Proc. Natl. Acad. Sci. USAVol. 90, pp. 9095-9099, October 1993Microbiology

Three-dimensional structure of a membrane-containing virusANGEL M. PAREDES*, DENNIS T. BROWN*t, ROSALBA ROTHNAGELt, WAH CHIU*, RANDAL J. SCHOEPP§,ROBERT E. JOHNSTON§, AND B. V. VENKATARAM PRASADt**Cell Research Institute and Department of Microbiology, The University of Texas at Austin, Austin, TX 78713; tVerna and Marrs McLean Department ofBiochemistry and W. M. Keck Center for Computational Biology, Baylor College of Medicine, Houston, TX 77030; and IDepartment of Microbiology andImmunology, University of North Carolina School of Medicine, Chapel Hill, NC 27599

Communicated by Max D. Summers, June 24, 1993

ABSTRACT The structure of Sindbis virus was deter-mined by electron cryomicroscopy. The virion contains twoicosahedral shells of viral-encoded proteins separated by amembrane bllayer of cellular origin. The three-dimensionalstructure of the Ice-embedded intact Sindbis virus, recon-structed from electron images, unambiguously shows thatproteins in both shelas are arranged with the same icosahedrallattice of trianglation number T = 4. These studies alsoprovide structural evidence ofcontact between the glycoproteinand the nucleocapsid protein across the membrane bilayer. Thestructural organization of Sindbis virus has profound implica-tions for the morphogenesis of the alphaviruses. The observedinteractions conflrm stoichiometric and speciflc protein asso-ciations that may be crucial for virlon stability and predict amechanism for assembly.

Membrane-containing viruses are assembled through theinteraction of virus-encoded proteins with a host-cell mem-brane. For most enveloped viruses, this process involves twopathways. In one of these pathways, virus-encoded mem-brane proteins are cotranslationally integrated into mem-branes of the cell endoplasmic reticulum (1). These proteinsare subsequently processed and delivered to a particular cellmembrane (usually the plasma membrane) by a sequence oftransport and processing events used by the cell in thematuration of its own membrane proteins. Virus membraneproteins, therefore, have proven to be important models forstudying the processes involved in the maturation and in thetargeting of cellular membrane proteins.While envelope proteins are transported to the plasma

membrane, other viral proteins are translated in the cellcytoplasm and subsequently attach to the modified cellularmembranes through the interaction of the cytoplasmic pro-tein with cytoplasmic domains of the viral membrane glyco-proteins (1). This association defines a critical step in themorphogenesis of the virus particle as it initiates and drivesthe process of envelopment of the core structure in themodified cellular membrane. The specific protein-proteininteractions that occur during the fmal stages of assemblyresult in the production of a mature virus particle thatmaintains its structural integrity until its functional compo-nents interact with a potential host cell, and virus disassem-bly occurs.

Sindbis virus, the prototype of the alphaviruses, achievesits mature structure in a distinctive fashion. Unlike manyother enveloped viruses, the two-membrane glycoproteins ofSindbis virus (El and E2) are organized on the surface of thevirus membrane as a precise triangulation number (T) = 4icosahedron (2-5). The structure of this icosahedral latticedepends upon intramolecular disulfide bridges residing in theEl glycoprotein (5, 6), and the integrity of the protein latticehas been demonstrated to determine the structure and sta-

bility of the membrane bilayer (6). Thus, whereas mostenveloped viruses are described as including a membranebilayer that contains virus-specified surface proteins andencloses the viral nucleocapsid, the alphaviruses might bebetter described as including distinct surface and nucleocap-sid protein lattices between which a membrane bilayer inter-venes.The alphavirus nucleocapsid is composed ofpositive-sense

single-stranded RNA and the capsid protein C, which isassembled into a three-dimensional structure having icosa-hedral symmetry. The structure of this icosahedron has beenproposed to have a triangulation number of 3 (5) or 4 (7-9).The specific interaction of the capsid structure with theenvelope glycoproteins in the modified cell membrane islikely responsible for assembly of the glycoproteins into theT = 4 icosahedral lattice as envelopment progresses. Thestructure of the nucleocapsid is critical to understanding howthis process occurs. A T = 3 icosahedral nucleocapsid impliesthat envelopment is driven by the association of 180 copies ofthe capsid protein with 240 copies ofeach ofthe two envelopeproteins (5). This process implies that nonequivalent associ-ations must occur between some of the envelope glycopro-teins and the nucleocapsid protein. However, the presence ofa T = 4 icosahedral lattice in the nucleocapsid implies aone-to-one correspondence between the envelope proteinsand the C protein in the nucleocapsid. To resolve thisproblem and to learn more about the structural organizationof the alphaviruses, we have reinvestigated the three-dimensional structure of Sindbis virus by electron cryomi-croscopy and computer image analysis.

MATERIALS AND METHODSVirus Production and Purification. The growth of the

AR339 and HR strains of Sindbis virus in BHK-21 cells hasbeen described (10, 11). Virions containing the AR339 struc-tural proteins were derived from a full-length cDNA clone(10). Viruses were purified by isopycnic density-gradientcentrifugation on linear potassium tartrate gradients, as de-scribed (4).

Electron Cryomicroscopy. Electron cryomicroscopy wasdone by using the protocols of Adrian et al. (12), Dubochetet al. (13), and Prasad et al. (14). Approximately 4 ,ul ofpurified virus suspension was placed on a carbon-coatedholey grid. The specimen was blotted from the grid with filterpaper and was then quickly plunged into liquid ethane. Thisprocedure embedded the virions within carbon holes in a thinlayer of vitrified ice. This technique avoids artifacts intro-duced to the specimen by fixatives and heavy metal stains.Because virions are preserved in a hydrated state, dryingartifacts are completely eliminated. Grids prepared in thismanner were subsequently stored in liquid nitrogen.

Abbreviation: T, triangulation number.tTo whom reprint requests should be addressed.

9095

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

9096 Microbiology: Paredes et al.

For EM, grids were transferred under liquid nitrogen to aGatan cryo-specimen holder, which maintains a -155°Ctemperature. The holder was transferred to a JEOL 1200 EM,and images were recorded at x 30,000 operating at an accel-erating voltage of 100 kV. To reduce radiation damage of thespecimen, the images were recorded by using an electrondose of 4-5 electrons per A2.A set of two images per field was recorded. The set

consisted of images recorded at =1.5 ,um and 2.5 Am under-focus, respectively. The closer-to-focus, higher-resolution1.5-,um image was recorded first and, thus, received theminimum 4-5 electrons per A2 electron dose. The higher-contrast 2.5-,m underfocus image was computer processedseparately to confirm the orientations of the higher resolutiondata, which were used in the three-dimensional reconstruc-tion.

Three-Dimensional Reconstruction. A set of electron mi-crographs were selected on the basis of virion concentration(e.g., >50 particles per field), uniform ice thickness, andabsence of both image astigmatism and specimen drift. Themicrographs were digitized on a Perkin-Elmer microdensi-tometer with a step size of 25 ,um x 25 ,um per pixel,representing 8.3 A in the specimen. The digitized image wasdisplayed on the Silicon Graphics workstation, and theindividual particles in the image were boxed into a 128 x 128pixel2 area using an X-window-based computer graphicsprogram (Hardt and B.V.V.P., unpublished work). Theboxed images were masked from the background at a radiusof 42 pixels and floated onto a new uniform background. Allsubsequent computations were run on Silicon Graphics com-puters.The three-dimensional reconstructions were performed by

using the established procedures of Crowther (15), Fuller (5),Prasad et al. (14), and Baker et al. (16). Virion centers weredetermined by the cross-correlation method (17). The orien-tations of individually captured particles were then deter-mined using the "common lines" method developed byCrowther (15). Only particles with phase residuals -55' wereselected into the data set.Once the data set was established, the orientation of each

particle was refined against all other particles in the set byusing the cross-common lines method (5). After a sufficientnumber of particles with unique orientations, adequatelysampling the asymmetric unit, had been obtained, theirtwo-dimensional Fourier transforms were combined to createa three-dimensional Fourier transform. This transform wasinterpolated by cylindrical expansion to place transformvalues at regularly spaced points required for the Fourierinversion (15). Fourier inversion of the three-dimensionaltransform generated a three-dimensional structure that wasviewed by using Silicon Graphics Explorer software.

RESULTS AND DISCUSSIONAn electron image of intact Sindbis virions embedded invitreous ice without negative stain and fixative is shown inFig. 1. Three-dimensional reconstructions were producedfrom such images for both the AR339 and the HR strains ofSindbis virus. Although these strains differ in their biochem-ical, immunological, and pathogenic properties (10, 18, 19),their three-dimensional maps are indistinguishable at 28-Aresolution. Views of the virus down the 5-fold and 3-fold axisare shown in Fig. 2. The three-dimensional reconstruction ofSindbis virus at 28-A resolution revealed the surface of thevirus to be composed of 80 distinct trimeric structures thatare located at the local and strict 3-fold axis of a T = 4icosahedral lattice (Fig. 2). The trimer is likely composed ofthree E1/E2 heterodimers (4) that are twisted about oneanother. Each trimer protrudes from the surface by 50 A andis flared at the distal end. The flaring of the trimers was not

FIG. 1. Electron micrograph of the Sindbis virions embedded ina thin layer of vitreous ice recorded with an underfocus of 1.5 ,um.(Bar = 700 A.)

resolved in a previously published reconstruction of Sindbisvirus (5) but was seen in a reconstruction of the relatedalphavirus Semliki Forest (20). Semliki Forest virus containsEl and both the E2 and E3 products of the proteolyticprocessing of the precursor PE2 protein, whereas Sindbisvirus contains only El and E2 (1). Therefore, the conclusionthat the flares seen in the Semliki Forest virus reconstructionwere from the presence of E3 (5) is unlikely. The trimersmeasure 115 A on the distal edge and converge to a diameterof 90 A at the base. The distal tips of the triangular-shapedtrimers point either toward the 5-fold or the quasi 6-fold axisof symmetry. The trimers are interconnected at their basesinto hexameric and pentameric arrays (4) (Fig. 2), and theyform large openings in the lattice with an average diameter of50 A at the center of the strict 2-fold axes. Smaller openingsare seen at the 5-fold axes (Fig. 2a), and depressions in theicosahedral lattice are seen at the local 2-fold axes.A 50-A thick cross-section of the mass density is presented

in Fig. 3a. The outer E1-E2 glycoprotein spikes extend froma radius of -235-340 A. The average mass density decreasessignificantly between the radii 205 and 235 A. This lowdensity is likely due to the scattering difference betweenprotein and lipid (21) and probably represents the location ofthe lipid bilayer consistent with previous findings (3). Thelipid bilayer is probably exposed through the openings at thestrict 2-fold axes. The nucleocapsid, which is composed ofRNA and the nucleocapsid protein (C), extends to a radius of=205 A. At particular positions in the structure (indicated byarrows) protein can be seen to span the putative membranebilayer, providing a continuum between the surface glyco-proteins and the internally situated nucleocapsid proteins.Biochemical studies have demonstrated that 33 amino acidsat the carboxyl terminus of the E2 glycoprotein extendbeyond the membrane and interact with the nucleocapsid

Proc. Nati. Acad. Sci. USA 90 (1993)

Proc. Natl. Acad. Sci. USA 90 (1993) 9097

FIG. 2. Stereoviews of the three-dimensional structure of Sindbis virus along the icosahedral 5-fold axis (a) and icosahedral 3-fold axis (b).In b, locations of 5-, 3-, and 2-fold symmetry axes are indicated on one of the facets of the icosahedral lattice. (x800,O00)

protein (22-25). Our observations offer structural evidencefor such a direct interaction between the nucleocapsid proteinand the glycoproteins.A surface representation of the mass density of the nucle-

ocapsid at 205 A radius is presented in Fig. 3b. The nucleo-capsid, like the envelope glycoproteins, exhibits a distinct T= 4 icosahedral symmetry. The mass density is clusteredaround the 5- and strict 2-fold axis. In a T = 4 lattice, the strict2-fold position should have a quasi 6-fold character. In ournucleocapsid structure, the quasi 6-fold feature is clearly seenaround the strict 2-fold axis (Fig. 3b), which further substan-tiates the presence of T = 4 symmetry.

In a T = 4 icosahedral lattice, there are four quasi-equivalentlocations in an asymmetric unit that a subunit can occupy.These positions are labeled as Al, A2, Bl, and B2 in Fig. 3b.Previous biochemical studies suggested that the C proteinsexisted as dimers in the native virion (7). In our structure,these dimers are likely the two quasi-equivalent dimers (Aland A2 and Bl and B2) located across the local 2-fold axis (Fig.3b). Each dimeric subunit (Al and A2 or Bl and B2) has anaverage length of =80 A and a width of =25 A, in agreementwith the dimensions of the dimer determined by x-ray crys-tallography (8). The fact that the dimensions of the proposeddimers seen in this reconstruction of intact virus resemblethose determined by Choi et al. (8) from purified capsid proteinsuggests that the dimeric nature ofthe capsid protein does notdepend on the association with RNA or the viral membrane.The C protein has a molecular size of 29 kDa and, assuming adensity of 1.30 g/cm3, we have to include mass density to aradius of 165 A in our reconstruction to accommodate 240molecules of the C protein. The radial extension of =40 A forthe nucleocapsid protein agrees with the crystallographic

studies (8); thus, the RNA protein interface is likely at a radiusof -165 A.The same T value for both outer and inner icosahedral

shells and the interconnecting mass density traversing thebilayer suggests a specific and one-to-one interaction be-tween the E2 and C proteins. We have determined, usingsite-directed mutagenesis (H. Lee and D.T.B., unpublishedresults), that amino acid substitutions in the capsid proteinsurrounding Tyr-180 destabilize the capsid protein-E2 tailassociation and block virus envelopment. We have used thedensity map produced by Choi et al. (8) to locate the proteindomain of C protein-containing Tyr-180 on the surface of thenucleocapsid in our reconstruction map. Fig. 3b shows theapproximate area of contact between the tail of the E2glycoprotein and the nucleocapsid dimer (asterisks).The nucleocapsid structure has four types of "holes" with

different shapes and sizes. The diameters ofthese holes rangefrom 30 A at the 5-fold positions and strict 3-fold positions to-60 A at the local 3-fold positions. The holes at the strict2-fold axes are of intermediate size. The holes at the 5-foldand strict 2-fold axis are in register with the openings seen atthe outer icosahedral lattice formed by the trimeric spikes.The holes seen in the reconstruction ofthe nucleocapsid mayexplain the sensitivity ofthe viralRNA to RNase degradationin purified nucleocapsids (16, 26, 27).The El and E2 glycoproteins appear to be organized into

discreet structural domains that may be related to theirdifferent functions. One would predict that functions such asinteractions with cellular receptors occur at the distal ends ofthe twisted glycoprotein structures. On the other hand, onewould anticipate that significant protein interactions thatmaintain the overall envelope structure are located within the

Microbiology: Paredes et al.

9098 Microbiology: Paredes et al.

FIG. 3. (a) A cross-section, 50 A thick, close to the center andperpendicular to the strict 3-fold axis is extracted from the three-dimensional density map. The outer envelope proteins are shown inyellow; the nucleocapsid protein is shown in blue. The location ofthelipid bilayer (M) is also indicated. Points of interaction between thespike proteins (S) and the nucleocapsid proteins (C) are shown byarrows. Location of the genomic RNA is indicated by R. (b) Surfacerepresentation of the three-dimensional structure of the nucleocap-sid, as viewed along the strict 3-fold axis. The quasi-equivalentsubunits, Al, A2, Bi, and B2, in the asymmetric unit of the T = 4icosahedron are shown. The subunits are so labeled as to indicate twotypes of dimers, A and B. The first type, A, is formed between Aland A2, and the second type, B, is formed between Bi and B2.Positions of the symmetry axes (positions 5, 3, and 2) in theicosahedral facet that is facing the viewer are indicated. *, Approx-imate point of association of nucleocapsid protein with E2 tail (seetext). (Bar = 50 A.)

icosahedral lattice at the base of the spikes. Such interactionsinclude previously demonstrated El/E2 associations thatmaintain the structure of individual heterodimers (4, 28) andEl/El associations that maintain the structure of the icosa-hedral lattice (4). We have previously demonstrated that thestructure of the icosahedral lattice of membrane glycopro-teins depends upon intramolecular disulfide bridges withinthe El glycoprotein (6). It is likely that the disassembly of thevirus membrane during cell penetration requires some mech-anism to destabilize this lattice. We have recently providedevidence that this lattice is disassembled by disulfide ex-change reactions occurring within the spikes, which areinitiated by conformational changes in the glycoproteinsinduced by receptor binding (29, 30). These conformationalchanges may facilitate the fusion of the cell membrane andthe viral membrane exposed through the openings at theicosahedral 2-fold axes of the struMcture.The structure of Sindbis virus presented above resolves

some important questions regarding the structure of alpha-viruses and has very important implications for the process

of virus assembly. The virion is clearly composed of a T = 4icosahedral membrane protein lattice surrounding a matchedT = 4 icosahedral nucleocapsid. Thus, the virion is composedof 240 copies each of the proteins El, E2 (envelope glyco-proteins), and C (nucleocapsid proteins). The stoichiometryof the C protein in the nucleocapsid agrees with the cross-linking studies of Coombs and Brown (7), the proposal ofChoi et al. (8), based on their crystallographic studies, and themass determination of Paredes et al. (9). Associations be-tween the envelope protein E2 and the nucleocapsid areapparent in this reconstruction (Fig. 3a). Evidence suggeststhat the trimers of El/E2 heterodimers are exported to theplasma membrane after assembly in the endoplasmic reticu-lum of infected cells (4). It has also been demonstrated thatthe amino acids closer to the carboxyl terminus of thecytoplasmic tail of the E2 glycoprotein are specifically rec-ognized by a domain in the C protein of the completelyassembled nucleocapsid (22, 23). Specific interactions be-tween the three E2 tails of each trimer and the tail-bindingsites on the nucleocapsid dimers would initiate the process ofenvelopment. These specific associations suggest that thenucleocapsid would direct the process of assembly, orientingthe trimers in the cell plasma membrane, and prepare themfor the specific lateral El-El and E2-E2 associations amongthe heterotrimers (4). Such a process would create theicosahedral lattice and draw the modified membrane aroundthe nucleocapsid as envelopment proceeds.

The initial technical assistance in electron cryomicroscopy of Ms.Evonne Marietta is greatly appreciated. This research was supportedby grants AI14710, AI19545 (D.T.B.); AI22186, NS26681 (R.E.J.);GM 41064 (B.V.V.P., W.C.); RR02250 (W.C.), all from the U.S.Public Health Service, the W. M. Keck Foundation, and by fundsappropriated to the Cell Research Institute by the State of Texas.

1. Schlesinger, M. T. & Schlesinger, S. (1986) in The Togaviridaeand Flaviviridae, eds. Schlesinger, M. T. & Schlesinger, S.(Plenum, New York), pp. 121-148.

2. von Bonsdorff, C.-H. & Harrison, S. C. (1975) J. Virol. 16,141-348.

3. Harrison, S. C. (1986) in The Togaviridae and Flaviviridae,eds. Schlesinger, M. T. & Schlesinger, S. (Plenum, New York),pp. 21-34.

4. Anthony, R. P. & Brown, D. T. (1991) J. Virol. 65, 1187-1194.5. Fuller, S. D. (1987) Cell 48, 923-934.6. Anthony, R. P., Parades, A. M. & Brown, D. T. (1993) Virol-

ogy 190, 330-336.7. Coombs, K. & Brown, D. T. (1987) J. Mol. Biol. 195, 359-371.8. Choi, H. K., Tong, L., Minor, W., Dumas, P., Boege, U.,

Rossmann, M. G. & Wengler, G. (1991) Nature (London) 354,37-43.

9. Paredes, A. M., Simon, M. L. & Brown, D. T. (1993) Virology187, 329-332.

10. Meyer, W. J., Gidwitz, S., Ayers, V. K., Schoepp, R. J. &Johnston, R. E. (1992) J. Virol. 66, 3504-3513.

11. Renz, D. & Brown, D. T. (1976) J. Virol. 19, 775-781.12. Adrian, M., Dubochet, J., Lepault, J. & McDowall, A. W.

(1984) Nature (London) 308, 32-36.13. Dubochet, J., Adrian, M., Chang, J. J., Homo, J. C., Lepault,

J., McDowall, A. W. & Schultz, P. (1988) Q. Rev. Biophys. 21,129-228.

14. Prasad, B. V. V., Prevelige, P. E., Marietta, E., Chen, R. O.,Thomas, D., King, J. & Chiu, W. (1993) J. Mol. Biol. 231,65-74.

15. Crowther, R. A. (1971) Philos. Trans. R. Soc. London B 261,221-230.

16. Baker, T. S., Drak, J. & Bina, M. (1988) Proc. Natl. Acad. Sci.USA 85, 422-426.

17. Schrag, J. D., Prasad, B. V. V., Rixon, F. J. & Chiu, W. (1989)Cell 56, 651-660.

18. Boggs, W. M., Hahn, C. S., Strauss, E. G., Strauss, J. H. &Griffin, D. E. (1989) Virology 169, 485-486.

19. Polo, J. M., Davis, N. L., Rice, C. M., Huang, H. V. &Johnston, R. E. (1988) J. Virol. 62, 2124-2133.

Proc. Natl. Acad. Sci. USA 90 (1993)

Microbiology: Paredes et al.

20. Vogel, R. H., Provencher, S. W., von Bonsdorff, C.-H.,Adrian, M. & Dubochet, J. (1986) Nature (London) 320,533-535.

21. Unwin, P. N. T. (1993) J. Mol. Biol. 229, 1101-1124.22. Metsikko, K. & Garoff, H. (1990) J. Virol. 64, 4678-4683.23. Liu, N. & Brown, D. T. (1993) J. Cell Biol. 120, 877-883.24. Scheefers, H., Scheefers-Borchel, U., Edwards, J. & Brown,

D. T. (1980) Proc. Natl. Acad. Sci. USA 77, 7277-7281.

Proc. Natl. Acad. Sci. USA 90 (1993) 9099

25. Hahn, C. S. & Strauss, J. H. (1990) J. Virol. 64, 3069-3073.26. Coombs, K., Brown, B. & Brown, D. T. (1984) Virus Res. 1,

297-302.27. Coombs, K. & Brown, D. T. (1989) J. Virol. 63, 883-891.28. Wahlberg, J. M., Boere, W. I. M. & Garoff, H. (1989) J. Virol.

63, 4991-4997.29. Brown, D. T. & Edwards, J. (1992) Semin. Virol. 3, 519-527.30. Abell, E. & Brown, D. T. (1993) J. Virol. 67, 5496-5501.