Three-dimensional structure of a membrane-containing virus

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 90, pp , October 1993 Microbiology Three-dimensional structure of a membrane-containing virus ANGEL 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 of Biochemistry and W. M. Keck Center for Computational Biology, Baylor College of Medicine, Houston, TX 77030; and IDepartment of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC Communicated by Max D. Summers, June 24, 1993 ABSTRACT The structure of Sindbis virus was determined by electron cryomicroscopy. The virion contains two icosahedral shells of viral-encoded proteins separated by a membrane bllayer of cellular origin. The three-dimensional structure of the Ice-embedded intact Sindbis virus, reconstructed from electron images, unambiguously shows that proteins in both shelas are arranged with the same icosahedral lattice of trianglation number T = 4. These studies also provide structural evidence of contact between the glycoprotein and the nucleocapsid protein across the membrane bilayer. The structural organization of Sindbis virus has profound implications for the morphogenesis of the alphaviruses. The observed interactions conflrm stoichiometric and speciflc protein associations that may be crucial for virlon stability and predict a mechanism for assembly. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact Membrane-containing viruses are assembled through the interaction of virus-encoded proteins with a host-cell membrane. For most enveloped viruses, this process involves two pathways. In one of these pathways, virus-encoded membrane proteins are cotranslationally integrated into membranes of the cell endoplasmic reticulum (1). These proteins are subsequently processed and delivered to a particular cell membrane (usually the plasma membrane) by a sequence of transport and processing events used by the cell in the maturation of its own membrane proteins. Virus membrane proteins, therefore, have proven to be important models for studying the processes involved in the maturation and in the targeting of cellular membrane proteins. While envelope proteins are transported to the plasma membrane, other viral proteins are translated in the cell cytoplasm and subsequently attach to the modified cellular membranes through the interaction of the cytoplasmic protein with cytoplasmic domains of the viral membrane glycoproteins (1). This association defines a critical step in the morphogenesis of the virus particle as it initiates and drives the process of envelopment of the core structure in the modified cellular membrane. The specific protein-protein interactions that occur during the fmal stages of assembly result in the production of a mature virus particle that maintains its structural integrity until its functional components interact with a potential host cell, and virus disassembly occurs. Sindbis virus, the prototype of the alphaviruses, achieves its mature structure in a distinctive fashion. Unlike many other enveloped viruses, the two-membrane glycoproteins of Sindbis virus (El and E2) are organized on the surface of the virus membrane as a precise triangulation number (T) = 4 icosahedron (2-5). The structure of this icosahedral lattice depends upon intramolecular disulfide bridges residing in the El glycoprotein (5, 6), and the integrity of the protein lattice has been demonstrated to determine the structure and stability of the membrane bilayer (6). Thus, whereas most enveloped viruses are described as including a membrane bilayer that contains virus-specified surface proteins and encloses the viral nucleocapsid, the alphaviruses might be better described as including distinct surface and nucleocapsid protein lattices between which a membrane bilayer intervenes. The alphavirus nucleocapsid is composed ofpositive-sense single-stranded RNA and the capsid protein C, which is assembled into a three-dimensional structure having icosahedral symmetry. The structure of this icosahedron has been proposed to have a triangulation number of 3 (5) or 4 (7-9). The specific interaction of the capsid structure with the envelope glycoproteins in the modified cell membrane is likely responsible for assembly of the glycoproteins into the T = 4 icosahedral lattice as envelopment progresses. The structure of the nucleocapsid is critical to understanding how this process occurs. A T = 3 icosahedral nucleocapsid implies that envelopment is driven by the association of 180 copies of the capsid protein with 240 copies of each of the two envelope proteins (5). This process implies that nonequivalent associations must occur between some of the envelope glycoproteins and the nucleocapsid protein. However, the presence of a T = 4 icosahedral lattice in the nucleocapsid implies a one-to-one correspondence between the envelope proteins and the C protein in the nucleocapsid. To resolve this problem and to learn more about the structural organization of the alphaviruses, we have reinvestigated the threedimensional structure of Sindbis virus by electron cryomicroscopy and computer image analysis. MATERIALS AND METHODS Virus Production and Purification. The growth of the AR339 and HR strains of Sindbis virus in BHK-21 cells has been described (10, 11). Virions containing the AR339 structural proteins were derived from a full-length cdna clone (10). Viruses were purified by isopycnic density-gradient centrifugation on linear potassium tartrate gradients, as described (4). Electron Cryomicroscopy. Electron cryomicroscopy was done by using the protocols of Adrian et al. (12), Dubochet et al. (13), and Prasad et al. (14). Approximately 4,ul of purified virus suspension was placed on a carbon-coated holey grid. The specimen was blotted from the grid with filter paper and was then quickly plunged into liquid ethane. This procedure embedded the virions within carbon holes in a thin layer of vitrified ice. This technique avoids artifacts introduced to the specimen by fixatives and heavy metal stains. Because virions are preserved in a hydrated state, drying artifacts are completely eliminated. Grids prepared in this manner were subsequently stored in liquid nitrogen. Abbreviation: T, triangulation number. tto whom reprint requests should be addressed.

2 9096 Microbiology: Paredes et al. For EM, grids were transferred under liquid nitrogen to a Gatan cryo-specimen holder, which maintains a -155 C temperature. The holder was transferred to a JEOL 1200 EM, and images were recorded at x 30,000 operating at an accelerating voltage of 100 kv. To reduce radiation damage of the specimen, the images were recorded by using an electron dose 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 underfocus, respectively. The closer-to-focus, higher-resolution 1.5-,um image was recorded first and, thus, received the minimum 4-5 electrons per A2 electron dose. The highercontrast 2.5-,m underfocus image was computer processed separately to confirm the orientations of the higher resolution data, which were used in the three-dimensional reconstruction. Three-Dimensional Reconstruction. A set of electron micrographs were selected on the basis of virion concentration (e.g., >50 particles per field), uniform ice thickness, and absence of both image astigmatism and specimen drift. The micrographs were digitized on a Perkin-Elmer microdensitometer with a step size of 25,um x 25,um per pixel, representing 8.3 A in the specimen. The digitized image was displayed on the Silicon Graphics workstation, and the individual particles in the image were boxed into a 128 x 128 pixel2 area using an X-window-based computer graphics program (Hardt and B.V.V.P., unpublished work). The boxed images were masked from the background at a radius of 42 pixels and floated onto a new uniform background. All subsequent computations were run on Silicon Graphics computers. 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 were determined by the cross-correlation method (17). The orientations of individually captured particles were then determined using the "common lines" method developed by Crowther (15). Only particles with phase residuals -55' were selected into the data set. Once the data set was established, the orientation of each particle was refined against all other particles in the set by using the cross-common lines method (5). After a sufficient number of particles with unique orientations, adequately sampling the asymmetric unit, had been obtained, their two-dimensional Fourier transforms were combined to create a three-dimensional Fourier transform. This transform was interpolated by cylindrical expansion to place transform values at regularly spaced points required for the Fourier inversion (15). Fourier inversion of the three-dimensional transform generated a three-dimensional structure that was viewed by using Silicon Graphics Explorer software. RESULTS AND DISCUSSION An electron image of intact Sindbis virions embedded in vitreous ice without negative stain and fixative is shown in Fig. 1. Three-dimensional reconstructions were produced from such images for both the AR339 and the HR strains of Sindbis virus. Although these strains differ in their biochemical, immunological, and pathogenic properties (10, 18, 19), their three-dimensional maps are indistinguishable at 28-A resolution. Views of the virus down the 5-fold and 3-fold axis are shown in Fig. 2. The three-dimensional reconstruction of Sindbis virus at 28-A resolution revealed the surface of the virus to be composed of 80 distinct trimeric structures that are located at the local and strict 3-fold axis of a T = 4 icosahedral lattice (Fig. 2). The trimer is likely composed of three E1/E2 heterodimers (4) that are twisted about one another. Each trimer protrudes from the surface by 50 A and is flared at the distal end. The flaring of the trimers was not Proc. Nati. Acad. Sci. USA 90 (1993) FIG. 1. Electron micrograph of the Sindbis virions embedded in a thin layer of vitreous ice recorded with an underfocus of 1.5,um. (Bar = 700 A.) resolved in a previously published reconstruction of Sindbis virus (5) but was seen in a reconstruction of the related alphavirus Semliki Forest (20). Semliki Forest virus contains El and both the E2 and E3 products of the proteolytic processing of the precursor PE2 protein, whereas Sindbis virus contains only El and E2 (1). Therefore, the conclusion that the flares seen in the Semliki Forest virus reconstruction were from the presence of E3 (5) is unlikely. The trimers measure 115 A on the distal edge and converge to a diameter of 90 A at the base. The distal tips of the triangular-shaped trimers point either toward the 5-fold or the quasi 6-fold axis of symmetry. The trimers are interconnected at their bases into hexameric and pentameric arrays (4) (Fig. 2), and they form large openings in the lattice with an average diameter of 50 A at the center of the strict 2-fold axes. Smaller openings are seen at the 5-fold axes (Fig. 2a), and depressions in the icosahedral 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 from a radius of A. The average mass density decreases significantly between the radii 205 and 235 A. This low density is likely due to the scattering difference between protein and lipid (21) and probably represents the location of the lipid bilayer consistent with previous findings (3). The lipid bilayer is probably exposed through the openings at the strict 2-fold axes. The nucleocapsid, which is composed of RNA and the nucleocapsid protein (C), extends to a radius of =205 A. At particular positions in the structure (indicated by arrows) protein can be seen to span the putative membrane bilayer, providing a continuum between the surface glycoproteins and the internally situated nucleocapsid proteins. Biochemical studies have demonstrated that 33 amino acids at the carboxyl terminus of the E2 glycoprotein extend beyond the membrane and interact with the nucleocapsid

3 Microbiology: Paredes et al. 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 evidence for such a direct interaction between the nucleocapsid protein and the glycoproteins. A surface representation of the mass density of the nucleocapsid at 205 A radius is presented in Fig. 3b. The nucleocapsid, like the envelope glycoproteins, exhibits a distinct T = 4 icosahedral symmetry. The mass density is clustered around the 5- and strict 2-fold axis. In a T = 4 lattice, the strict 2-fold position should have a quasi 6-fold character. In our nucleocapsid structure, the quasi 6-fold feature is clearly seen around the strict 2-fold axis (Fig. 3b), which further substantiates the presence of T = 4 symmetry. In a T = 4 icosahedral lattice, there are four quasi-equivalent locations 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 proteins existed as dimers in the native virion (7). In our structure, these dimers are likely the two quasi-equivalent dimers (Al and 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 an average length of =80 A and a width of =25 A, in agreement with the dimensions of the dimer determined by x-ray crystallography (8). The fact that the dimensions of the proposed dimers seen in this reconstruction of intact virus resemble those determined by Choi et al. (8) from purified capsid protein suggests that the dimeric nature of the capsid protein does not depend on the association with RNA or the viral membrane. The C protein has a molecular size of 29 kda and, assuming a density of 1.30 g/cm3, we have to include mass density to a radius of 165 A in our reconstruction to accommodate 240 molecules of the C protein. The radial extension of =40 A for the nucleocapsid protein agrees with the crystallographic studies (8); thus, the RNA protein interface is likely at a radius of -165 A. The same T value for both outer and inner icosahedral shells and the interconnecting mass density traversing the bilayer suggests a specific and one-to-one interaction between the E2 and C proteins. We have determined, using site-directed mutagenesis (H. Lee and D.T.B., unpublished results), that amino acid substitutions in the capsid protein surrounding Tyr-180 destabilize the capsid protein-e2 tail association and block virus envelopment. We have used the density map produced by Choi et al. (8) to locate the protein domain of C protein-containing Tyr-180 on the surface of the nucleocapsid in our reconstruction map. Fig. 3b shows the approximate area of contact between the tail of the E2 glycoprotein and the nucleocapsid dimer (asterisks). The nucleocapsid structure has four types of "holes" with different shapes and sizes. The diameters ofthese holes range from 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 strict 2-fold axes are of intermediate size. The holes at the 5-fold and strict 2-fold axis are in register with the openings seen at the outer icosahedral lattice formed by the trimeric spikes. The holes seen in the reconstruction ofthe nucleocapsid may explain the sensitivity of the viral RNA to RNase degradation in purified nucleocapsids (16, 26, 27). The El and E2 glycoproteins appear to be organized into discreet structural domains that may be related to their different functions. One would predict that functions such as interactions with cellular receptors occur at the distal ends of the twisted glycoprotein structures. On the other hand, one would anticipate that significant protein interactions that maintain the overall envelope structure are located within the

4 9098 Microbiology: Paredes et al. FIG. 3. (a) A cross-section, 50 A thick, close to the center and perpendicular to the strict 3-fold axis is extracted from the threedimensional density map. The outer envelope proteins are shown in yellow; the nucleocapsid protein is shown in blue. The location ofthe lipid bilayer (M) is also indicated. Points of interaction between the spike proteins (S) and the nucleocapsid proteins (C) are shown by arrows. Location of the genomic RNA is indicated by R. (b) Surface representation of the three-dimensional structure of the nucleocapsid, as viewed along the strict 3-fold axis. The quasi-equivalent subunits, Al, A2, Bi, and B2, in the asymmetric unit of the T = 4 icosahedron are shown. The subunits are so labeled as to indicate two types of dimers, A and B. The first type, A, is formed between Al and A2, and the second type, B, is formed between Bi and B2. Positions of the symmetry axes (positions 5, 3, and 2) in the icosahedral facet that is facing the viewer are indicated. *, Approximate point of association of nucleocapsid protein with E2 tail (see text). (Bar = 50 A.) icosahedral lattice at the base of the spikes. Such interactions include previously demonstrated El/E2 associations that maintain the structure of individual heterodimers (4, 28) and El/El associations that maintain the structure of the icosahedral lattice (4). We have previously demonstrated that the structure of the icosahedral lattice of membrane glycoproteins depends upon intramolecular disulfide bridges within the El glycoprotein (6). It is likely that the disassembly of the virus membrane during cell penetration requires some mechanism to destabilize this lattice. We have recently provided evidence that this lattice is disassembled by disulfide exchange reactions occurring within the spikes, which are initiated by conformational changes in the glycoproteins induced by receptor binding (29, 30). These conformational changes may facilitate the fusion of the cell membrane and the viral membrane exposed through the openings at the icosahedral 2-fold axes of the strumcture. The structure of Sindbis virus presented above resolves some important questions regarding the structure of alphaviruses and has very important implications for the process Proc. Natl. Acad. Sci. USA 90 (1993) of virus assembly. The virion is clearly composed of a T = 4 icosahedral membrane protein lattice surrounding a matched T = 4 icosahedral nucleocapsid. Thus, the virion is composed of 240 copies each of the proteins El, E2 (envelope glycoproteins), and C (nucleocapsid proteins). The stoichiometry of the C protein in the nucleocapsid agrees with the crosslinking studies of Coombs and Brown (7), the proposal of Choi et al. (8), based on their crystallographic studies, and the mass determination of Paredes et al. (9). Associations between the envelope protein E2 and the nucleocapsid are apparent in this reconstruction (Fig. 3a). Evidence suggests that the trimers of El/E2 heterodimers are exported to the plasma membrane after assembly in the endoplasmic reticulum of infected cells (4). It has also been demonstrated that the amino acids closer to the carboxyl terminus of the cytoplasmic tail of the E2 glycoprotein are specifically recognized by a domain in the C protein of the completely assembled nucleocapsid (22, 23). Specific interactions between the three E2 tails of each trimer and the tail-binding sites on the nucleocapsid dimers would initiate the process of envelopment. These specific associations suggest that the nucleocapsid would direct the process of assembly, orienting the trimers in the cell plasma membrane, and prepare them for the specific lateral El-El and E2-E2 associations among the heterotrimers (4). Such a process would create the icosahedral lattice and draw the modified membrane around the nucleocapsid as envelopment proceeds. The initial technical assistance in electron cryomicroscopy of Ms. Evonne Marietta is greatly appreciated. This research was supported by grants AI14710, AI19545 (D.T.B.); AI22186, NS26681 (R.E.J.); GM (B.V.V.P., W.C.); RR02250 (W.C.), all from the U.S. Public Health Service, the W. M. Keck Foundation, and by funds appropriated to the Cell Research Institute by the State of Texas. 1. Schlesinger, M. T. & Schlesinger, S. (1986) in The Togaviridae and Flaviviridae, eds. Schlesinger, M. T. & Schlesinger, S. (Plenum, New York), pp von Bonsdorff, C.-H. & Harrison, S. C. (1975) J. Virol. 16, Harrison, S. C. (1986) in The Togaviridae and Flaviviridae, eds. Schlesinger, M. T. & Schlesinger, S. (Plenum, New York), pp Anthony, R. P. & Brown, D. T. (1991) J. Virol. 65, Fuller, S. D. (1987) Cell 48, Anthony, R. P., Parades, A. M. & Brown, D. T. (1993) Virology 190, Coombs, K. & Brown, D. T. (1987) J. Mol. Biol. 195, Choi, H. K., Tong, L., Minor, W., Dumas, P., Boege, U., Rossmann, M. G. & Wengler, G. (1991) Nature (London) 354, Paredes, A. M., Simon, M. L. & Brown, D. T. (1993) Virology 187, Meyer, W. J., Gidwitz, S., Ayers, V. K., Schoepp, R. J. & Johnston, R. E. (1992) J. Virol. 66, Renz, D. & Brown, D. T. (1976) J. Virol. 19, Adrian, M., Dubochet, J., Lepault, J. & McDowall, A. W. (1984) Nature (London) 308, Dubochet, J., Adrian, M., Chang, J. J., Homo, J. C., Lepault, J., McDowall, A. W. & Schultz, P. (1988) Q. Rev. Biophys. 21, Prasad, B. V. V., Prevelige, P. E., Marietta, E., Chen, R. O., Thomas, D., King, J. & Chiu, W. (1993) J. Mol. Biol. 231, Crowther, R. A. (1971) Philos. Trans. R. Soc. London B 261, Baker, T. S., Drak, J. & Bina, M. (1988) Proc. Natl. Acad. Sci. USA 85, Schrag, J. D., Prasad, B. V. V., Rixon, F. J. & Chiu, W. (1989) Cell 56, Boggs, W. M., Hahn, C. S., Strauss, E. G., Strauss, J. H. & Griffin, D. E. (1989) Virology 169, Polo, J. M., Davis, N. L., Rice, C. M., Huang, H. V. & Johnston, R. E. (1988) J. Virol. 62,

5 Microbiology: Paredes et al. 20. Vogel, R. H., Provencher, S. W., von Bonsdorff, C.-H., Adrian, M. & Dubochet, J. (1986) Nature (London) 320, Unwin, P. N. T. (1993) J. Mol. Biol. 229, Metsikko, K. & Garoff, H. (1990) J. Virol. 64, Liu, N. & Brown, D. T. (1993) J. Cell Biol. 120, Scheefers, H., Scheefers-Borchel, U., Edwards, J. & Brown, D. T. (1980) Proc. Natl. Acad. Sci. USA 77, Proc. Natl. Acad. Sci. USA 90 (1993) Hahn, C. S. & Strauss, J. H. (1990) J. Virol. 64, Coombs, K., Brown, B. & Brown, D. T. (1984) Virus Res. 1, Coombs, K. & Brown, D. T. (1989) J. Virol. 63, Wahlberg, J. M., Boere, W. I. M. & Garoff, H. (1989) J. Virol. 63, Brown, D. T. & Edwards, J. (1992) Semin. Virol. 3, Abell, E. & Brown, D. T. (1993) J. Virol. 67,