Virology 284, 223–234 (2001) doi:10.1006/viro.2000.0914, available online at http://www.idealibrary.com on Structural Transitions of Satellite Tobacco Mosaic Virus Particles Yurii G. Kuznetsov, Steven B. Larson, John Day, Aaron Greenwood, and Alexander McPherson1 University of California, Irvine, Department of Molecular Biology and Biochemistry, Room 560, Steinhaus Hall, Irvine, California 92697-3900 Received November 6, 2000; returned to author for revision January 15, 2001; accepted March 15, 2001 Satellite tobacco mosaic virus (STMV) can undergo at least two physical transitions that significantly alter its mechanical and structural characteristics. At high pH the 17-nm STMV particles expand radially by about5Åtoyield particles having diameters of about 18 nm. This pH-induced transition is further promoted by aging of the virions and degradation of the RNA, so that swollen particles ultimately appear even at neutral pH. While the native 17-nm particles crystallize as orthorhombic or monoclinic crystals which diffract to high resolution (1.8 Å), the enlarged 18-nm particles crystallize in a cubic form which diffracts to no better than 5 Å. In the transition, not only do the capsid protein subunits move radially outward, but the helical RNA segments with which they interact do as well. This is noteworthy because it demonstrates that the RNA and the protein shell are capable of coordinated movement, and that neither structure is rigidly defined or independent of the other. Using atomic force microscopy, it can be shown that STMV particles, upon drying, lose their mechanical rigidity and undergo deformation. Virions initially 17 nm in diameter shrink to more uniform final sizes than do 18 nm, initially swollen particles. This transition appears to be irreversible, as the particles do not reassume their former size nor structural rigidity upon rehydration. Evidence is also presented that preparations of native virus and their crystals are naturally somewhat heterogeneous and contain a variety of particles of anomalous size. © 2001 Academic Press INTRODUCTION the particle (Caspar and Klug, 1962; Horne, 1974). From the 1.8 Å resolution, refined STMV structure (Larson et al., ϭ Satellite tobacco mosaic virus (STMV) is a T 1 1998), it was possible, with a high degree of certainty, to particle of about 17 nm diameter containing a single- describe the distribution of solvent molecules both in- stranded RNA genome of 1058 bases (Mirkov et al., 1989) side and outside the capsid and to define their roles in inside a capsid composed of 60 identical copies of a maintaining intermolecular interactions. In addition, the ϭ coat protein having 159 amino acids and Mr 17,542 structural association between the capsid protein and (Valverde and Dodds, 1986, 1987; Valverde et al., 1991). the visible portions of the RNA were delineated in detail. The structure of STMV was solved by X-ray crystallog- In the course of additional X-ray diffraction and atomic raphy and reported at 1.8 Å resolution (Larson et al., force microscopy (AFM) studies of STMV, it became 1998) for the orthorhombic form. The coat protein was evident that the virus particles could undergo certain shown to be a “Swiss roll” beta barrel of 123 carboxyl- structural transitions. These occurred as a consequence terminal residues and a long, extended amino terminal of age, alteration of the pH, and as a consequence of strand of 36 amino acids. The first 12 amino terminal drying. The transitions in structure are accompanied by residues were not visible and apparently in the interior of changes in the mechanical properties of the particles as the virus. well as their sizes. Structural changes of this nature have An unanticipated feature of the STMV structure was long been known to occur in T ϭ 3 icosahedral plant the appearance of nearly 45% of the total viral RNA in the viruses (Kaper, 1975). electron density maps. This was visualized as 30 double- helical segments of seven base pairs with an additional base stacked at either 3Ј terminus (Larson et al., RESULTS 1993a,b). The double-helical RNA segments were closely If freshly prepared STMV samples are deployed for associated with dimers of coat protein and were oriented crystallization at 12–20% saturated ammonium sulfate so that viral twofold axes were perpendicular to the using vapor diffusion below pH 8.0, they consistently helical axes of the segments and coincident with their form orthorhombic crystals above 12°C, and monoclinic central dyad. Thus the RNA segments were disposed in crystals at colder temperatures. Above pH 8.0, at any a manner consistent with the icosahedral symmetry of temperature greater than 2°C, cubic crystals grow. Or- thorhombic crystals of STMV diffract to at least 1.8 Å resolution, and the structure of STMV was refined to that 1 To whom correspondence and reprint requests should be ad- resolution limit (Larson et al., 1998). The monoclinic dressed. Fax: (949) 824-1954. E-mail: [email protected]. STMV crystals diffract to about 2.7 Å, possibly higher, 0042-6822/01 $35.00 223 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. 224 KUZNETSOV ET AL. STRUCTURAL TRANSITIONS OF STMV PARTICLES 225 FIG. 2. Central cross section of STMV based on the model refined at 1.8 Å resolution by X-ray crystallography (Larson et al., 1998). This model is derived from the orthorhombic crystal form which yields a 17-nm particle by AFM and quasi elastic light scattering. The protein is shown in ribbon format in red, and the RNA as all atoms in white. Tabulated below are relevant values for the native and swollen particles. FIG. 1. (a) Variation of the correlation coefficient (CC) and R value as a function of radial displacement from the particle center based on 5–20 Å data cut at F Ն 5␴F. The darker curves are for the orthorhombic STMV model placed in the cubic cell and rotated 10.8° about the body diagonal and then expanded in 10 Å by 0.1 Å increments. Clearly, the optimum radial expansion is slightly more than 5 Å. For comparison (light curves), a similar expansion of the STMV model in the orthorhombic cell was performed starting from a particle shrunken by 5 Å and then expanded 10 Å by 0.1 Å increments. (b) Variation of CC and R as a function of the rotation angle about the body diagonal of the cubic cell using 10–20 Å data (dark curves) and 5–20 Å data (light curves), both with F Ն 5␴F. The particle was expanded by 5 Å from the original orthorhombic model and then rotated 120° in 0.1° increments about the diagonal. The optimum rotation is about 10.8°, which further refinement showed to be 11.19°. (c) Variation of CC and R as a function of particle translation along the body diagonal using 5–20 Å data cut at F Ն 5␴F. The particle was expanded by 5.38 Å and rotated by 11.11° before the 5 Å translation along the diagonal was executed using 0.1 Å increments. Clearly the correct particle position is the origin. 226 KUZNETSOV ET AL. and their structure is now also solved (unpublished STMV model composed of protein, helical RNA, and data). Cubic STMV crystals, however, diffract to no more sulfate anion was placed at the origin in the orthorhom- than 5 Å resolution even in the best of cases (Koszelak et bic orientation and noncrystallographic symmetry oper- al., 1995). ators with a variable skew matrix and skew translation As the STMV ages through storage or standing, how- were employed. The model was expanded along the ever, the pH at which cubic crystals appear decreases, radial vector through the centers of mass of the model so that after several years, when the RNA is fragmented, components, rotated about, and translated along the as clearly shown by polyacrylamide gel electrophoresis, body diagonal. The search was monitored by the corre- cubic crystals appear even at pH 7. Often in aged prep- lation coefficient based on F. Figure 1 illustrates the arations, near neutral pH, both orthorhombic and cubic variation in CC and R with respect to these operations. crystals coexist in the same crystallization samples. The Clearly the particle is centered at the origin and ex- RNA of STMV, although it does eventually break down, is panded by about 5 Å. The skew angle (equivalent to the remarkably stable. If, for example, STMV particles are rotation angle) was estimated to be about 10.8°. Contin- exposed to pH 9.0 for 24 to 36 h, their RNA, when ued refinement of these parameters with rigid body re- analyzed by agarose gel electrophoresis, is almost en- finement of the model gave final values of 11.19° for the tirely intact. Other experiments (Day et al., 2001) show skew angle and ϳ5.7 Å for the expansion. Figure 2 is an that even heating to 90°C produces little disruption of the illustration of the STMV virion showing the protein coat encapsidated RNA conformation in particles extensively and the double-helical RNA inside. The change in parti- digested with proteinase K. cle size is also tabulated as are results from the rigid ϭ Cubic STMV crystallizes in space group P213 with a body refinement. It should be noted that the 5.7 Å radial 257.25 Å. This implies that the STMV particle must have expansion of the particle is a 7.8% change in the mean its center on the body diagonal (a threefold axis) with an radius and translates directly to a 7.8% increase in tan- icosahedral threefold axis coincident with the body di- gential surface distances at the mean radii.