Eur. J . Biochem. 102, 589-594 (1979)

Crystallization of Bacteriophage MS2 Willy MIN JOU, Alex RAEYMAEKERS, and Walter FIERS Laboratory of Molecular Biology, State University of Ghent (Received July 3, 1979)

Crystals of bacteriophage MS2 have been obtained by slowly cooling a 1 yi virus solution from 23 C to 0 "C in the presence of poly(ethy1ene glycol) 6000. The crystals were colorless, needle-like, anisotropic and very fragile. Electron microscopic observation of the crystals revealed a twodimensional lattice of particles with RNA phage morphology and dimensions. Preliminary X-ray examination of the crystals confirmed their viral nature.

Since the discovery of RNA bacteriophages by Loeb and Zinder [l], this class of small, spherical, RNA-containing viruses has been intensively studied at the genetic, biochemical and structural level (for reviews, see [2-41). The genetic information of bacteriophage MS2 is encoded in a single RNA molecule containing 3569 nucleotides; it was the first viral genome whose complete primary structure was established [5 - 81. Three proteins are specified by this MS2 RNA genome: the A protein, the coat protein and the virus-coded replicase subunit. The primary structure of these polypeptides can be deduced from the nucleotide sequence; the amino acid sequence of the coat protein ([9] and the above-mentioned reviews) and of most of the A protein [lo] was also established by direct procedures. Each mature virion consists of one molecule of viral RNA, about 180 copies of coat protein and a single copy of the A protein. The diameter of the particle is approximately 26.3 nm and the protein shell is only 2-3 nm thick [l 1,121. Tentative models based on experimental observations and on theoretical considerations have been proposed for the secondary structure of MS2 RNA [5-81 as well as for the folding of the coat protein (J.-P. Vingerhoedt, personal communication). A conclusive and detailed elucidation of the threedimensional structure of the virus particles, however, has to come from X-ray crystallographic studies. By 1935, W. M. Stanley [13] had obtained (pseudo)crystals of tobacco mosaic virus, a rod-shaped plant pathogen, and several spherical plant viruses were subsequently crystallized without apparent difficulty [14-161. Since the first X-ray studies of tobacco This is paper XXXIX in our series Studies on the Bacteriophage M S 2 . Paper XXXVIII was by Devos et al. (1979) J . Mol. Biol. 128, 621 -632.

mosaic virus and tomato bushy stunt virus by Bernal and Fankuchen [17], X-ray data have been collected for several plant viruses [18-201 and detailed structures have been reported down to a resolution of 0.29 nm for tomato bushy stunt virus [21] and 0.28 nm for the protein disk of tobacco mosaic virus [22]. Crystals of an RNA bacteriophage have so far not been obtained in vitro, even though these spherical viruses have a molecular complexity comparable to that of many known plant viruses. But viral aggregates in what appear to be crystalline arrays have often been observed in infected, unlysed cells [23,24]. In the present communication we report the successful crystallization of purified bacteriophage MS2. In contrast to other viruses crystallized so far, which are all isometric (except tobacco mosaic virus, but this virus does not form real crystals but well-oriented gels suitable for X-ray analysis), the MS2 particle is anisometric, due to the presence of a single A protein molecule. This element might well determine the orientation of the RNA molecule within the particle. If crystals could be obtained in which the virus particle is accomodated in the crystal lattice according to this anisometric element, it would probably be possible to study also the RNA within the particle and not solely the protein moiety, as is the case with isometric viruses. MATERIALS AND METHODS

Virus Growth and Purification A 10-1 culture of Escherichia coli D11 [25] was grown at 37 "C in a medium containing 32 g/1 bactotryptone, 20 g/1 yeast extract (both from Difco, Detroit, Mich., U.S.A.), 5 g/l sodium chloride, and

Crystallization of Bacteriophage MS2

5 90

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.-c + 50 .> .~ X 40 0 .-D

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4 5 6 7 8 Poly (ethylene glycol) (%)

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Fig. 1. Soluhility of hucteriophagc M S 2 al I virus concentrution with poly(ethy1ene glycol) precipitants of dqferent molecular weight as n,function of the solvent composition (n,/v). ( 0 )4'c, poly(ethy1ene glycol) 4000; (0) 23°C. poly(ethy1ene glycol) 4000; (A) 4'C, poly(ethy1ene glycol) 6000; (A) 23 "C, poly(ethy1ene glycol) 6000; (+) 4"C, poly(ethy1ene glycol) 20000; (0) 23 'C, poly(ethy1ene glycol) 20 000

Fig. 2. Soluhility of hacteriophagc M S 2 U I 17; virus concentration as a function of the concentration of ammonium .sulphate. (A) 4°C; ( 0 )23 ' T ; (+) 37 'C. There is prdCtiCally no difference between virus solubility at 23 "C and 37 ' C at a given ammonium sulphate concentration, but the solubility is markedly lower at 4°C

0.7 g/l sodium hydroxide, until a density of 6 x lo8 bacteria/ml was reached. Calcium chloride was added to a final concentration of 5 mM and the cells were infected with phage MS2 at a multiplicity of 5 - 10, and incubated for another 4 h. Purification of the virus included the following steps : (a) precipitation with poly(ethylene glycol) (lo%, w/w) and 0.5 M NaCl; (b) lysozyme treatment followed by DNase digestion ; (c) low-speed centrifugation ; (d) differential precipitation with ammonium sulphate: 0.57 M for non-viral material; 1.9 M for precipitation of the virus; (e) Freon-1 1 extraction; (f) overnight dialysis against buffer A (0.1 M NaCI, 0.01 M Tris-C1, pH 7.4, 0.001 M MgC12, 0.0001 M EDTA) ; (8) low-speed centrifugation followed by high-speed centrifugation ; (h) CsCl gradient centrifugation; and (i) dialysis against buffer A. The purified virus solution was stored at 4 "C. I4C-labeled bacteriophage MS2 was prepared by growing the virus in minimal medium supplemented with ['4C]~ridine (16500 dis. min-' pg virus-').

various concentrations of one of the following precipitants : ammonium sulphate pro analysi (Merck, Darmstadt, F.R.G.), poly(eth1ene glycol) 1500, 4000, 6000 or 20000 (B.D.H. Chemicals Ltd, Poole, England), or 2-methyl-2,4-pentanediol (Aldrich-Europe, Beerse, Belgium). loo-@ samples of these solutions were stored in Beckmann microfuge tubes at 4"C, 23 "C, or 37 *C. After equilibrium was reached (two days), the precipitate, if present, was collected by centrifugation and the amount of virus was determined both in the supernatant and in the redissolved pellet by measuring the radioactivity in Triton X-lOO/toluene scintillation cocktail [26].

Soluhility Curves MS2 virus solutions were made up of the following: MS2 virus (final concn 1 %, w/v) containing 3000 counts/min I4C label in crystallization buffer (0.6 M NaCI, 0.01 M Tris-HC1, pH 7.6, 0.5 mM spermidine, 0.1 mM MgC12, 0.01 mM EDTA) and

Crystallization 1 MS2 solution containing crystallization buffer and 4 (w/v) poly(ethy1ene glycol) 6000 was sucked into 1.5-1.8 x 100-mm pyrex capillaries (Bie Sr Berntsen A/S, Arhus, Denmark). The ends were sealed and the capillaries were placed in an insulated bucket containing about 4 1 water and kept at 23 ' C for two days. The bucket was transferred to a 15'C room for two days and subsequently kept at 4 'C for another two to three days. The capillaries were then placed on melting ice. Crystals began to appear after the water bath was transferred to the 4 ° C room. Crystals for observation under a light microscope were grown in cuvettes with a 2-mm light path.

W. Min Jou, A . Raeymaekers, and W. Fiers

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Fig. 3 . Boc,teriophage M S 2 crystals as viewed under the light microscope

Microscopy

RESULTS A ND DISCUSSION

Crystals were examined under a Leitz Diavert inverted microscope (magnification 25 x ) using phasecontrast optics. The cuvettes were kept at 0°C in a wateriice mixture.

Solubility Curves

Electron Microscopy The contents of a capillary containing MS2 crystals in crystallization buffer and 4 (w/v) poly(ethylene glycol) 6000 were spread out on a microscope plate and the mother liquor removed with absorbing paper. In order to obtain thin sections, we broke the crystals by rubbing the surface of the plate with a second microscope plate. The wet crystals were stained for 20 s with 0.1 % uranyl acetate in crystallization buffer containing 5 % poly(ethy1ene glycol) 6000. A drop of this suspension was placed on carboncoated electron microscope grids and the crystals were allowed to sediment for 30 s. Liquid was then removed with absorbing paper. All procedures were carried out at 4°C. The samples were then examined in a Siemens Elmiskop 101 electron microscope and photographs were taken at a crystal magnification of about 60000 x .

The solubility of bacteriophage MS2 was determined at 1 % virus concentration as a function of the solvent composition and as a function of the temperature. It was decided to try inter alia poly(ethy1ene glycols) as precipitants as this procedure is often used in the purification of viruses [27] and as it has been shown by McPherson [28] that they are well suited for crystallization of proteins. The solubility curves obtained with poly(ethy1ene glycol) precipitants of different molecular weight are shown in Fig. 1. Since the results at 37 "C were indistinguishable from those at 23 "C for poly(ethy1ene glycol) 6000 and 4000, the series at 37 "C was omitted for poly(ethy1ene glycol) 1500 and 20000. In general, the higher molecular weight poly(ethy1ene glycols) precipitate the virus at lower polymer concentration. With a poly(ethy1ene glycol) of defined molecular weight, the virus is precipitated at a lower concentration as the temperature is reduced. However, this temperature effect is very limited in the case of poly(ethy1ene glycol) 20000, thus making the latter practically worthless for producing crystals by programmed cooling. A similar set of curves was obtained when ammonium sulphate

Crystallization of Bacteriophage MS2

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Fig.4. Mechunicully disrupted MS2 cry.stuls us viewed in the eliJctronmicroscope. The crystals were stained for 20 s with 0.1 uranyl acetate in crystallization buffer containing 5 poly(ethy1ene glycol) 6000. The pictures reveal a regular two-dimensional lattice of particles having the appearance and dimension of RNA phage

‘x

was used as the precipitant (Fig. 2); however, no precipitation was observed when 2-methyl-2,4-pentanediol was used. At a lower virus concentration (0.5% instead of 1 %) a slightly higher percentage of poly(ethylene glycol) 6000 was required to initiate precipitation.

Crystallization Attempts to crystallize MS2 were made with a virus concentration of 1 in crystallization buffer, and the concentration of the precipitant was chosen from the solubility curves (Fig. 1 and 2) in such a way that the virus remained completely soluble at the higher

W. Min Jou, A . Raeymaekers, and W. Fiers

temperature and was largely insoluble at the lower temperature. Capillaries were prepared with a precipitant concentration of 4 (w/v) poly(ethy1ene glycol) 6000 or 0.9 M ammonium sulphate and slowly cooled to 4 ' C . Amorphous precipitates were obtained with the latter precipitant, but with poly(ethy1ene glycol) 6000, small, colorless, needle-like crystals were found in almost all the capillaries. McPherson [28] has already noted that poly(ethy1ene glycol) may be the best initial trial reagent for the crystallization of proteins. This probably holds also for viruses which have an outer protein coat. Fig. 3 shows some representative pictures of such crystals as observed under the light microscope; the crystals are 1-2 mm long and 0.1 -0.2 mm thick; the corners are often rounded and the surfaces appear irregular and etched. The crystals are very fragile and therefore difficult to handle. Cliaracttkation of the Cvystals Preliminary evidence that the crystals do indeed consist of MS2 particles is the fact that they appear under conditions predicted by the solubility curves of the virus. Furthermore, similar crystals were obtained when spermidine or magnesium chloride was omitted from the crystallization buffer. Crystals also formed when glycine-NaOH (pH 9.0) or cacodylateHCI (pH 6.0) was substituted for Tris-HCI (pH 7.6). It seems, therefore, unlikely that one of the components of the crystallization buffer was specifically involved in crystal formation. Electron microscope observation of the mechanically disrupted crystals (Fig. 4) revealed a regular two-dimensional lattice composed of particles having the appearance and dimensions of RNA phage (a center-to-center distance of approx. 23.5 nm as measured from one of the pictures, but without internal length standards). Many dislocations can be seen; these may have been caused by mechanical distortion of the crystals during the preparation for electron microscopy. The particle packing in the plane is hexagonal. One can also discern more than one layer of viruses in certain areas of the pictures, but it is not possible to correlate this with the ordering of the particles in the crystal at this stage. Further examination under polarized light showed that the crystals were bi-refringent, thus excluding a cubic symmetry. Preliminary X-ray diffraction pictures confirmed the viral nature of the crystals, but their quality did not yet allow high resolution. It is our hope that this crystallographic quality can be improved, e.g., by purifying the virus under milder conditions, by changing the crystallization conditions, etc. Since each MS2 virion contains a single A protein molecule located at least partly on the outside of the particle [29], it might be possible to obtain crystals

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in which the virus particles are in a fixed orientation with respect to this element of asymmetry. If so, there is also a good chance that the RNA within each virion would have the same orientation, especially as the A protein has been shown to bind to specific regions on the MS2 RNA molecule (our unpublished results). This would bring the MS2 RNA into the realm of detailed X-ray crystallographic analysis. We thank D r E. Gillis for critical discussions, Lydia de Grave for taking the electron microscope pictures, Daisy Dewitte for preparing the virus, and Dr B. Strandberg for the X-ray examination of the crystals. This research was supported by grants from the Fonds voor Kollektief Fundamenteel Onderzoek and from the Geconcerteerrle Akties of the Belgian Ministry of Sciencc.

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W. Min Jou, A . Raeymaekers, and W. Fiers, Laboratorium voor Moleculaire Biologie, Fakulteit der Wetenschappen, Rijksuniversiteit te Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium