Crystallization of cowpea chlorotic mottle virus

.J. Mol.

Hid.

(1981) 146, 635-640

Crystallization

of Cowpea

Chlorotic

Mottle

Virus

(lowpea chlorotic mottle virus belongs to the brome mosaic virus group of the small spherical plant viruses. It contains I80 protein subunits, which are arranged on a T = 3 icosahrdral surface lattice. The virus crystallizes in orthorhombic space group P2,2,2 (a = 394 A, b = 382 A, and c = 397 8). The unit cell contains four virus particles, while the crystallographic asymmetric unit consists of one complete virion. X-ray diffraction data from the crystals extend to nearly 3.0 A resolution. Cowpea chlorotic mottle virus (N’MV) is a small spherical RNA plant virus. which belongs to the bromovirus group (Harrison et nl., 1971). CCMV infection requires the presence of three nucleoprotein particles whose identical protein capsids consist, of 1X0 polypeptide chain copies each with a molecular weight of 19,600 (Bancroft et nl., 196X; Agrawal Nr Tremaine, 1972). The protein subunits form an icosahedrally symmetric virion shell with triangulation number 7’ = 3 (Casper & Klug. 1962). The RNA of the virus consists of four distinct species with respective molecular weights of 1.2, 1.1, 0.7 and 0.3 x lo6 (Bancroft, 1971 ; Bancroft dz Flack, 1972). The first two RNA components are individually encapsidated, while a third virion contains the third and fourth RNA components. This multipartite genome is r*haractrristic of the bromoviruses (Lane, 1974). The average virion molecular weight is nea,r 4-6 x IO6 with the RNA accounting for about 25:/, of the particle weight. Extensive chemical studies of the virus have been performed. The isolated coat protein can aggregate in several quaternary states dependent on environmental et ~1.. 1967: conditions such as ionic strength, pH and temperature (Bancroft Adolph & Butler, 1974,1977). The viral particles increase their radial extension (“swelling”) atI a pH greater than 7.5 and in the presence of 10 mM-EDTA and 0.2 m-KC’1 (Bancroft et al.. 1967; Jacrot, 1975). At higher ionic strengths, the swollen particles disassemble (Adolph & Butler, 1974). In the case of thra alosel) related brome mosaic virus (BMV), the particle radius increases from 13.4 nm at pH 5.0 to 15.5 nm at pH 7.5 (Jacrot et al., 1977 ; Chauvin et al., 1978). Dye-binding studies indicate that about 50% of the in sitlr RNA is in the double-helical configuration (Adolph. 1975). Electron micrographs of CCMV confirm the prot,ein capsid icosahedral symmetry and show its diameter to be approximately 26 mn (Home et al.. 1975: Steven et al., 1978). Furthermore, the coat protein molecule is suggchsted to be ellipsoidal in shape with a radially directed long axis of 5.0 nm and a shorter diameter of 3.5 nm (Steven et cd.. 1978). The complete amino acid seyuencr of the WMV coat protein has been determined (Tremaine et nl.. 1972: tires rt ul., 1979). Several (‘(WV crystal morphologies have been reported. Only the cubic* (Rossman rt al., 1973) and orthorhombic (Rayment et a.Z., 1977) forms displa) useful S-ray diffraction power: however, t,he resolution extends to no more t,han x .A. 635

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For the present crystallization experiments, CCMV was grown in cowpea plants (Vigna zcnquiculata (I.)) and isolated according to the techniques of Bancroft et al. (1972). A further purification step involving banding of the virus by cesium chloride density-gradient centrifugation was also included. No attempts were made to separate the tripartite genomic virions. Crystals were produced with the use of the vapor diffusion technique (McPherson, 1976). The reservoir medium consisted of 0.1 iv-sodium acetate at pH 4-O with a polyethylene glycol (PEG) 6000 concentration of 4%. The diffusion well contained virus at 50 mg/ml in a 0.075 Msodium acetate buffer at pH 4.2 and with the PEG 6000 concentration set at 1.3%. The CCMV crystals appeared within one to four days: their longest dimension of growth is generally between 1.0 and 2.5 mm. The crystals were examined with CuKr radiation produced by an Elliot rotatinganode X-ray generator; the X-ray beam was focused by two perpendicularly oriented mirrors (Harrison. 1968). The diffraction data from the crystals extend to nearly 3.0 A resolution. Zero-level precession photographs along the principal zones displayed an orthorhombic unit cell (a = 394 A, b = 382 A, and e = 397 A) with space group P2,2,2. Figure 1 shows two-degree precession photographs for the (MlZ) and (OkE)zones. If four complete virus particles are assumed to be within the unit cell, the value of V,,, (Matthews, 1968) is 3.3 A3/dalton, which is in good agreement with values found for other small spherical plant viruses (cf. Akimoto et aE., 1975). One complete virion would occupy the crystallographic asymmetric unit, thus allowing 60-fold molecular-replacement averaging of the electron density (cf. Unge et al., 1980). Satellite tobacco necrosis virus is the only other example of a plant virus whose crystals diffract to atomic resolution and yet contain a complete particle in the crystallographic asymmetric unit (Akervall et al., 1971). The particle positions and approximate orientations of the icosahedral symmetry axes within the unit cell can be proposed from the following observations and deductions. Extra systematic absences of all reflections (h = 2n,+ 1) were observed on the two-degree (h02) precession photograph (Fig. 1). Furthermore, pseudoabsences were observed for the low-resolution (k = 2n + 1) reflections on the twodegree (hk0) precession photograph. Since virus particles can be regarded as uniformly dense spheres from a very low-resolution perspective, the centric space group consistent with the aforementioned intensity absences would be Pmab. This space group would require a particle e-fold axis to lie along the unit cell c-axial direction and the particle centers to be at x = l/4 and x = 314 along the a-axis. A similar situation exists for the type III southern bean mosaic virus (SBMV) crystals with orthorhombic space group C222,. A systematic search of all possible virion positions within the cell with the constrained x-axis centers resulted in only one site in such a way that the spherical particle centers were uniformly separated by 265 A, a value consistent with the 260 A virion diameter observed from electron microscopy data. This position given in fractional cell co-ordinates is x = 0250, y = 0.255 and z = 0.270. The particle spatial distribution generated by the crystallographic symmetry would thus correspond to a distorted cubic close-packed arrangement (Fig. 2). If icosahedral2-fold axes were also aligned with the a- and h-axial directions, the

LETTERS

TO

THE

EDITOR

637

(b) FI (:. 1. (a) A screenless precision photograph of the CCMV (hO1) zone. The precession angle was 2” surh that data extend to 22 A resolution for the zero-level reflections. The h-axis is vertical and the 1-axis hori: zontal. (b) A screenless precession photograph of the CCMV (O/cl) zone. The precession angle WHLS2’. The k-axis is vertiml and the Z-axis horizontal.

638

K

I,

HE

FIG. 2. Proposed packing arrangements of the CCMV virions as viewed down the unit cell c-axis. The 4 particles within the cell are shown with their icosahedral %fold axes parallel to the c-axis. The exact 5fold contacts lie on a line in the (110) plane; the approximate 5-fold contacts are also visible.

space group would be cubic and the principal zone precession photographs would show the same intensity distribution. This was not observed. However, since the orthorhombic cell has nearly equal axial lengths, some small misalignment of the 2fold axes with respect to the a- and b-axial directions would be expected. A rotation about the c-axis of approximately 13” would result in colinear particle 5-fold axes in the (110) plane and require the same intensity distribution on the four-body diagonal zero-level precession zones. Furthermore, very low-resolution intensities would be expected to show S-fold symmetry in these zones due to the approximate alignment of the icosahedral S-folds along the body diagonal. These intensity distribution constraints were observed on the diagonal precession photographs. The particle separation along the line of exact 5-fold contacts showed a maximum virion diameter of 274 8. thus suggesting 5-fold protrusions. Such extensions have also been observed in SBMV (Johnson et al., 1976: Abad-Zapatero et al., 1980). Atomic resolution structures are presently known for SBMV (AbadZapatero et al., 1980) and TBSV (Harrison et al.. 1978). The SBMV protein subunit and the surface domain of the TBSV capsid protein show an almost exact similarity in the topology and extent of their secondary structural elements. As suggested by Kaper (1972), simple plant viruses can be categorized according to the dominance of protein-protein or protein-RNA interactions that stabilize the viral particle. In the case of TBSV and SBMV, both protein-protein and protein-RNA interactive forces are essential for the integrity of the virion. For the tymoviruses, protein-protein interactions dominate. while in the bromoviruses the proteinnucleic acid forces are strongest. Crystals that diffract to atomic resolution now exist for belladonna mottle virus (a t’ymovirus) and CCMV (a bromovirus). Their tertiary structure determination and subsequent comparison with the architecture of TBSV and SBMV will, hopefully. result in a better comprehension of the infective processes of all small spherical plant virus groups.

LETTERS

TO THE

EI)ITOK

ww I.

The authors are deeply grateful for many helpful discussions with Drs John Johnson. Michael Rossmann and Sherin Abdel-Meguid. Drs Andrew Jackson and Richard Lister supplied the CCMV virus and were most helpful in achieving its purification. Appreciation is expressed to Marilyn Anthony for help in the manuscript preparation. ]‘.A. wishes to acknowledge financial support from the National Science Foundation (no. PCM77-20287 and no. PCME@-04575) &9 well as the American Cancer Society (Faculty Research Award no. FRA173).

ljepartment of Biological Sciences I’urdutl lTniversit,y West Lafayette, Ind. 47967, U.S.A.

KATHLEEN L. HE~ISS ,I. K. MOHANA RAO PATRICK Atu:ost

Recrivt~d 13 Xovem brr 1980 t To whom reprint, requests should be addressed.

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