Small Compounds Targeted to Subunit Interfaces Arrest Maturation in a Nonenveloped, Icosahedral Animal Virus

Nudaurelia ${omega}$ capensis virus (N ${omega}$ V) capsids were previously characterizedin two morphological forms, a T=4, 485-Å-diameter roundparticle with large pores and a tightly sealed 395-Å icosahedrallyshaped particle with the same quasi-symmetric surface lattice.The large particle converts to the smaller particle when thepH is lowered from 7.6 to 5, and this activates an autocatalyticcleavage of the viral subunit at residue 570. Here we reportthat both 1-anilino-8 naphthalene sulfonate (ANS) and the covalentattachment of the thiol-reactive fluorophore, maleimide-ANS(MIANS), inhibit the structural transition and proteolysis atthe lower pH. When ANS is exhaustively washed from the particles,the maturation proceeds normally; however, MIANS-modified particlesare still inhibited after the same washing treatment, indicatingthat covalent attachment targets MIANS to a critical locationfor inhibition. Characterization of the low-pH MIANS productby electron cryo-microscopy (cryo-EM) and image reconstructiondemonstrated a morphology intermediate between the two formspreviously characterized. A pseudoatomic model of the intermediateconfiguration was generated by rigid body refinement of theX-ray structure of the subunits (previously determined in theassembled capsid) into the cryo-EM density, allowing a quantitativedescription of the inhibited intermediate and a hypothesis forthe mechanism of the inhibition.

INTRODUCTION

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Introduction

Protein capsids of many icosahedral viruses respond dynamicallyto environmental signals such as DNA or RNA packaging, receptorbinding, and changes in pH and ionic composition. In so doing,they undergo conformational changes that involve the concertedreorganization of hundreds of protein subunits and nucleic acids.In many viruses, large-scale conformational transformationsoccur during assembly and maturation. Such transformations havebeen observed in several bacteriophages (7, 9, 11, 16, 19, 23,30), herpesvirus (15, 33), and a single-stranded RNA insectvirus (1, 4). Icosahedral virus particles with T numbers higherthan 1 apparently do not always directly assemble into a highlystable form. Rather, capsid proteins initially organize intoprocapsid shells that place subunits into proper relative locationsbut with, in many cases, sparse interfacial contacts (4, 9,16). These precursor particles are poised for reorganizationinto the stable mature particle when the proper signal is receivedby the protein or nucleo-protein aggregate (for examples, seereferences 4 and 9). Nudaurelia ${omega}$ capensis virus (N ${omega}$ V), an insecttetravirus, was the first single-stranded RNA animal virus forwhich three-dimensional structures were determined for the procapsidand mature capsid conformations (4, 22). The procapsid structurewas discovered for N ${omega}$ V virus-like particles (VLPs) expressedin a recombinant baculovirus system. These particles containheterologous cellular RNA in a ratio to protein that is similarto that of authentic virions (4). A cryo-electron microscopy(cryo-EM) reconstruction at 26 Å revealed a nearly sphericalprotein shell perforated by large holes at all symmetry axes(4). These holes were absent in the mature, authentic virionstructure, determined by crystallography at 2.8-Å resolution(4, 22). Maturation of procapsid nucleoprotein VLPs inducedby acidification to pH 5 resulted in icosahedral capsid particlesindistinguishable at 26-Å resolution from authentic virions(4). This transformation has also been monitored using small-angleX-ray scattering experiments, which reported a decrease in averagediameter, from 485 to 395 Å, when procapsids were convertedto capsids (5). The reorganization activates an autoproteolyticcleavage between residues 570 and 571 of the 70-kDa ${alpha}$ proteininto 62- and 8-kDa ß and ${gamma}$ polypeptides. The resultingmature structure contains 240 copies of the ß and ${gamma}$ polypeptides and the same nucleic acid composition as procapsid(4, 22). Comparison of the procapsid and capsid structures withthe X-ray coordinates as a reference demonstrated that the conversiondepends largely on quaternary reorganization of subunits andnot large-scale refolding of tertiary or secondary structure(4, 5). The transition occurs in a highly cooperative mannerover a 0.7 pH unit range between pH 6.7 and 6.0 (5). Above andbelow this range, particles exist as stable procapsid and capsid,respectively.

In order to further characterize the conformational transition,we labeled N ${omega}$ V procapsid particles with the thiol-reactive fluorophore2-(4'-maleimidylanilino)naphthalene-6-sulfonate (MIANS). MIANSexhibits a pronounced sensitivity to its local environment,fluorescing strongly only when sequestered at hydrophobic surfaces(2, 18). We reasoned that the covalently attached MIANS situatedat subunit interfaces might report on the conformational transitionas it is buried during maturation. Instead, we found that uponacidification, the labeled particles were trapped at an intermediatestage of maturation and that the autocatalytic cleavage, believedto be essential for infectivity, was virtually inhibited.

The results presented in this communication provide supportfor the strategy of targeting subunit interfaces with antiviralcompounds as a means of disrupting the viral life cycle (24,32, 34). Most previous studies of small-compound inhibitionof other viruses have focused on interrupting polymerizationof capsid subunits. For example, a compound that is structurallyrelated to MIANS, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate(bis-ANS), was observed to inhibit in vitro assembly of hepatitisB virus capsids (34). In that case, the fluorophore was foundto prevent assembly of complete capsids by binding to solubledimers. It was postulated that bis-ANS acts as a molecular "wedge"that prevents formation of correct capsid geometry. Similarly,bis-ANS was observed to inhibit procapsid assembly in the bacteriophageP22 system (32) and to contribute to inactivation of vesicularstomatitis virus (3). Capsid-targeted compounds from the heteroaryldihydropyrimidinefamily also have been demonstrated to inhibit hepatitis B viruscore particle formation (10); in vivo, this is accompanied byan increase in core protein degradation. Similarly, a recentstudy of human immunodeficiency virus type 1 assembly demonstratedthat a small molecule (CAP-1) binds to the human immunodeficiencyvirus capsid protein and inhibits infectivity in a dose-dependentmanner (28). The compound gives rise to abnormal core particlesby interfering with interactions between capsid proteins thatare critical for proper maturation. By contrast, our resultsdemonstrate that a later stage of virus maturation, after theprotein shell has fully polymerized, can be effectively disruptedby small compounds.

MATERIALS AND METHODS

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Materials and Methods

Purification of N ${omega}$ V procapsid VLPs. Purification of VLPs was performed following previously describedprocedures (12, 31). Briefly, Sf21 cells were infected witha recombinant baculovirus vector containing the N ${omega}$ V wild-type(WT) coat protein. After 4 days of incubation at 27°C, cellswere lysed with 0.5% (vol/vol) NP-40 detergent. Supernatantand debris were separated by centrifugation. Supernatant containingthe VLPs was treated with 10 µg of RNase A/ml in orderto degrade ribosomal material that would otherwise copurifywith the VLPs. VLPs were pelleted through a 30% (wt/vol) sucrosecushion at pH 7.6. The high-speed pellets were resuspended in400 µl of pH 7.6 buffer containing 250 mM NaCl-50 mM Tris(buffer A). The soluble resuspended material was then sedimentedthrough a 10-to-40% sucrose gradient (wt/vol) prepared usingpH 7.6 buffer A. Standard centrifugation conditions were 113,000x g for 1.25 h at 11°C in a Beckman SW41 rotor. Fractionswere collected using an ISCO fractionator running at 0.75 min/fractionwith continuous measurement of absorbance at 280 nm.

The primary sequence of the capsid protein subunit used in thesestudies differs from the sequence deposited in GenBank. Thedifferences are isolated to three point mutations. Specifically,in our clone residue 37 is cysteine (rather than lysine), residue235 is valine (rather than cysteine), and residue 283 is valine(rather than glutamate).

Labeling reaction. N ${omega}$ V procapsid particles were labeled at a 50-fold molar excessconcentration of MIANS (Molecular Probes) to capsid protein([MIANS] = 700 µM; [capsid protein] = 14 µM) inbuffer A with 10% dimethyl sulfoxide (DMSO). N ${omega}$ V particles maturenormally and are insensitive to DMSO concentrations in thisrange. The reaction mixture was incubated at 4°C for 18h. Excess dye was removed by centrifugal filtration through100-kDa MWCO membranes (Millipore Corp.). The first three washeswere performed using a mixture of 10% DMSO and 90% buffer A.This step was essential for removing all excess, unconjugateddye. Typically, five additional washes with only buffer A weredone to remove all DMSO. Samples were protected from exposureto ambient light throughout labeling and purification and duringstorage.

Quantitation of labeling efficiency involved accurate determinationof concentrations of both covalently bound fluorophore and capsidprotein. Determination of capsid protein concentration is nottrivial, however, due to the contributions of the nucleic acidto absorbance measurements at 280 nm. Moreover, the extinctioncoefficient available in the literature for N ${omega}$ V of A₂₆₀ = 5 correspondingto 1 mg/ml has never been rigorously verified, and this relationpresumably gives total VLP concentration (including RNA andprotein) rather than protein capsid concentration alone. Therelative mass fractions of RNA and protein have never been establishedfor this virus, however, and thus it is not possible to directlydetermine protein concentration by using the A₂₆₀ = 5 value.

In order to accurately determine capsid protein concentrationand correlate A₂₆₀ with actual capsid protein concentration,we developed a fluorescence assay that relates the intrinsicfluorescence intensity of a VLP sample to a known capsid proteinconcentration based upon a standard curve determined for pureN ${omega}$ V capsid protein in the absence of nucleic acid and under denaturingconditions. Because RNA does not fluoresce, the fluorescenceintensity measured for a VLP sample containing both the capsidprotein and encapsidated RNA denatured in 6 M guanidine HClreports on the protein concentration alone. By this assay, wehave found that an A₂₆₀ value of 2.8 corresponds to a capsidprotein concentration of 1 mg/ml. For virus samples labeledwith MIANS, the A₂₆₀ was further corrected to account for absorbanceby MIANS, thus yielding the actual capsid protein concentration.The concentration of covalently bound MIANS was determined bymeasuring the absorbance at 325 nm (the absorbance maximum forMIANS shifts from 443 to 325 nm upon conjugation to sulfhydrylgroups).

Cleavage maturation assayed by gel electrophoresis. Purified procapsids in buffer A were converted to capsids byrapid dilution into pH 5.0 buffer B (250 mM NaCl, 50 mM Na-acetate;pH 5.0). Maturation was allowed to progress for a minimum of4 h at 25°C. Previous studies have demonstrated that cleavageis essentially complete within 1 h under these conditions (1,5). Identical conditions were used in attempting to mature MIANS-conjugatedN ${omega}$ V (M-N ${omega}$ V) particles. Maturation inhibition by noncovalentlybound 1-anilino-8 naphthalene sulfonate (ANS) was examined usingsolutions containing 0.1, 0.3, and 1.0 mM ANS (Sigma) made withbuffer B. After 4 h of incubation at pH 5.0, half of each samplewas removed and washed by centrifugal filtration (100-kDa MWCOmembrane; Millipore Corp.). Three washes were performed usinga 10% DMSO-90% buffer B, ANS-free solution followed by threeadditional washes with buffer B. Both washed and unwashed sampleswere allowed to incubate for an additional 2 h at pH 5.0 at25°C. All samples were analyzed for cleavage maturationby running them on 4-to-12% denaturing acrylamide gradient gels(Invitrogen) according to standard procedures.

Velocity sedimentation of labeled VLPs. M-N ${omega}$ V VLPs and unlabeled WT N ${omega}$ V capsid were sedimented at 113,000x g for 1.25 h through a 10-to-40% (wt/vol) sucrose gradientprepared with pH 5.0 buffer B. Unlabeled WT N ${omega}$ V procapsid wassedimented at 113,000 x g for 1.25 h through a 10-to-40% (wt/vol)sucrose gradient prepared with pH 7.6 buffer A. Sedimentationconditions and fractionation methods were performed as describedabove for particle purification.

EM and image reconstruction. Samples for negative-stain transmission EM were prepared at0.5 mg/ml. Staining and microscopy were performed as describedpreviously (31) using 1% uranyl-acetate solutions. Preliminarythree-dimensional image reconstruction studies were carriedout with negative-stained samples, and final reconstructionswere calculated with frozen hydrated specimens. We note thatnegative stain and cryo-EM reconstructions of the M-N ${omega}$ V particlesat pH 5.0 showed very good agreement with each other, underscoringthe apparent robustness of the labeled particles even underthe relatively harsh conditions of negative staining (data notshown). Three-hundred-mesh copper grids were used for makingthe holey film. Five-microliter samples at the concentrationof 0.5 mg/ml were applied onto the holey grid before plunge-freezingin liquid ethane. A Gatan cryo-holder was used to insert thespecimen into the electron microscope. The micrographs weretaken at a magnification of x45,000 on a Philip CEM 120 electronmicroscope equipped with decontaminating blades. Images weredigitized using a ZEISS flat bed scanner with a step size of21 µm corresponding to 4.67 Å/pixel. After digitizingthe micrographs, virus particles were selected with the boxingprogram X3D from James Conway (8). The reconstructions werecalculated with SPIDER (14). The resolution of the reconstruction(899 selected particles) was estimated with a Fourier shellcorrelation coefficient of 27 Å.

Fitting models into cryo-EM density. The crystal structure of the mature N ${omega}$ V model was docked intothe electron density calculated from the M-N ${omega}$ V particles. Becausethis particle is larger than the mature virus, the coordinateswere moved radially outwards to fit the reconstruction. Then,the four subunits were treated as rigid bodies to fit the electrondensity map calculated from the cryo-EM reconstruction. Smallportions of the subunits were not used in the pseudoatomic modeling,because these residues exhibit the greatest subunit-to-subunitdifferences in the capsid structure and are likely to be insubstantially different conformations in procapsid and M-N ${omega}$ Vstructures, which exhibit less subunit differentiation (4).Only residues 61 to 599, 44 to 590, 42 to 591, and 44 to 590out of 644 residues in each subunit were used in the modelingfor the A, B, C, and D subunits, respectively. The fitting wasoptimized by a modified version (for cryo-EM application) (29)of the real-space refinement program Rsref (6).

The four subunits (A, B, C, and D) in the N ${omega}$ V T=4 capsid asymmetricunit were analyzed individually to determine the movement duringthe conformational transition from procapsid to the M-N ${omega}$ V andthen to the mature capsid (see Table 1). Each subunit in theprocapsid model was compared with its corresponding subunitin the M-N ${omega}$ V to find the translational and rotational transformationfor superimposing them by the program LSQMAN from the UppsalaSoftware Factory. Similarly, the translational and rotationaltransformations from M-N ${omega}$ V to the matured capsid were analyzedfor each subunit by LSQMAN.

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TABLE 1. Translation of subunits between procapsid, M-N ${omega}$ V (pH 5.0), and capsid

RESULTS

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Results

MIANS labeling of N ${omega}$ V procapsid. MIANS reacts with the viral capsid protein by forming a covalentthio-ether linkage with the thiol group of cysteine residues.In order to quantify the efficiency of labeling, we determinedthe number of MIANS molecules attached to labeled procapsidVLPs by measuring absorbances at 325 and 260 nm (see Materialsand Methods). We consistently obtained an average (±standard deviation) of 4.2 ± 0.8 MIANS molecules conjugatedper subunit. Labeling of each subunit was inferred to be uniform,because all four quasi-equivalent subunit environments in theprocapsid were similar (4). Labeling studies with maleimide-functionalizedpropionic acid followed by proteolytic digestion and matrix-assistedlaser desorption ionization-mass spectrometry showed that thethree most reactive cysteines are CYS-135, CYS-507, and CYS-552(B. Bothner and J. E. Johnson, unpublished data). It is notclear whether the bulkier, aromatic MIANS probe accesses thesame sites to the same degree. Mass spectrometry has not provenfeasible for identifying the residues labeled with MIANS, possiblydue to the influence of the negatively charged fluorophore uponpeptide ionization. However, based on the known subunit structurein which two cysteines (CYS-247 and CYS-507) are buried in theß-sandwich domain, we infer that these cysteines maybe inaccessible to the MIANS dye. Furthermore, since the reactionconditions should have saturated the available sites, we believethe figure of four MIANS per subunit that we measured indicatesthat all other cysteines (37, 135, 516, and 552) were completelylabeled.

Autoproteolytic cleavage is greatly diminished. N ${omega}$ V subunits normally undergo an autoproteolytic cleavage betweenresidues 570 and 571 (1, 22); this converts the 70-kDa ${alpha}$ proteininto 62-kDa ß and 8-kDa ${gamma}$ components. The reactionhas a half-life on the order of an hour and only is initiatedonce the compact capsid state has been attained (5). Based onthe structural similarities with nodaviruses such as Flock housevirus, cleavage of the ${gamma}$ peptide in N ${omega}$ V is believed to be essentialfor infection (26). N ${omega}$ V VLPs labeled with MIANS showed almostno cleavage after 4 h of incubation in the pH 5.0 buffer at25°C, while WT unlabeled VLPs were cleaved (Fig. 1a). Similarly,cleavage in unlabeled N ${omega}$ V VLPs incubated at pH 5.0 in the presenceof a 1 mM concentration of the non-thiol-reactive MIANS analogANS was significantly inhibited (see Fig. 3); lower concentrationsof ANS, however, were not as effective at inhibiting maturationand preventing cleavage. Once the ANS was exhaustively washedaway, the particles matured fully, exhibiting normal levelsof cleavage and thus demonstrating that the ANS itself was notdisrupting or irreversibly altering the particles and cleavagemechanism.

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FIG. 1. Labeled particles do not exhibit cleavage and sediment similarly to procapsid N ${omega}$ V during velocity sedimentation. (a) Labeled and unlabeled N ${omega}$ V samples run on a 4-to-12% denaturing acrylamide gradient gel. Lane assignment from left to right: 1, Invitrogen SeeBlue Plus 2 molecular weight standards; 2, WT procapsid pH 7.6; 3, WT capsid pH 5.0; 4, M-N ${omega}$ V pH 7.6; 5, M-N ${omega}$ V pH 5.0. (b) Fractionation of WT procapsid, capsid, and M-N ${omega}$ V particles sedimented through a 10-to-40% sucrose gradient. Ten-to-40% sucrose gradients (top of gradient on the left) prepared with 250 mM NaCl, pH 7.6 buffer for WT procapsid (buffer A) and 250 mM NaCl, pH 5.0 buffer (buffer B) for M-N ${omega}$ V and WT capsid. The curves presented are continuous measurements of the A₂₈₀ for gradient fractionations.

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FIG. 3. Noncovalently bound 1,8-ANS can inhibit maturation at pH 5.0 when present in high concentrations. If particles are washed with a 10% DMSO-90% buffer B solution, the ANS is removed and the particles mature normally. (a) Four-to-12% denaturing acrylamide gradient gel assay for subunit cleavage. Lanes from left to right: 1, Invitrogen SeeBlue Plus 2 molecular weight markers (sizes indicated in kilodaltons); 2, WT procapsid; 3, WT capsid (no ANS); 4, 1.0 mM ANS unwashed; 5, 1.0 mM ANS washed; 6, 0.3 mM ANS unwashed; 7, 0.3 mM ANS washed; 8, 0.1 mM unwashed; and 9, 0.1 mM washed. (b) Negative-stain EM micrograph of particles in 1.0 mM ANS at pH 5.0, revealing that the particles are intact but penetrable by uranyl-acetate stain. (c) Negative-stain EM micrograph of particles in 0.3 mM ANS at pH 5.0, revealing that particles are intact and appear to have matured to capsid.

M-N ${omega}$ V sediments similarly to unlabeled procapsid. Velocity sedimentation through sucrose gradients provides asensitive assay of hydrodynamic character for an entire populationof particles. Sedimentation of unlabeled procapsid and capsidthrough a 10-to-40% sucrose gradient at 113,000 x g for 1.25h consistently results in procapsid at fractions 17 to 18 andcapsid at fractions 19 to 20 (31). Sedimentation of labeledM-N ${omega}$ V particles, at pH 5.0, generated a single peak at fractions17 and 18 (Fig. 1b). By this assay, the labeled particles exhibitedhydrodynamic properties similar to those of unlabeled procapsid,even at acidic pH.

M-N ${omega}$ V particles are permeable to negative stain at neutral and acidic pH. Unlabeled procapsid and capsid N ${omega}$ V VLPs are distinguishable bynegative-stain transmission EM (Fig. 2). Procapsids exhibita round shape and are permeable to stain, whereas capsid particlesare polyhedral and are mostly impermeable to stain. Significantpopulations of procapsids break apart, presumably due to thelow-pH shock produced by uranyl-acetate staining, while capsidparticles maintain their integrity. Figure 2 also shows thatM-N ${omega}$ V particles were permeable to stain at both neutral and acidicpH. Yet, compared with the fragile N ${omega}$ V procapsid particles (Fig.2c), the labeled particles appeared regular and robust at lowpH (Fig. 2b). M-N ${omega}$ V particles at high pH also appeared to besomewhat less prone to breaking apart than the unlabeled procapsid.

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FIG. 2. Negative-stain EM micrographs. (a) M-N ${omega}$ V in pH 7.6 buffer A; (b) M-N ${omega}$ V in pH 5.0 buffer B; (c) WT N ${omega}$ V pH 7.6 buffer A; (d) WT N ${omega}$ V pH 5.0 buffer B. Magnification, x52,000.

Particles incubated in 1 mM ANS at pH 5.0 likewise were permeableto the stain, whereas particles in 0.3 mM ANS appeared to havematured fully to form mostly stain-impermeable capsids (Fig.3). These results corroborate the findings that VLPs in 1 mMANS are unable to undergo autoproteolytic cleavage maturation(Fig. 3), while those in 0.3 mM ANS mature normally. Negative-stainEM micrographs also confirmed that ANS does not break apartthe particles.

EM reconstructions and pseudoatomic model of M-N ${omega}$ V at pH 5. The three-dimensional cryo-EM reconstruction of labeled VLPsat pH 5.0 is shown in Fig. 4, with reconstructions of unlabeledN ${omega}$ V procapsid (pH 7.6) and capsid (pH 5.0) adapted from Canadyet al. (4) for comparison. The unlabeled procapsid and capsidexhibit distinct surface morphologies due to different configurationsof the immunoglobulin (Ig) domains and lack of close-packedß-sandwich interfaces in procapsid (4). Ig domainsin the procapsid form dumbbell-shaped surface protrusions correspondingto dimer pairs between the A and B and the C and D subunits.The dimers differentiate into two categories of trimer (ABCand DDD) in mature capsid, dramatically altering the particleshape and surface morphology. The internal surface of the procapsidreconstruction exhibits a trefoil-shaped density at the three-foldand quasi-three-fold axes. This density is absent in the capsidand may denote a region of the subunit that refolds during thetransition (4).

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FIG. 4. Cryo-EM reconstructions for procapsid, capsid, and M-N ${omega}$ V at pH 5.0 particles. (a, b, and c) Outer surface view of the procapsid, capsid, and M-N ${omega}$ V particle, respectively. (d, e, and f) Inner surface view with the trefoil feature highlighted in red. Capsid and procapsid reconstructions were adapted from Canady et al. (4).

The reconstruction of M-N ${omega}$ V particles at pH 5.0 (Fig. 4) resemblesprocapsid despite the fact that at pH 5.0 unlabeled N ${omega}$ V existsin the compact capsid form. Closer examination revealed thatM-N ${omega}$ V at pH 5.0 was intermediate in size between procapsid andcapsid, with an approximate diameter of 440 Å. The sizedetermination by EM contrasted with the results of a sedimentationassay that suggested the M-N ${omega}$ V particles at pH 5 were similarin size to procapsid. This indicates that the sucrose gradientsedimentation assay is more sensitive to the hydrodynamic surfaceproperties and porous nature of the particles than to the relativelysmall change in particle diameter. Indeed, the exterior of thelabeled particles at pH 5 was very procapsid-like (Fig. 4).The Ig domains were still dimeric dumbbells, and close packingof interfaces had not yet occurred. The trefoil densities observedon the inner surface of procapsid persisted, but densities atthree-fold and quasi-three-fold axes differed, with the lattershowing a weaker density, suggesting that subunits are differentiatedto a greater extent than in procapsid. Holes present in theprocapsid structure at quasi-six-fold and five-fold axes wereclosed, but pores still penetrated at the three-fold and quasi-three-foldaxes (see Fig. 5). The existence of the open pores likely wasresponsible for the penetration of negative stain into M-N ${omega}$ Vparticles (Fig. 2). The N ${omega}$ V subunit X-ray coordinates were dockedinto the cryo-EM density and refined as four rigid bodies inthe icosahedral asymmetric unit. The excellent fit to the densitywas consistent with limited refolding between the capsid-basedX-ray structure and the inhibited, low-pH, procapsid-like structure(Fig. 4).

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FIG. 5. (a) Location of five of the six cysteine residues in the N ${omega}$ V D subunit; CYS-37 is not observed in the crystallographic structure. The Ig-like domain is orange, the ß-sandwich domain is cyan, and the helical internal domain is green. (b) View of the pseudoatomic model for M-N ${omega}$ V at pH 5.0 down the threefold and quasi-threefold axes; only the polypeptide backbone trace for the ß-sandwich domains is depicted. Cysteines 135, 247, 507, and 516 are rendered as space-filling models. Subunit interfaces are open and not tightly packed. (c) Same view as in panel b, but of the internal helical domain and CYS-552.

DISCUSSION

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Discussion

N ${omega}$ V morphogenesis and maturation involve three main stages: (i)an assembly event in which capsid protein subunits and nucleicacid coalesce to form a fragile particle with subunits looselyarranged on a T=4 quasi-equivalent lattice (procapsid), (ii)a pH-dependent conformational compaction in which subunits undergoa dramatic rearrangement to form the more robust, closed capsid,and (iii) an autoproteolytic cleavage event that locks the particlesinto their capsid states (4). Comparing particle dimensionsand the refined pseudoatomic coordinates of M-N ${omega}$ V with thoseof the capsid and procapsid (Table 1), we estimate that M-N ${omega}$ Vhas progressed halfway along the trajectory between procapsidand capsid. Indeed, the pH 5.0 M-N ${omega}$ V reconstruction was consistentwith data obtained from X-ray scattering experiments for a structuralintermediate that becomes populated during the course of thistransition (5). We conclude that M-N ${omega}$ V at pH 5.0 represents atrapped intermediate along the maturation pathway between procapsidand capsid. This result underscores the fact that labeling ofN ${omega}$ V with MIANS did not disable the pH-triggering event that initiatesthe conformational compaction of procapsid. Instead, it appearsthat the particles were prevented from attaining their fullycompact conformation that is required for autoproteolytic cleavage.

The crystal structure of the N ${omega}$ V capsid revealed that the structuralcontrol elements required for the formation of the T=4 latticereside in the N- and C-terminal helical regions of the subunitand that these are internal in the particles (22). Canady etal. suggested that a trefoil of internal density located aticosahedral and quasi-three-fold symmetry axes in the procapsidbut absent in the capsid may correspond to segments of the subunitthat become unstructured at low pH (Fig. 4) (4). Taylor et al.have also suggested that this portion of the subunit domainsmay represent the conformational switch that directs the transformationas a whole (31). We attribute the trefoil-shaped density tothe clustered internal domain helices of the N ${omega}$ V subunit (Fig.5), which form trimeric interactions around the three-fold andquasi-three-fold vertices. The trefoils are present in the M-N ${omega}$ Vintermediate structure determined at pH 5.0, although at reduceddensity around the quasi-three-fold axes. This reduction intrefoil density around the quasi-three-fold axes (A, B, andC subunits) is accompanied by the appearance of additional densityat the five-fold vertex. This may indicate that the procapsid-capsidtransition is initiated by movement of subunits around the five-foldvertex, namely the A subunit which is at the vertex and theB subunits that interact closely with the A subunits. Trefoilscentered on the true three-fold axes (D subunits) are foundwith nearly the same density as in the procapsid structure,suggesting that these portions of the icosahedral assembly matureat a later stage.

The trefoil structures may persist at lower pH because the MIANSas well as ANS bind directly to portions of this domain andinhibit its dissolution by unfolding of helices or dissociationof paired helices at low pH. ANS and the closely related compoundbis-ANS have been observed to influence protein conformationand oligomerization in many other systems. In some cases, bindingof the fluorophore to proteins induces tightened conformationalstates, particularly when the proteins are initially denaturedor only partly structured (17, 21). In N ${omega}$ V, binding of ANS orattachment of MIANS to the helical domain may stabilize thetrefoil conformation, thus restraining the switching mechanismthat drives the conformational maturation to completion. TheEM reconstruction suggests this inhibition is most effectivefor the D subunits that ring the three-fold axes.

Based upon the pseudoatomic structure we have determined, thelargest gaps between subunits in the maturation-arrested particlesare located along the three-fold and quasi-three-fold axes (Fig.5). Of the six cysteine residues present in each subunit, CYS-552is the only residue located along the three-fold and quasi-three-foldaxes. CYS-552 is also the only cysteine that is located on theinternal helical domain of the subunit which, as described above,is believed to contribute to the trefoil density observed byEM. The locations of the other cysteines make them less likelyto be involved in arresting the conformational change. CYS-247and CYS-507 are buried in the ß-sandwich domain ofthe virus; as such they are unlikely to be readily accessibleto covalent modification by bulky molecules such as MIANS. CYS-37is not crystallographically ordered, and thus its precise locationis not known. However this residue is surrounded by severalarginine and lysine residues that are believed to interact withthe encapsidated RNA. CYS-135 is the only cysteine situatedon the exterior surface of the mature capsid. While this residuesits near a subunit interface, compounds bound to this siteprobably are not sterically restrained, and thus they are probablyincapable of preventing the subunit interfaces from sealingshut. CYS-516 is located along the face of the ß-sandwichdomain, relatively distant from the three-fold and quasi-three-foldvertices. Our working hypothesis is that labeling of CYS-552may be critical for inhibition of the conformational maturation.

Is MIANS unique in its ability to inhibit particle maturation?We have demonstrated that noncovalently bound ANS alone willinhibit the transition at sufficient concentrations. However,we do not know the location of the specific site(s) at whichANS binds to the capsid protein; indeed, ANS may interact withcapsid proteins through generalized, nonspecific associationat numerous hydrophobic surface patches. The covalent attachmentof MIANS is significant, because it localizes the inhibitorycompounds to specific sites that also successfully arrest thetransition. Preliminary studies involving a variety of othercompounds conjugated to the cysteines in N ${omega}$ V also revealed inhibitionof the maturation. These compounds include an uncharged, 252-Daspin label (TEMPO-maleimide), as well as bulkier fluorophoressuch as PyMPO and Oregon Green 488-maleimide that bear oppositeelectrostatic charges (K. K. Lee, B. Bothner, and J. E. Johnson,unpublished data). Although the role of charge burial cannotbe ruled out (13, 27), one feature these compounds have in commonwith MIANS is a bulky apolar moiety. The specific structuresof this moiety vary widely. This suggests that the protein-proteininteractions that must undergo rearrangement during maturationare highly sensitive to perturbation at their interfaces, particularlywhen it is localized to the pressure points marked by the cysteineresidues.

Whereas studies of disruption of virus function by capsid-directedcompounds in other systems have demonstrated inhibition of properassembly (24, 28, 32, 34), in the case of the M-N ${omega}$ V capsids thecorrect assembly has already been attained, and the interferingmolecule does not break down the assembly. Instead, it appearsthat a later stage involving a large-scale conformational reorganization,required for maturation, has been inhibited by the introductionof the inhibitory compounds at interfaces. In this respect,the actions of the MIANS and ANS compounds on N ${omega}$ V maturationare similar to the action of the WIN compounds that inhibitpicornavirus infectivity by binding to fully formed virus capsidsand inhibiting dynamic quaternary breathing motions that arenecessary for infection (20, 25). Clearly, viral capsids canbe targeted at several stages of the viral life cycle: by disruptingsubunit polymerization (24, 28, 32, 34) and by inhibiting dynamiccapsid breathing (20, 25), as well as by preventing maturationof the already-assembled capsid.

Lastly, the covalently modified N ${omega}$ V particles appear to be stableand more robust than procapsid N ${omega}$ V. We are, thus, presented withan opportunity to characterize an alternative state of subunitorganization in this virus by using moderate (EM) and high-resolution(X-ray crystallography) methods. This will provide insight intothe mechanisms of large-scale conformational transformationsin these massive macromolecular complexes.

ACKNOWLEDGMENTS

This work was supported by NIH grant R01-GM54076. K. K. Leeis supported by a postdoctoral fellowship grant from the NationalInstitutes of Health (F32-GM65013).

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Biology, The Scripps Research Institute, MB-31, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 784-9705. Fax: (858) 784-8660. E-mail: jackj{at}scripps.edu.

K.K.L. and J.T. contributed equally to this work.

REFERENCES

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