the fissure of the associated membrane (20,22,31).

Multiple copies of the same protein form the capsid of many nonenveloped viruses that infect invertebrates and vertebrates; however, it is not known which of the quasi-equivalent subunits in the capsid actually provide the active peptides. This question ad-dresses fundamental issues associated with nonenveloped, quasi-equivalent viruses; i.e., do subunits with the same sequence but in different quasi-equivalent capsid positions have different matura-tion properties, and do their liberated peptides have different functions? Because just one protein sequence forms the virus cap-sid, classic approaches such as mutagenesis cannot be easily em-ployed to address differential roles of quasi-equivalent subunits.

Nudaurelia capensis omega virus(N␻V) capsids allow these

questions to be addressed for the first time. N␻V is a T⫽4, ssRNA insect virus with a crystal structure determined at 2.8 Å resolution (13,21). The capsid is formed by 240 copies of the same protein organized in four quasi-equivalent positions, named A, B, C, and D. A subunits form pentamers, while 2-fold related B, C, and D subunits form quasi-hexamers (Fig. 1). VLPs are made by express-ing the N␻V capsid protein in a baculovirus system, and they are purified at pH 7.6 as immature procapsids that can later be ma-turedin vitro(7). Maturation is initiated by lowering the pH to

Received2 May 2012Accepted28 June 2012

Published ahead of print3 July 2012

Address correspondence to John E. Johnson, jackj@scripps.edu.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.01089-12

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5.0, which results in a change in the particle dimension from 490 to 410 Å and initiation of the autocatalytic cleavage reaction at residue 570 to form␤protein (residues 1 to 570) and␥peptide (residues 571 to 644) (Fig. 1A). Following cleavage ofⱖ10% of the subunits, the particle remains in the compact form even when the pH is returned to 7.6 (8). The irreversibility is promoted by the stabilization of a molecular switch that supports the flat contacts within the icosahedral particle. The switch is the C-terminal helix of the␥peptide that is ordered only in subunits C and D (residues 627 to 639) and is placed between the flat contacts of the capsid (Fig. 1) (28). The kinetics of cleavage after procapsids are changed to pH 5.0 is unusual, with 50% of the subunits cleaved in 30 min while it takes several hours for all of the subunits to be cleaved (17). Taking advantage of the slow kinetics of maturation of N␻V, Matsui et al. (18) used cryoelectron microscopy (cryo-EM) to study the structure of particles at intermediate stages of matura-tion, namely, 3 min, 30 min, and 4 h, at pH 5.0. Because the size of the particles is the same throughout the maturation process, it is possible to use difference cryo-EM density maps␳(fully mature) and⫺␳ (partially mature) and the X-ray model to determine which autocatalytic sites reached a final conformation similar to that of the fully mature particle and which had significant differ-ences at that location at a particular stage of maturation. It was shown that subunits surrounding icosahedral 5-fold (A subunits, seeFig. 1B) and 3-fold icosahedral symmetry axes (D subunits) are quickly stabilized and cleave in 30 min, while the subunits not adjacent to these axes (B and C) cleave slowly, with B subunits requiring at least 4 h and C subunits taking more than a day to complete cleavage (18). The rate of subunit-specific cleavage can be disturbed by mutations that affect subunit contacts. It was pre-viously shown that an E73Q mutant undergoes normal A and D subunit cleavage but takes longer to stabilize the autocatalytic site at positions B and C, resulting in a lower rate of cleavage of these subunits. Here we used previous knowledge of the

subunit-spe-cific cleavage rate, the liposome lysis assay, and the N␻V near-atomic-resolution structure to attribute the observed membrane-lytic activity to the peptides coming from A subunits.

MATERIALS AND METHODS

VLP production and purification.Purification of wild-type (WT) N␻V VLPs and the N570T and E73Q mutants was described previously (17). Briefly, Sf21 cells infected with recombinant baculovirus were harvested at 6 days postinfection. The cell suspension was incubated for 15 min with 0.5% NP-40 and centrifuged for 15 min at 10,000⫻gat 4°C. The super-natant was further centrifuged at 100,000⫻gfor 3 h against a 30% sucrose cushion at 11°C. The pellet containing VLP procapsids was suspended in 50 mM Tris-HCl (pH 7.6)–250 mM NaCl. The VLP suspension was fur-ther purified through sedimentation in a 10 to 40% sucrose gradient at 100,000⫻gfor 2.5 h. The fraction containing VLPs was dialyzed in 10 mM Tris-HCl–250 mM NaCl (pH 7.6). In order to obtain mature capsids, procapsids were dialyzed in 10 mM sodium acetate–250 mM NaCl (pH 5.0).

Liposome preparation and membrane disruption assay.Liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, Inc.) containing the entrapped fluorescent dye sulforhodamine B (SulfoB; Invitrogen/Molecular Probes) were prepared as previously de-scribed (24). For the membrane disruption assay, a liposome suspension in 10 mM HEPES buffer (pH 7.0) was diluted 100⫻in the appropriate assay buffer, i.e., 50 mM Tris–250 mM NaCl adjusted to pH 7.5 to 9.5 or 50 mM sodium acetate–250 mM NaCl adjusted to pH 5.0 to 7.0.

N␻V procapsids or capsids were added to the diluted liposome sus-pension to the desired final concentration and incubated for 10 min at room temperature. Fluorescence intensity was measured with a Cary Eclipse fluorescence spectrophotometer (Varian) using an excitation wavelength of 535 nm and an emission wavelength of 585 nm. Triton X-100 was added to liposomes to a final concentration of 0.1% to deter-mine 100% dye release. The percentage of dye release (DR%) was calcu-lated as follows: DR%⫽100⫻[(FVLPFB)/(FTX100FB)], whereFVLP

is the measured fluorescence intensity after VLP addition,FTX100is the intensity recorded after Triton X-100 addition, andFBis the fluorescence

FIG 1The N␻V T⫽4 capsid is formed by 240 copies of the same protein organized into four quasi-equivalent positions. (A) Structure of the D subunit determined by X-ray crystallography of mature N␻V (13). Arrows indicate the site of the autoproteolytic cleavage at residue N570 and the location of residue E73, whose replacement with a glutamine gives rise to an abnormal maturation phenotype (17). (B) Schematic drawing of the T⫽4 subunit arrangement in the mature N␻V particle. Quasi-equivalent subunits A, B, C, and D are colored blue, red, green, and yellow, respectively. The A subunits form pentamers, while the B, C, and D subunits form quasi-hexamers positioned at the icosahedral 2-fold axes. Thick black lines represent bent contacts (⬃138°), and dashed lines highlight the flat contacts of the capsid. Flat contacts are stabilized by the C-terminal helix of the␥peptide coming from the C and D subunits, which function as a molecular switch. Solid colors (A and D) represent the subunits whose autocatalytic site is structurally stabilized within 3 min and therefore show the highest rate of␥ cleavage. Gradient intensity and different cleavage site symbols in subunits B and C reflect the times required for autocatalytic site stabilization in WT particles, i.e., 30 min for subunit B and more than 4 h for subunit C (18).

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measured in the absence of VLPs or detergent. All experiments were done in triplicate with independent VLP preparations.

VLP maturation assay.Procapsids at pH 7.6 were diluted at least 10⫻ to obtain 1 mg/ml in 100 mM sodium acetate–250 mM NaCl (pH 5.0). The maturation reaction was carried out at 20°C. Aliquots were taken at different time points, immediately mixed with SDS-PAGE sample buffer, and frozen in liquid nitrogen. Another aliquot corresponding to the same time point was quickly collected and diluted to reach a final VLP concen-tration of 10␮g/ml in a solution containing liposomes for the membrane disruption assay. This concentration was chosen because it is within the linear range observed with dose-dependent liposome assays with fully cleaved capsids. The liposome assay was carried out as described above, but we included a further normalization step for the dye release of matur-ing particles. Percent lytic activity⫽100⫻(DR%PC/DR%Cap), where DR%PCis the dye release calculated for each partially cleaved time point and DR%Capis the dye release observed for the 100% cleaved capsids assayed after 1 day of incubation at pH 5.0.

The kinetics of cleavage was calculated from SDS-PAGE gels stained with Coomassie blue. The percentage of cleavage was determined from a digitized gel image by using the program ImageJ (http://rsbweb.nih.gov /ij/). Results are the means⫾standard deviations of three independent maturation assays performed with different VLP preparations.

Structural analysis.N␻V crystal structure coordinates (Protein Data Bank [PDB] code 1ohf) (13) and mature N␻V cryoelectron micrograph reconstructions (VIPER EMDB code em_2nwv) (18) were used for anal-ysis of the structural environment of␥peptides. Specific oligomers were generated by using the Oligomer-generator tool from the VIPER database (http://viperdb.scripps.edu/) (9). Molecular graphic images were pro-duced by using the UCSF Chimera package from the Resource for Bio-computing, Visualization, and Informatics at the University of California, San Francisco (http://www.cgl.ucsf.edu/chimera/) (25).

RESULTS

Lysis of artificial membranes requires covalently independent

peptides.Although the structural similarities between tetraviruses

and nodaviruses suggested that N␻V would present lytic activity against membranes (27), this assumption has never been experi-mentally tested. To determine if N␻V can disrupt lipid mem-branes, liposomes at pH 7.6 filled with fluorescent dye at quench-ing concentration were incubated with WT N␻V capsids. As shown inFig. 2A, N␻V induced a dose-dependent lytic activity, reaching values close to the total liposome disruption induced by Triton X-100 that defines 100% dye release. We also tested the activity of WT N␻V procapsids (no␥peptide cleavage) and the noncleaving N␻V N570T mutant in both the procapsid expanded form and the capsid-like compact conformation. In contrast to the cleaved mature WT N␻V capsids, WT procapsids and N570T mutant VLPs, in both the procapsid and capsid conformational states, did not elicit dye release, even at high concentrations. These experiments showed that␥peptide cleavage is required for N␻V membrane disruption activity.

Lytic activity of mature capsids is pH dependent.We

ob-served that free␥peptide could be isolated in the soluble fraction of boiled mature capsids (Fig. 2B). This protocol allowed us to investigate if the lytic activity of mature capsid and free␥peptide is controlled by pH.Figure 2Cshows that the lysis of liposomes by 10␮g/ml of mature capsids was pH dependent, being completely inhibited below pH 6.5 and strongly potentiated by alkaline pH. Soluble␥peptide, obtained by boiling the same concentration of mature capsids and removing all other remnants of the particle by high-speed centrifugation, induced strong lytic activity, as ob-served for the entire capsid (Fig. 2C, dashed lines). On the basis of gel quantification analysis, less than 50% of the␥peptide present

in the capsid was released into the supernatant. Regardless, com-parable or greater lytic activity was observed when the same amount of the entire capsid preparation and supernatant contain-ing␥peptide released after boiling was incubated with liposomes. This result corroborates the data inFig. 2A, confirming that␥ peptide is the capsid component responsible for liposome disrup-tion. Although pH also modulated the lytic activity of isolated peptide, free␥peptide was clearly active at pH 5. Therefore, the capsid suppresses␥peptide activity at acidic pH.

Capsids with half of the subunits cleaved achieve 100%

lipo-some lytic activity.N␻V autocatalysis is precisely initiated and

sufficiently slow (17) that lytic activity can be correlated with cleavage extent.Figure 3Ashows the kinetics of cleavage of WT particles after maturation triggering by dilution of procapsids in pH 5.0 buffer (black lines). The same experiment was performed with the E73Q mutant (gray lines), which cleaves 50% of the sub-units at the same rate as the WT and is then exceedingly slow to complete maturation (17). The maturation curve was derived from an SDS-PAGE image by quantifying the intensity of the␣ (uncleaved) and␤(cleaved) protein bands at each time point (17).

FIG 2N␻V␥peptides show lytic activity against artificial membranes. (A) Liposomes at pH 7.6 were treated with increasing concentrations of mature WT N␻V capsids (WT Cap), WT N␻V procapsids (WT Procap), and the noncleaving N570T mutant in the capsid form (N570T Cap) or in the procap-sid form (N570T Procap). Dye release was calculated relative to the increase in fluorescence achieved by the addition of Triton X-100. A clear dose-dependent membrane-lytic activity was observed with mature VLPs but not in the absence of␥cleavage. (B) SDS-PAGE gel stained with Coomassie blue showing that free␥peptide can be isolated in the soluble fraction of boiled particles. Lane 1, mature capsid preparation before boiling (␤protein,⬃60 kDa;␥peptide,⬃10 kDa); lane 2,␥peptide observed in the soluble fraction after boiling; lane 3, procapsids (protein␣,⬃70 kDa; no autocatalytic cleavage); lane 4, soluble fraction of boiled procapsids with no detectable␥peptide; lane MW, molecu-lar mass markers. (C) Mature N␻V VLPs (solid lines) were added to liposomes in buffers with pHs ranging from 5.0 to 9.5 (final VLP concentration, 10␮g/ ml). Dashed lines correspond to liposome lysis induced by free “gamma” pep-tide present in the soluble fraction of boiled particles. The␥peptide released by the same concentration of mature capsid was enough to induce potent mem-brane leakage.

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Liposome disruption measures were taken throughout the time course of maturation of N␻V VLPs and compared to the lytic activity obtained with fully mature particles at the end of the ex-periment (Fig. 3B).

Note that despite its aberrant maturation kinetics, the E73Q mutant can complete cleavage (Fig. 3A, inset) and reach the same lytic potential as WT particles (Fig. 3B, inset, dye release values normalized only by Triton X-100-induced leakage). Figure 3C

shows a control experiment done to verify that there was no evi-dent increase in cleavage during the time frame required to get a dye release measurement (no more than 10 min). Lytic activity and extent of cleavage were analyzed immediately after 10 min of capsid maturation at pH 5.0 and after the same sample was incu-bated at pH 7.6 for an additional 20 min. Because the pH in the liposome disruption assay was adjusted to 7.6, the maturation rate was lowered considerably, allowing reproducible and stable time-resolved measurements of lytic activity for WT and also E73Q mutant partially mature particles (Fig. 3C).

The effect of the percentage of subunits cleaved on lytic activity is shown inFig. 3B, where lysis was measured at the same time points when cleavage progress was recorded inFig. 3A. The rela-tionship between the percentage of subunits cleaved and lysis ac-tivity is plotted explicitly inFig. 3D. The results show that the maximum lytic potential of maturing WT and E73Q mutant VLPs (defined as percent lytic activity) was acquired with less than 50% of the subunits cleaved, as evidenced by the sharp increase in ac-tivity within the first 15 min of maturation.

When completely mature particles, obtained at the end of the maturation experiment, were serially diluted and the␤-protein intensity was correlated with the lytic activity, an approximately linear correlation was observed, demonstrating that the liposome assay was not saturated. Therefore, the shape of the curve obtained with maturing particles is dictated by the subunit-specific cleavage rates during maturation, with subunits contributing to the lytic activity cleaving earlier and lytic-inactive subunits cleaving later (Fig. 3D).

DISCUSSION

Previous studies of FHV showed that autocatalytic cleavage pro-duces a covalently independent␥peptide that is required for both infectivity and liposome lysis activity (4). Likewise, VP0 in picor-naviruses must be autocatalytically cleaved into VP4 and VP2 to generate infectious particles (14) and a comparable autocleavage reaction that generates the peptide␮1N in reovirus is required for cell membrane penetration (23). Thus, there is ample precedent for the necessity of a covalently independent lytic peptide derived from a capsid subunit to generate an infectious particle. Previ-ously, lytic activity of FHV␥peptides was tightly correlated with maturation and infectivity (4). Similar observations were made for reovirus␮1N peptide that is formed after a maturation step and has lytic activity against red blood cellsin vitroand promotes reovirus virulencein vivo. (26). Thus, the requirement for N␻V cleavage to generate lytic activity is in accordance with previously observed patterns of nonenveloped virus membrane disruption requiring a maturation cleavage event.

Chemical cues for activation of lytic activity (after cleavage has occurred) have been characterized for FHV, minor and major group rhinoviruses, parvovirus, polyomavirus, and adenovirus (20). In each case, acidic pH led to membrane disruptionin vitro. This is proposed to mimic the environment of late endosomes, where low pH would result in capsid-mediated membrane trans-location of the viral genome (20). N␻V is strikingly different in its pH-dependent lytic activity profile, and this is the first example of basic pH being the trigger for membrane disruption. We address

FIG 3N␻V acquires full lytic activity with less than 50% of the subunits cleaved. Controlled maturation of NV procapsids (WT and E73Q mutant) was triggered by changing the pH from 7.6 to pH 5.0. Samples were taken throughout the mat-uration time course for determination of the extent of cleavage (A) and lytic activ-ity at pH 7.6 with a final VLP concentration of 10␮g/ml (B). (A) The percentage of subunits cleaved was determined by densitometry analysis of SDS-PAGE gels stained with Coomassie blue (shown in the inset). (B) Percent lytic activity repre-sents the dye release recorded for the same time points shown in panel A normal-ized to the dye release achieved by the fully mature particles, obtained after 1 day at pH 5.0. The inset shows that fully mature WT and E73Q mutant particles induce the release of equivalent amounts of dye when the data are normalized just by Triton X-100 activity. (C) Cleavage (top) and lytic activity (bottom) remain un-changed during the time required to take dye release measurements at pH 7.6. Capsids matured for 10 min at pH 5.0 were analyzed immediately (solid bars) or after 20 min of incubation at pH 7.6 (open bars). (D) Correlation between amounts ofprotein and lytic activity observed for maturing particles (solid lines) and fully mature capsids (dashed lines). The correlation curve obtained with ma-turing particles was generated by combining the data shown in panels A and B. Fully cleaved capsids obtained at the end of the maturation experiment were seri-ally diluted, and the lytic activity and relativeprotein concentration were deter-mined for each dilution step by liposome assay and SDS-PAGE band densitome-try, respectively. The distinct curve shape observed for maturing particles in comparison with the serially diluted mature particle reflects the subunit-specific rate of cleavage and shows thatpeptides contributing to lytic activity are gener-ated early during the maturation process.

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two aspects of this observation: first, the chemical mechanism of basic pH dependence, and second, the biological implications.

N␻V maturation is triggered at pH 5 with the capsid reorgani-zation driven by the protonation of negatively charged residues that dominate the subunit interfaces, allowing the formation of noncovalent interactions that shape the compact capsid structure (19). In the absence of␥peptide cleavage, as is observed with the N570T mutant, the entire process is reversible and the particle re-expands to the procapsid form (29). Cleaved mature particles, however, are restrained in the compact form even at pH values above 7.0, despite the increasing electrostatic repulsion. We pro-pose that this repulsion perturbs the stability of the mature parti-cles at alkaline pH, facilitating the exposure of␥peptides. Our data suggest that N␻V enters susceptible cells not through acidic vesicles of the endocytic pathway but directly through the plasma membrane. Little is known about the life cycle of N␻V, but

Heli-coverpa armigera stunt virus, a closely related tetravirus, is known

to proliferate exclusively in mid-gut cells of lepidopteran larvae (3). The pH within the mid-gut of lepidopteran larvae is usually alkaline (11), enabling the pH-dependent membrane-lytic activity observed. Tetraviruses display a remarkable use of a large pH range in their life cycle, with procapsid assembly occurring at ap-proximately neutral pH, followed by maturation at⬃pH 5 (7,19), probably promoted by apoptosis of infected cells (6,30) and then release into the basic mid-gut environment, where particle activa-tion and infecactiva-tion may occur.

Our data also reveal another important feature of the viral

maturation process. Even though the procapsid can take days to mature into a fully cleaved particle, the virion would be ready for a new cycle of infection only a few minutes after maturation begins (Fig. 3). Figure 3D shows that the same amount of cleaved subunits in the early steps of capsid matu-ration results in higher lytic potential than that amount of cleaved␤protein from fully mature particles. This observation supports the notion that just a subset of all␥peptides, ulti-mately present in the mature capsid, are actually involved in membrane lysis and also that these active␥peptides are specif-ically generated within the first 30 min of maturation.

The acquisition of lytic activity correlates with the time re-quired for cleavage of A and D subunits of the capsid, as shown by Matsui et al. (18). This conclusion is further corroborated by the results obtained with the E73Q mutant (Fig. 3). This strain showed a WT membrane-lytic activity phenotype despite the severe retardation of the maturation rate after 30 min at pH 5.0. The E73Q mutation disrupts the structural environment around the switch helix, affecting the stabilization of the flat contacts of the particle. Hence, the substitution specifically im-pacts the cleavage rate of subunits B and C, which depends on the stabilization of the flat contacts in order to form their au-tocatalytic site (Fig. 1and3). However, A and D subunits of the E73Q mutant show normal cleavage kinetics and structure (18), resulting in a lytic activity comparable to that of the WT. Examination of the␥peptide structures in subunits A and D suggests that they are involved in different structural roles

FIG 4␥peptides from pentamers form a helical bundle that is implicated in lytic activity. (A) N␻V␥peptide sequence and the secondary structure visible in the crystal structure for each of the quasi-equivalent positions (13). Spirals indicate alpha-helices, wavy lines represent loops, and dots represent disordered regions. The color code is the same in all of the panels. (B) Cutaway view of the capsid protein shell at 20 Å resolution, as calculated from crystallographic data (gray). The subunit positions are the same as those shown inFig. 1B. Internal RNA densities were derived by cryoelectron micrograph reconstruction (brown), and the positions of cleaved␥peptides are based on the X-ray coordinates. Switch helices are shown as rods, and atoms from the first␥peptide residue (F571) are shown as white spheres. (C) Lateral view of the pentamer formed by A subunits (blue) and D trimers (yellow). One of the A subunits was eliminated to reveal the internal helical bundle formed by␥peptides. (D) The capsid as seen from the top with the capsid protein densities made transparent to show the internal orientation of

␥peptides.

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(Fig. 4). D-␥peptide is almost fully ordered in the crystal struc-ture and is stabilized by several intermolecular interactions with subunits C and D. The␥peptide C-terminal helix is the molecular switch responsible for the stability of the flat con-tacts of the capsid. Therefore, the fast cleavage of D subunits is important to promptly ensure particle stability in different en-vironments and to make the maturation process irreversible. In fact, it is known that cleavage can proceed at lower rates even at neutral pH, provided that at least 10% of the subunits are cleaved during soaking at pH 5.0 (17).

While the D-␥peptide is mostly tangentially oriented and deeply buried within the capsid, the A-␥peptide is vertically oriented and organized in a helical bundle (Fig. 4). The bundle defines a central channel that is separated from the external environment by only a thin aperture formed by flexible turns from the shell domains of the A pentamer. Almost all of the extension of the A-␥peptide helix visible in the crystal struc-ture is facing the central channel of the 5-fold axis and there-fore would be readily exposed to the external environment with the breathing dynamic movements or opening of the upper region of the channel due to repulsion between subunits. In-deed, mass spectrometry analysis of peptides generated by trypsin digestion of mature N␻V capsid showed that the ␥ peptide is highly accessible to proteolysis, compared to other regions of the capsid protein (5).

We propose that the late-cleaving B and C subunits are dis-pensable for lytic activity and may be implicated in other func-tional roles. Just a short segment of the B-␥peptide is ordered in the structure, and it is tangentially oriented in the capsid and extensively overlaid by the jelly roll and Ig-like subunit motifs (Fig. 4). The B-␥peptide helix seems to follow the shape of the internal RNA cage visible in cryo-EM reconstructions (Fig. 4B), suggesting a potential role in nucleic acid interaction and organi-zation. The RNA stabilization may be driven by the C terminus of the␥peptide, which is enriched in positively charged residues, suggesting a dual role for identical sequences, with the B-␥peptide involved in RNA interactions and the A-␥peptide involved in phospholipid binding. The␥peptide of subunit C, the last subunit to complete cleavage, shows the same fold observed in the D-␥ peptide, and the cleavage of these subunits must be important to enhance overall particle stability. Note that even though the D-␥ peptide is cleaved within the same time frame that the capsid is acquiring membrane-lytic activity, its structure and position in the capsid are virtually identical to those of the lytic-inactive C-␥ peptide, strengthening the conclusion that the pentamers formed by A subunits are providing the lytic peptides.

Therefore, pentamers formed by A subunits can be considered a region of the capsid specialized for membrane disruption (Fig. 5). This conclusion can be extended not only to the evolutionarily linked T⫽3 nodaviruses (27), which show a similar organization of pentamers and␥peptides, but also to more remotely associated viruses such as the double-stranded RNA (dsRNA) T⫽13

Infec-tious bursal disease virus(IBDV) (10), a member of the

Birnaviri-daefamily. IBDV capsids are formed by a VP2 protein that also undergoes cleavage and show an internal bundle of amphipathic helices associated with pentamers (15). Recently, it was demon-strated that this noncovalently linked peptide is active against membranes (12). Thus, pentamers harboring noncovalently asso-ciated helical bundles may be a common motif in nonenveloped virus capsids and the recognition of this structural pattern in other

viruses can help us to understand their cell entry mechanism and design therapeutic strategies.

ACKNOWLEDGMENTS

This work was supported by NIH grant 5R01 GM 54076 to J.E.J., the PEW Latin American Fellowship Program, and the Conselho Nacional de Desenvolvimento Cientifico (CNPq) Brasil (to T.D.). Portions of this re-search were carried out at the Stanford Synchrotron Radiation Light-source, a directorate of the SLAC National Accelerator Laboratory and an Office of Science user facility operated for the U.S. Department of Energy Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Envi-ronmental Research and by the NIH, National Center for Research Re-sources, Biomedical Technology Program (P41RR001209).

The contents of this publication are solely our responsibility and do not necessarily represent the official view of NCRR or NIH.

We thank Andrew Routh and David Veesler for critically reading the manuscript and Jeff Speir, Mandy Janssen, and Megan Guelker for stim-ulating discussions, as well as providing laboratory reagents and technical support.

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FIG 5Lytic peptide helical bundles at the pentons of different virus species. Panels show ribbon representations of the 5-fold axes with covalently indepen-dent peptides highlighted in blue, as viewed from inside the capsid. N␻V, PDB code 1ohf; FHV (an example of an ssRNA nodavirus), PDB code 2q25; Prov-idence virus (PrV; an example of an ssRNA betatetravirus), PDB code 2qqp; IBDV (an example of a dsRNA birnavirus), PDB code 1wce. IBDV peptides are not ordered in the crystal structure, but cryo-EM reconstruction (15) revealed internal densities corresponding to the membrane-active amphipathic helix (approximate positions are indicated by blue circles).

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[image:6.585.301.542.72.317.2]
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Figure

FIG 1 The Ndetermined by X-ray crystallography of mature N�V T�4 capsid is formed by 240 copies of the same protein organized into four quasi-equivalent positions
FIG 2 NLiposomes at pH 7.6 were treated with increasing concentrations of matureWT N�V � peptides show lytic activity against artificial membranes
FIG 3 N�V acquires full lytic activity with less than 50% of the subunits cleaved.Controlled maturation of N�V procapsids (WT and E73Q mutant) was triggeredby changing the pH from 7.6 to pH 5.0
FIG 4 � peptides from pentamers form a helical bundle that is implicated in lytic activity
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