Aura Virus Structure Suggests that the T=4 Organization Is a Fundamental Property of Viral Structural Proteins

Aura and Sindbis viruses are closely related alphaviruses. Unlikeother alphaviruses, Aura virus efficiently encapsidates bothgenomic RNA (11.8 kb) and subgenomic RNA (4.2 kb) to form virusparticles. Previous studies on negatively stained Aura virusparticles predicted that there were two major size classes withpotential T=3 and T=4 capsid structures. We have used cryoelectronmicroscopy and three-dimensional image reconstruction techniquesto examine the native morphology of different classes of Auravirus particles produced in BHK cells. Purified particles separatedinto two components in a sucrose gradient. Reconstructions ofparticles in the top and bottom components were computed toresolutions of 17 and 21 Å, respectively, and comparedwith reconstructions of Sindbis virus and Ross River virus particles.Aura virus particles of both top and bottom components havesimilar, T=4 structures that resemble those of other alphaviruses.The morphology of Aura virus glycoprotein spikes closely resemblesthat of Sindbis virus spikes and is detectably different fromthat of Ross River virus spikes. Thus, some aspects of the surfacestructure of members of the Sindbis virus lineage have beenconserved, but other aspects have diverged from the SemlikiForest/Ross River virus lineage.

INTRODUCTION

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Introduction

Alphavirus is a genus within the family Togaviridae that consistsof about 26 members. Alphaviruses are enveloped, positive-sense,single-strand RNA viruses, are transmitted by arthropods (primarilymosquitoes), and cause fever, rash, or encephalitis in humansor domestic animals (24). The alphaviruses can be grouped intothree lineages based on comparisons of their genome sequences:the VEE/EEE (Venezuelan equine encephalitis/Eastern equine encephalitisvirus) lineage, the SFV/RRV (Semliki Forest/Ross River virus)lineage, and the SINV (Sindbis virus) lineage (18). Aura virus,first isolated from Aedes serratus in Brazil and northern Argentina,is a member of the SINV lineage (21). There is no evidence forhuman disease caused by the virus.

The 11.8-kb alphavirus genome contains two domains. The 5'-terminaltwo-thirds encodes four nonstructural proteins required forRNA replication. The 3' one-third encodes the structural proteins,which are expressed by means of a 4.2-kb subgenomic mRNA. Thesestructural proteins are translated as a polyprotein that issubsequently cleaved into five proteins: the nucleocapsid protein(CP) ( $~$ 30 kDa); two envelope glycoproteins, E1 ( $~$ 52 kDa) and E2( $~$ 49 kDa); and two small peptides, E3 ( $~$ 10 kDa) and 6K ( $~$ 6 kDa)(19). The Aura virus structural proteins and those of SINV haveamino acid sequence identity of 77% for CP, 56% for E2, and61% for E1, for an average identity of 62% (21). These structuralproteins have only 46% sequence identity with those of RRV orSFV, although the structural proteins of RRV and SFV have 75%sequence identity.

No atomic structure of an alphavirus has been determined becausethe best crystals thus far diffract X-rays to a resolution ofonly $~$ 30 Å (10). Low-resolution (20- to 30-Å) andmoderate-resolution (9-Å) structures of several alphaviruseshave been determined by means of cryoelectron microscopy (cryo-EM)and three-dimensional image reconstruction techniques (5, 13,15, 16, 27). Cryo-EM studies on RRV (5), SFV (27), and SINV(16) demonstrated that alphaviruses are spherical, multilayeredstructures with an outer diameter of $~$ 710 Å. The viralgenome is encapsidated in a closed protein shell, called thenucleocapsid core, and is composed of 240 copies of the CP proteinarranged in a T=4 icosahedral lattice (26). The CP subunitsare organized as pentameric and hexameric capsomers, which extendto a radius of 215 Å and project $~$ 45 Å above thebase of the protein shell. This base contains the N terminusof the CP protein, which interacts with the viral RNA. It isnot known if the base contains surface-accessible RNA or whetherthe RNA is simply encapsidated by this protein shell. However,the RNA in isolated nucleocapsid cores is sensitive to RNasedigestion (11). Hence, the protein shell may not completelyshield the genome. In virions, the viral nucleocapsid core istightly enveloped by a lipid bilayer derived from the host.Glycoproteins penetrate the bilayer and form a one-to-one associationwith CP (5). Glycoproteins E1 and E2 form heterodimers thatare present in a T=4 icosahedral lattice, organized as 80 trimericspikes on the viral surface. Glycoprotein E2 forms the externalprotruding portion of each spike, whereas E1 lies roughly parallelto the membrane, forming an icosahedral scaffold (12, 17).

The assembly of alphavirus capsids requires RNA, as no exampleof nucleic acid-free virus assembly has been reported (25).In most alphaviruses, packaging of RNA is specific, and thegenomic RNA is preferentially packaged because an RNA packagingsignal is present only in the genomic RNA (23, 29). However,both the genomic RNA and subgenomic RNA of Aura virus are efficientlyencapsidated to form virion particles, suggesting that a packagingsignal for Aura virus RNA may lie in the region of the genometranscribed into subgenomic RNA (22). Two major size classesof Aura particles were found by electron microscopy in negativelystained preparations (20). One class contained particles witha diameter of $~$ 720 Å which are similar in size to the virionsof other alphaviruses, whereas the second class contained particleswith a diameter of $~$ 620 Å. The 720-Å-diameter particleswere postulated to be T=4 particles that contain one copy ofgenomic RNA or up to three copies of subgenomic RNA. The 620-Å-diameterparticles were postulated to contain only a single copy of subgenomicRNA and possibly to have T=3 symmetry. In addition, a smallfraction of very small particles ( $~$ 360-Å diameter) wasalso observed (20).

In this paper we report the use of cryo-EM and three-dimensionalimage reconstruction (3) to examine the native morphology ofAura virus particles produced in BHK cells. We found that Auravirus particles separated into two components in a sucrose gradient.The upper component consisted of wild-type virions, the majorityof which contain genomic RNA. In contrast, the lower componentcontained a mixture of genomic and subgenomic RNA-containingparticles. Although 10 to 30% of the particles in the lowerfraction appeared smaller in stained preparations, cryo-EM reconstructionsrevealed that Aura virus particles from both the top and bottomcomponents of the sucrose gradient (Aura_T and Aura_B, respectively)are similar T=4 structures that resemble those of other alphaviruses.

MATERIALS AND METHODS

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Materials and Methods

Virus propagation and purification. Baby hamster kidney cells (BHK-15) used for propagation of Auravirus were grown in Eagle minimal essential medium supplementedwith 10% fetal bovine serum. Large-scale production of viruswas made by infecting BHK cells with virus at a multiplicityof infection of 1. Virus was harvested at 25 h postinfectionand was first precipitated from the supernatant with polyethyleneglycol after a brief centrifugation (5,000 x rpm for 10 minat 4°C in a Sorvall GSA rotor). The virus was resuspendedin 1.5 ml of TNE buffer (50 mM Tris, 200 mM NaCl, 1 mM EDTA)with a pH of 7.6 and was sedimented on a freeze-thaw sucrosegradient for 1.25 h at 38,000 rpm at 4°C in a Beckman SW41rotor. The purified virus was prepared by centrifugation ona 15 to 30% sucrose gradient at 36,000 rpm for 1.5 h at 4°Cin a Beckman SW41 rotor. Two viral bands were discernible. Thetop and bottom bands, corresponding to Aura_T and Aura_B, respectively,were collected by side puncture. Each band was then separatelypelleted by centrifugation in a Beckman 50.2 rotor for 2 h at36,000 rpm at 4°C, and each pellet was resuspended in 20to 40 µl of TNE buffer.

Cryo-EM and three-dimensional image reconstruction. Small aliquots (3.5 µl) of Aura virus (Aura_T and Aura_B),SINV, and RRV samples were prepared for cryo-EM as describedpreviously (1, 2, 5, 14). Images were recorded in a PhilipsCM200 field emission gun transmission electron microscope underlow-dose conditions (10 to 13.5 e^-/Å²) at a nominal magnificationof x38,000. Micrographs of Aura_T, Aura_B, SINV, and RRV, allof which displayed a uniform distribution of particles and icethickness, were digitized at 7-µm intervals on a Zeiss-SCAIscanner. Each image was bin averaged to 14-µm pixels,corresponding to intervals of 3.68 Å at the specimen.

Images of individual virus particles were boxed and floatedas is customary (3). The common-lines method (9) was used tocompute initial reconstructions of each of the virus samples.These reconstructions were then used as models to determineand refine orientation and origin parameters for each virusimage by means of the model-based polar-Fourier transform method(1). Orientation and origin refinement were monitored with real-and reciprocal-space correlation coefficients. The Fourier transformof each image was modified to correct in part for the effectsof the microscope contrast transfer function. Eigen value spectrawere used to assess the conditioning of the linear, least-squaresequations. The resolution of each map was estimated by splittingthe image data into two roughly equal sets and comparing thestructure factors, calculated independently for each data set.

Difference maps of Aura_T minus Aura_B and of Aura_T minus SINVwere computed as described previously (17). The three-dimensionalreconstructions of Aura_B, SINV, and RRV were compared to theAura_T reconstruction by means of correlation coefficient analysiscomputed as a function of radius.

The absolute hand of the Aura virus structure was determinedby means of tilt experiments (4). Simian virus 40 (T=7d lattice)was used as a control to establish a protocol for recordingtilt-pair images on a Philips CM200 microscope at a magnificationof x38,000. Each particle image from the tilt data set was cross-correlatedwith the corresponding projections of a reconstruction and itsenantiomorph. At 32-Å resolution, the reconstruction ofAura_T of the correct hand exhibited an average correlation coefficientof 0.474 ± 0.161, compared to 0.128 ± 0.062 forthat of the reconstruction of the incorrect hand. The correspondingcorrelation coefficients for the Aura_B reconstructions withthe correct or incorrect hands were 0.458 ± 0.208 and0.112 ± 0.006, respectively.

RESULTS

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Results

Cryo-EM reconstruction of Aura_B and Aura_T. Aura virus propagated in BHK cells is composed of a mixtureof virus particles containing both genomic and subgenomic viralRNAs. These particles separate differentially in sucrose gradientsinto faster-sedimenting particles (Aura_B component), which containboth 49S genomic and 26S subgenomic RNA, and slower-sedimentingparticles (Aura_T component), which contain only the genomicRNA (20). Negative staining and EM revealed two size populationsof virions present in the faster-sedimenting band (20; alsodata not shown). Particles with a diameter of 710 Å wereobserved, which is consistent with other T=4 alphaviruses. Asubset of smaller particles with a diameter of 620 Å wasalso observed, which was previously hypothesized to be T=3 particlesthat contain subgenomic RNA. However, this size discrepancywas not apparent in images of the frozen-hydrated Aura virussamples (either the top or bottom bands of the 15 to 30% sucrosegradient) (Fig. 1). Images of Aura_T and Aura_B particles werevisually indistinguishable. In both samples, the viruses exhibita consistent spherical morphology and a diameter of $~$ 710 Å.Glycoprotein spikes are discernible at the particle peripheries,and membrane features can be seen on close inspection of closeto focus micrographs. The diversity of the projected viral imagesindicated that virions were randomly orientated in the vitreousice.

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FIG. 1. Frozen-hydrated samples of Aura_T (A) and Aura_B (B) viruses. Bar, 1,000 Å.

Image reconstruction maps of Aura_T and Aura_B virus were computedto 17- and 21-Å resolution (Table 1). Two independentpreparations of Aura_T virus and three independent preparationsof Aura_B virus were imaged by cryo-EM and gave consistent results.

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TABLE 1. Data collection and image processing

Organization of Aura virus. The Aura virus structure appears similar to that of other alphavirusesexamined by cryo-EM methods (Fig. 2 and 3). The Aura_T structureshows clear T=4 quasi-symmetry and a multilayered organization,which includes a glycoprotein shell (263 to 355 Å), alipid bilayer (215 to 263 Å), a nucleocapsid protein shell(170 to 215 Å), and viral RNA. The outer glycoproteinshell is subdivided into three distinct layers: the spike projections(53 Å in height) that form the predominant feature onthe virus surface, the thin shell of density or skirt (22 Åthick) that, with the exception of holes at the icosahedraltwofold axes, covers most of the virus surface and connectsthe projecting spikes, and the subskirt region (18 Å thick)that connects the skirt to the top of the lipid bilayer. Previousstudies of SINV deglycosylation mutants (17) and fitting theatomic structure of SFV E1 into the cryo-EM reconstruction map(12) indicate that the spikes consist primarily of glycoproteinE2, whereas the skirt is attributed to a tangential arrangementof E1 glycoproteins. The nucleocapsid core proteins are organizedas 42 capsomers, of which 30 hexamers are situated at the icosahedraltwofold axes and 12 pentamers occupy the fivefold axes. Bothcapsomer types project radially outward from the base of a 15-Å-thickshell. A lipid bilayer membrane tightly envelopes the core,and the inside layer of phosphate head groups bends by approximately7 Å toward the center of each capsomer. Putative genome-capsidconnections are not observed in the reconstructions, thoughthis may be a consequence of enforcing icosahedral symmetryon the entire reconstruction.

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FIG. 2. Image reconstruction of Aura_T virus at 17-Å resolution, viewed along a twofold axis. (A) Surface-shaded representation. The yellow triangle demarks one icosahedral asymmetric unit. (B) Surface-shaded representation as in panel A but with the front half removed to reveal interior features. Arrows identify putative transmembrane connections. Aura virus exhibits a multilayered structure similar to that of SINV, which consists of glycoproteins (blue), a lipid bilayer (green), nucleocapsid proteins (orange), and the genome (pink). (C) Electron density map of an equatorial section. Icosahedral twofold (i2), threefold (i3), quasi-threefold (q3), and fivefold (i5) axes are labeled in panels A and C. Bar, 200 Å.

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FIG. 3. Comparison of Aura_T, Aura_B, SINV, and RRV reconstructions. Surface-shaded representations of the exteriors (top row), spikes (second row) (threefold [i3] and quasi-threefold [q3] axes), and equatorial cross sections (third row) from Aura_T and Aura_B, SINV, and RRV allow similarities and differences between these reconstruction maps to be compared. Reconstruction maps of Aura_T, Aura_B, and SINV are remarkably similar, whereas the RRV spike differs both on its exterior and its interior, where a cavity is present (see arrows on RRV cross section). The two panels on the bottom left are the central sections of the Aura_T-Aura_B and Aura_T-SINV difference maps. The bottom right panel plots the correlation coefficients between different pairs of reconstructions (Aura_T-Aura_B, Aura_T-SINV, and Aura_T-RRV) as a function of radius. The color bar at the top of the panel coincides with the color scheme used in the equatorial cross sections (third row): viral genome (pink), nucleocapsid core (orange), lipid membrane (green), and envelope glycoproteins (blue).

Structural comparisons of Aura_T, Aura_B, SINV, and RRV. The structures of Aura_T, Aura_B, SINV, and RRV were all renderedat 22-Å resolution to permit qualitative and quantitativecomparisons (Fig. 3). Careful comparisons of the sizes and shapesof internal and external features reveal that Aura_T and Aura_Bare virtually identical. The Aura_T minus Aura_B difference mapshows no significant peaks and confirms the similarity of thetwo reconstructions (Fig. 3). In addition, correlation coefficientanalysis, computed as a function of radius, between the Aura_Tand Aura_B reconstructions, indicated that the glycoprotein andnucleocapsid protein layers are almost identical in these Auraviruses (Fig. 3). Therefore, despite differences in RNA content,migration in sucrose gradients, and particle sizes observedusing negative staining and EM, Aura_T and Aura_B are indistinguishableat 22-Å resolution.

At first glance, the Aura_T and SINV structures appear quitesimilar if not identical (Fig. 3). However, the Aura_T minusSINV difference map shows strong positive and negative peaks,which may reflect genuine structural differences. Radial densitycorrelation coefficient analysis shows that SINV is less similarto Aura_T than Aura_T is to Aura_B. Superimposed wire-frame representationsof the two maps demonstrate that Aura_T hexamers and pentamersin the core and the transmembrane bundles are rotated $~$ 6°counterclockwise relative to that observed in SINV, whereasdensities at the upper surface of the membrane coincide (Fig.4). Such observations suggest that the CP proteins in Aura andSINV might bind to their respective glycoproteins in slightlydifferent orientations. Similar observations have been madeon the basis of recent cryo-EM studies of Venezuelan equineencephalitis virus in which the nucleocapsid capsomeres werefound to be rotated counterclockwise with respect to their SINVcounterparts (15).

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FIG. 4. Stereo view of the electron density maps of Aura virus (red) and SINV (blue), displayed at a high contour level, with the capsid appearing at the bottom and the glycoprotein at the top. The finger-like projections pointing downwards from the glycoproteins and upward from the capsid, and corresponding to the transmembrane densities of Aura and SINV, are indistinguishable at the upper surface of the membrane but have slightly different orientations at the lower surface.

The spike morphology in RRV differs noticeably from that seenin Aura_T, Aura_B, and SINV (Fig. 3). The most prominent differenceis that RRV lacks a small nodule that occurs on the distal endof the spike in the other alphaviruses. On SINV, this smallknob was recently shown to be the sugar moiety at residue 196of the E2 glycoprotein (17). The RRV E2 glycoprotein has a similarglycosylation site but at residue 200 (E2-200). Consistent withthis difference, extra density is found on the inner side ofthe RRV spike petal, close to the center (threefold or quasi-threefoldaxis) of each spike. A cavity lies beneath the center of eachRRV spike, because in RRV the E1 molecules have no glycosylationsite at residue 245, as the E1 glycoproteins of SINV and Auravirus do. Additional densities observed at the skirt level andbetween adjacent spikes in RRV correspond to the two carbohydratemoieties attached to each of the E2-262 residues that bridgethe two spikes. The locations of the RRV E1-141 and E2-200 glycosylationsites were discussed in an earlier study (17).

Glycosylation sites in Aura virus. The glycoproteins of Aura virus, like SINV, have four glycosylationsites. Two of these sites are located on the same residues asSINV (E1-139 and E1-245). The other two sites occur at E2-197and E2-319 on Aura virus instead of E2-196 and E2-318 in SINV.All of the glycosylation sites in SINV have recently been mappedthrough difference map analyses computed between wild-type SINVand each of four, single-site glycosylation mutants (E1-139,E1-245, E2-196, and E2-318) (17). Given the close similaritybetween the Aura virus and SINV structures, difference mapswere calculated by subtracting reconstructions of the individualSINV deglycosylation mutants (17) from the Aura_T reconstruction.As expected, on the basis of similarity of the Aura_T and SINVstructures and conservation in amino acid number for glycosylationsites in E1 and E2, the locations of the sites in Aura viruswere comparable to those found in SINV.

The T=4 icosahedral symmetry of the virion means that four uniquecopies of each glycosylation site are present in each asymmetricunit of the particle. These four, quasi-symmetric positionsare listed for Aura_T (Table 2) and SINV (17). The HOMOlogy programwas used to assess the accuracy with which each carbohydratedifference peak within the icosahedral asymmetric unit obeysthe T=4 quasi-symmetry (Table 3). The carbohydrate moiety positionsin Aura virus obeyed the T=4 quasi-symmetry to within 1.5 Åfor the E2-197 and E2-319 sites and to within 3.1 and 5.4 Åfor the E1-245 and E1-139 sites, respectively (Table 3). Asthe E1-245 sites are located near the threefold and quasi-threefoldaxes, they should exhibit the smallest deviation from T=4 quasi-symmetry.The large variation detected in the E1-245 sites is mainly attributedto measurement error, because three carbohydrate molecules aretoo close to be resolved and therefore, their densities mergeinto one larger feature. The three remaining Aura virus sitesshow much smaller deviations from T=4 quasi-symmetry than thecorresponding SINV sites, whose carbohydrate moieties obey thequasi-symmetry to within 1.6 Å for the E1-245 and E2-318sites and to within 3.4 and 7.4 Å for the E2-196 and E1-139sites, respectively.

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TABLE 2. Three-dimensional locations of carbohydrate sites in Aura virus^a

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TABLE 3. Superposition of individual glycosylation sites from Aura virus around threefold (i3) and quasi-threefold (q3) axes

Modeling studies have shown that the long, thin E1 moleculesfit nicely onto the skirt region and form an icosahedral scaffold(12, 17) (Fig. 5A). These same studies assigned the E2 moleculesto all unoccupied densities, the majority of which occur inthe protruding spikes. However, the association between E1 andE2 remains unclear. We have performed statistical analyses todetermine which E2-319 sites and E1-139 sites belong to a particularheterodimer based on the locations of the carbohydrate densities.Three E2-319 sites (A1, A2, and B1) surround each B1* site ofE1-139 (Fig. 5). Each of these three possible heterodimer assignments(B1*-A1, B1*-A2, or B1*-B1) gives rise to a unique set of four,quasi-symmetric dimers. The distances between E1-139 and E2-319for each of the four, quasi-symmetric dimers as well as thestandard deviations were measured (Fig. 6). Assignment I (Fig.5B), which links B1* of E1-139 and A1 of E2-319, gives the smallestvariation (i.e., ±1.0 Å). The variations foundfor the other two assignments are ±4.6 Å and ±2.4Å for B1*-B1 and B1*-A2, respectively. Similar resultswere found for SINV: ±0.3, ±3.5, and ±3.2Å for B1*-A1, B1*-B1, and B1*-A2, respectively (17). Therefore,for Aura virus, at a threefold spike, the A1 site of E2-319and the B1* site of E1-139 probably belong to the same heterodimer.

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FIG. 5. Stereo views of the Aura virus glycosylation sites. (A) Asymmetric unit of the Aura virus glycoproteins. Four SFV E1 molecules (blue) were fitted into the Aura_T density map at the lower portion of the spike and in the skirt (violet). The Aura virus carbohydrate densities are shown and were determined by generating difference maps between Aura_T and each of the four SINV deglycosylation mutants (17). The Aura_T carbohydrate densities are colored (E1-139 in brown, E1-245 in red, E2-197 in light green, and E2-319 in magenta). (B) Electron density of Aura virus and the shift in position between the Aura carbohydrate densities and those of SINV (green for E1-139 and blue for E2-318). The broken lines indicate possible pairs of Aura E1-139 and E2-319 carbohydrate sites which belong to one heterodimer (assignment I in Fig. 6).

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FIG. 6. Distances between E1-139 and E2-319 sites in the asymmetric unit in Aura virus. Distances are given in angstroms. The standard deviation (SD) for each assignment is also given. d_{B1_*-A1}, distance between B1_* and A1.

DISCUSSION

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Discussion

EM of Aura_T and Aura_B. Cryo-EM images of Aura_T and Aura_B are indistinguishable. Asrevealed in the three-dimensional reconstruction analyses, particlesin both fractions are 710 Å in diameter and they obeyT=4 icosahedral symmetry. The surface features of the glycoproteinsare essentially indistinguishable from those of SINV virions.Thus, the findings that these particles separate into two componentsduring sucrose gradient sedimentation and that many negativelystained particles in the bottom component appear to be smaller,uniform particles with a diameter of 620 Å are puzzling.These differences presumably arise as a result of differencesin the nucleic acid content in the two components. Given thetechnical difficulties in separating top and bottom components,it appears that the top component consists of particles thatcontain mostly genomic RNA and that the bottom component consistsof particles that contain primarily subgenomic RNA. We suggestthat the smaller particles seen by negative staining and EMhave a suboptimal content of RNA, perhaps only a single copyof the subgenomic 4.2-kb RNA, and that the structure collapsesupon exposure to the negative stain. Perhaps particles thatdo not collapse have more RNA, or perhaps they survive intactupon addition of stain. The fraction of particles that collapsecould depend upon the vagaries of exactly how they are exposedto the stain, and we have found that different preparationsfor EM observation differ in the fraction of smaller particlesobserved. If these assumptions are correct, it is still puzzlingwhy particles that contain less RNA would sediment faster. Perhapsthe high concentration of sucrose induces a reversible contractionof the particles that contain less RNA, causing them to sedimentfaster. However, other explanations may be just as plausible.

These results have significant implications for understandinghow the structure of the virion forms. They predict that thefinal T=4 structure is not dependent upon RNA content but isa fundamental property of the proteins that form the structure.Two forces are believed to contribute to the T=4 structure.With most alphaviruses, nucleocapsids form in the cytoplasmindependent of interactions with glycoproteins, and nucleocapsidscan be assembled in vitro in the absence of glycoproteins (25,30). Although the precise structure of these nucleocapsids hasnot been determined, they do possess the requisite number (240)of CP molecules for a T=4 structure. Significantly, when assembledin vitro, the size of the nucleocapsids is independent of thesize of the nucleic acid used to initiate assembly (25). Assemblyusing polynucleotides of length 14 gives rise to the same-sizestructure as does the viral genomic RNA. The glycoproteins,in particular E1, are also important for forming a stable T=4structure. The glycoproteins can form a regular lattice in theabsence of nucleocapsids (28) and can assist the formation ofnucleocapsids in mutants that are otherwise unable to form nucleocapsids(7). Thus, formation of a T=4 structure appears to be an innateproperty of both the CP protein and the glycoproteins. In thisregard, it is noteworthy that RNA molecules that are more than10 to 20% larger than the wild-type genome cannot be incorporatedinto virions, which is consistent with the hypothesis that thecomposition of the structure formed by the viral proteins ispredetermined (8).

If this analysis is correct, the assembly pathways of the alphavirusesdiffer from those used by flaviviruses in a number of fundamentalproperties. The E1 protein of alphaviruses has been shown recentlyto have a structure very similar to that of E protein of flavivirusesand to fulfill a similar role in the structure of the maturevirion (12, 17). However, the flavivirus virion is a T=1 structurein which the component E proteins are not all present in quasi-equivalentpositions (11a), whereas the alphavirus virion is a T=4 structurewith all E1 molecules present in quasi-equivalent positions.Of interest is the fact that E and prM of flaviviruses willform a T=1 structure in the absence of nucleocapsids (6), whereasan equivalent structure has never been seen in alphaviruses.Thus, the E protein of flaviviruses can participate in alternativestructures, whereas the E protein of alphaviruses appears tobe predetermined to a T=4 structure.

Structure of Aura virus at 17-Å resolution. The structure of the Aura virion is very similar to that ofSINV. Based on the difference map analyses, the details of theskirt and subskirt regions resemble those of SINV rather thanSFV or RRV. Also the locations of the carbohydrate chains inAura virus are strikingly similar to those in SINV but differfrom those in RRV. The conservation of this feature within theSINV lineage is consistent with the hypothesis that the carbohydratemoieties have a structural role in the assembly or disassemblyof the virion and are not present simply to increase the solubilityof the proteins during folding. Also consistent with this hypothesisis the fact that of the four carbohydrate chains present onE1 and E2, only one chain (at E2 position 197 in Aura [SINVposition 196]) is on the distal surface of the glycoproteinspike where it might interact with the external solvent. Theother three chains reside in the skirt region where they appearto interact with domains within the glycoprotein, with eachother, or with bound solvent, rather than with the externalsolvent.

The carbohydrate chains in the SFV/RRV lineage occupy detectablydifferent positions from those in the SINV lineage, suggestingthat the structures of the virions differ in ways that may beimportant for function. In this regard, it is notable that thesequences of the E2 proteins of SINV and Aura virus can be alignedthroughout their length without the introduction of gaps, withthe exception that Aura virus has one extra amino acid at theN terminus. In contrast, alignment of SINV and SFV or RRV E2proteins requires the insertion of several gaps. The ectodomainsof E1 of Aura virus, SINV, SFV, and RRV can all be aligned withoutgaps. This may reflect the importance of the structural roleof this protein in forming the skirt and imposing a T=4 structureon the virion.

ACKNOWLEDGMENTS

We thank Michael Rossmann, Ellen Strauss, and Rushika Pererafor useful discussions.

This work was supported in part by NIH grants to T.S.B. (AI45976),R.J.K. (GM56279), and J.H.S. (AI20612) and an NSF shared instrumentationgrant (BIR-9112921) to T.S.B. We also thank Purdue Universityfor an instrumentation reinvestment grant to the Purdue StructuralBiology Group.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907. Phone: (765) 494-5645. Fax: (765) 496-1189. E-mail: tsb{at}bragg.bio.purdue.edu.

REFERENCES

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