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Studies on the assembly of a spherical plant virus
J. Mol. Biol. (1977) 109, 345-357
Studies on the Assembly o f a Spherical Plant Virus H I . t Reassembly o f Infectious Virus U n d e r Mild Conditions KENNETH W. ADOLPH~ AND P. J. G. BUTLER
Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge CB2 2QH, England (Received 2 July 1976) Mild solution conditions are described for the direct reassembly of a simple spherical vimm, cowpea chlorotic mottle virus (CCMV), from its isolated protein and RNA components. Previously the reconstitution of spherical viruses has only been accomplished in conditions which are clearly non-physiological. The reassembly proceeds efficiently near neutral pH, at ionic strength 0.1 or 0.2 and 25~ and requires the protein to be in a non-equilibrium state. Addition of Mg 2+ is not required. At pH 6, about 70% of the RNA is eneapsidated as infectious virus. The 3 S protein aggregate {which assembles into the capsid) is monodisperse and consists of dimers of the subunit. The material is indistinguishable from native CCMV by physicochemical and structural characterization and also shows similar infectivity to native CCMV in a dilution assay on cowpea plants. However, the specificity of the protein for its homologous RNA is not strong and a number of plant virus RNAs and synthetic pol)~mcleotides can be encapsidated with effieiencies comparable to that found for the CCMV RNA. 1. Introduction Many simple viruses have been reconstituted from their isolated protein and nucleic acid components, but the reassembly of spherical viruses has previously only been accomplished under conditions which are clearly non-physiological. For example, reassembly of infectious bacteriophage R17, containing the A-protein, requires the components to be mixed under denaturing conditions and renatured from 5"7 M-urea (Roberts & Steitz, 1967). Similarly, the reconstitution of bacteriophage Qfl was accomplished by renaturation from 8 M-guanidine hydrochloride (Hung & Overby, 1969). The previously reported reassembly of cowpea chlorotic mottle virus involved dialysis from a high salt concentration, and had to be carried out at a low temperature (Bancroft & Hiebert, 1967; Hiebert & Bancroft, 1969). Cucumber mosaic virus can be reversibly dissociated with lithium chloride (Kaper & Geelen, 1971). The reassembly of simple spherical viruses thus differs considerably from t h a t of the rod-shaped tobacco mosaic virus, which was the first virus to be shown to selfassemble from its isolated protein and nucleic acid components (Fraenkel-Conrat & Williams, 1955). Investigations of the states of aggregation of tobacco mosaic virus -~Paper I I in this series is Jeerer (1975). :~Present address: Department of Biochemical Sciences, Princeton University, Princeton) N.J. 08540, U.S.A. 23* 345
346
K. W. A D O L P H A N D P. J. G. B U T L E R
protein and of the control of polymerization into the protein helix (Durham et aL, 1971; Durham & Klug, 1971) have led to an understanding of the physiologically plausible conditions for the reproducible and rapid formation of complete virions (Butler & Klug, 1971). Physiologically significant assembly processes are likely to occur at p H values near neutrality, ionic strengths of 0.1 or 0.2, and temperatures of 20 to 37~ A n y reassembly which requires the presence and subsequent removal of denaturing agents or of high concentrations of salt at low temperature clearly departs from the normal assembly of the virus. I n this paper we discuss the reassembly of CCMVt under mild conditions of pH, ionic strength and temperature, which are plausible for the in vivo assembly. A preliminary account of these results has been presented (Adolph & Butler, 1975). This procedure for reassembly shows clear differences in its properties from the earlier procedure of Bancroft and his co-workers (Bancroft & Hiebert, 1967; Hiebert & Bancroft, 1969). 2. M a t e r i a l s a n d M e t h o d s
(a) Virus growth and purification Cowpea ehlorotic mottle virus was grown in cowpea plants (Vigna unguiculata (L.) Walp. var. Blackeye), and purified from the sap by polyethylene glycol precipitation followed by differential centrifugation. The purified virus was stored in sodium acetate, pH 5, ionic strength 0.1 at 4~ Concentrations of CCMV were estimated ushlg an extinction coefficient E~ ~ = 5.87 (Bancroft et al., 1968). (b) Preparation of protein Two methods were used to isolate CCMV protein from the purified virus. One procedure, which we had previously used in investigating the states of aggregation of the protein (Adolph & Butler, 1974), involving dialyzing CCM-V against 1 M-calcium chloride at pH 8 to disassemble the virus and precipitate its RNA. In this procedure, any residual, degraded RNA was removed by ion exchange chromatography using a small cohmm of DEAE-eel]ulose. A second method allowed the protein to be isolated more rapidly, by avoiding lengthy dialyses to eliminate the Ca 2+. Here the virus was dialyzed against 1 M-KC1, 0.01 ~-Tris, 10 -3 M-dithiothreitol (pH 7.5) for 2 h at 5~ and then the RNA was removed by sedimentation (Hiebert & Bancroft, 1967). This method was used to provide the very fresh protein which, as previously reported by Hiebert and Bancroft, yielded better reassembly of virus. Concentrations of CCMV protein were estimated spectrophotometrically using an extinction coefficient E~ ,~ = 1.2 (Hiebert et al., 1968). (c) Preparation of R N A CCMV RNA is only extracted in low yield with phenol, and so a procedure was used which involved dissociating the virus in SDS and then separating the RNA from the protein by sedimentation into a sucrose gradient (Adolph, 1975a). The virus was dissociated at a concentration of about 5 mg/ml in sodium acetate, pI-I 5"5, ionic strength 0.1 with 2% SDS for 3 to 5 rain at 50~ The mixture was layered on a 5% to 200/o sucrose gradient in 0.1 M-sodium acetate, 0.01 M-Tris (pI-I 7.5), 1% SDS, and centrifuged for 18 h at 20,000 revs/min and 20~ in a Beckman SW27 rotor. Fractions could either be collected to isolate the individual RNA species of the multieomponent CCMV genome, or, more frequently, the entire central portion of the gradient removed to recover the mixed RI~IA. The mixture t Abbreviations used: CCMV, cowpea chlorotio mottle virus; SDS, sodium dodecyl sulphate; BM'V, brome mosaic virus; BSV, tomato bushy stunt virus; TCV, turnip crinkle virus; TYM~r turnip.yellow mosaic virus; TMV, tobacco mosaic virus.
REASSEMBLY
OF CCMV UNDER
MILD
CONDITIONS
347
of the RNA species was precipitated with 2.5 vol. ethanol at --20~ for 18 h and resuspended in 0.1 m-sodium acetate, 0.01 M-EDTA, 0.01 ~-Tris, pH 7.5. The ethanol precipitation was repeated once or twice to remove residual sucrose, and the RNA was then taken up in an appropriate buffer and stored at -- 20~ The other plant virus RNAs were purified by extraction with phenol. For each virus, bentonite was added to the virus solution (5 mg/10 mg of virus), which was then made 2~/o in SDS. The mixture was shaken at room temperature for 20 rain with an equal volume of phenol (saturated with 0.01 ~-EDTA, 0.01 ~-Tris, pH 7-5), following which the aqueous, RNA-containing phase was separated from the phenol phase by centrifugation. After twice repeating the extraction procedure, the RNA was precipitated with ethanol and stored in an appropriate buffer at --20~ The CCMV RNA concentration was determined from an extinction coefficient E~~ ---- 33.84 (Bancroft et aL, 1968), while the extinction coefficients of the other RNAs were taken to be E~ % -~ 25-0. (d) Buffers The buffers used to obtain the required pH values were" formate from pH 3.5 to 3.75, acetate from pH 4-0 to 6"0, phosphate from p H 6.25 to 7.5, and Tris at p i t 8.0. Buffers were prepared to an ionic strength of 0.1 and made up to higher ionic strengths by the addition of 3 ~-KC1.
(e) Analytical ultracent~rlfugation Sedimentation coefficients, buoyant densities and molecular weights were measured with an MSE analytical ultracentrifuge mark II, equipped with an ultraviolet scanning system. A least-squares program was used to calculate the sedimentation coefficients from values of ln(r) against t, and the sedimentation coefficients were corrected to give values of S2o.w. The sedimentation coefficients were generally determined at 15,000 revs/min at 25~ The percentage encapsidation was estimated from the ratio of the absorbance (at 260 nm) of material sedimenting at the rate of virus to the total absorbance by the sample at the beginning of the centrifugation, with allowance made for radial dilution. (f) Electron microscopy A Philips EM300 electron microscope was used to examine samples of the reassembled virus. Grids were prepared by adding a drop of reassembled virus to a "holey" carbon grid, washing with several drops of water after adsorption for about 5 min, and staining with a drop of 1~/o (w/v) uranyl acetate in water. The particles that were photographed were in a film of stain over holes in the carbon.
3. Results (a) Reassembly of infectious virus Reassembly of CCM-V involves the co-aggregation of the protein and the R•A and can occur under mild conditions which are plausible for the in rive assembly. A high yield of reassembled virus can be obtained, for example, b y mixing a weakly buffered protein solution (at p H 7.5, ionic strength 0.2) with a strongly buffered R N A solution (at p i t 6-0, ionic strength 0.2) to give a final p i t near 6. An alternative procedure is to mix the protein and R N A at p H 7.5 and then dialyze to p i t 6. I n both cases, with a 4:1 weight ratio of protein-to-RNA, nearly 70% of the R N A is encapsidated as infectious CCMV. (The solution conditions for reassembly arc discussed in greater detail in the following section.) Under these conditions for the reassembly, the isolated CCMV protein exists at equilibrium primarily (over 90%) as a low molecular weight aggregate (Adolph & Butler, 1974), which is found to be a dimer (see below), while the remaining protein
348
K.W.
ADOLPH
A N D P . J . G. B U T L E R
(under 10~o at the total protein concentration employed) occurs as capsid. Recent potentiometric titration experiments (Jaerot, 1975) have demonstrated hysteresis through the pH region where the dimer aggregates into the eapsid. Direct mixing of the equilibrium protein and RNA led primarily to the formation of small amounts of material sedimenting at the rate of capsid and not of reassembled virus. A high yield of virus could be obtained, however, by taking advantage of the hysteresis and supplying the protein in the non-equilibrium form, obtained by rapidly reducing the pH. (b) Effect of conditions on reassembly The effect of varying the final pH of the reassembly solution at ionic strength 0.2 was studied. CCMV was reassembled with a 5 : 1 weight ratio of protein-to-RNA for 16 hours at 5~ Three classes of material were found: reassembled virus sedimenting at about 80 S, non-encapsidated RNA, and heterogeneous material which sediments faster than reassembled virus, and their proportions varied with pH (Fig. 1). At pH 6.0 almost 70~o of the material sediments at the rate of virus, and at pH 6.5 50% of the RNA was encapsidated. For lower pH values, even greater percentages of
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o
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-
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I
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FIG. 1. Effect of varying the final p H of the reassembly solution upon the percentage of reassembled virus at ionic strength 0.2. --O--O--, Reassembled virus ( ~ 8 0 S); - - A - - A - - , non-encapsidated R N A (<<80 S); "" I " " I " ", rapidly sedimenting material (>> 80 S).
the optical density (at 260 nm) is found as virus-like material; by pH 5-0, nearly 90% of the material is in this form. But the interpretation of the results at pH 5.5 and below is complicated by the fact that the isolated protein itself aggregates into a shell below pH 5.5 (Adolph & Butler, 1974), and so in a reassembly at these low pH values the RNA may be being trapped by the aggregating protein rather than causing the assembly. At pH values of the final reassembly solution above pH 6.5, the percentage of reassembled virus plateaus at about 30~/o. The non-eneapsidated RNA varies from undetectable amounts at pH 5.0 to about 60~/o at pH 7.0 and above, while throughout the range of pH, approximately 13% of the material is in a form which sediments much faster than virus. The overall picture from these experiments
R E A S S E M B L Y OF CCMV U N D E R MILD C O N D I T I O N S
349
is that near neutral pH, at ionic strength 0.2, substantial co-aggregation of CCMV R N A with the protein occurs to form virus. Reassembly, as previously described, requh'ed low temperatm'es (4~ low ionic strengths (0.01 M-KCI, 0"01 M-Tris), and the presence of divalent cations (normally as 5 • 10 -3 M-MgC12) (Bancroft & Hiebert, 1967; Hiebert & Bancroft, 1969; Bancroft, 1970). The effect of these parameters was therefore tested under the present reassembly conditions. Reassembly at p H 6.0 with ionic strengths other than 0.2 was investigated and the results are shown in Table 1. Over the range from 0.1 to 1"0, ionic strength has little effect on the reassembly. The main difference is the slight decrease in the sedimentation coefficient, s20.w, with increasing ionic strength. A similar effect was found with native CCMV at p H 4.5 (Adolph, 1975b).
TABLE 1
Effect of solution conditions on reassembly of CCM V
pH 6, I = 0.1 pH 6, I = 0.5 pI-I 6, I = 1.0 pH 6, I = 0.2 25~ pH 6, I = 0.2 +0.01 ~-Mg2+ pH 5'75, I = 0.2 + 0"01 ~1-spermine
su0.w(S) of product
Percentage eneapsidation of RNA
81 76 72 76
53 58 57 60
7(.)
56
82
51
Reassembly was performed at 5~ (unless otherwise indicated) for 20 h. After reassembly at 25~ (pH 6.0, ionic strength 0.2), the percentage encapsidation and sedimentation coefficient, Seo.w, of the reassembled virus were the same as those for reassembly at 5~ (Table 1). A low, and unphysiological temperature is therefore not required for this reassembly. Although divalent cations need not be added to the reassembly solutions, any effect they might have was tested by adding 0-01 M-MgC12 or 0.01 M-spermine (which acted in a manner similar to Mg 2+ in reassembly, Hiebert & Bancroft, 1969) to the reassembly solutions. The results (Table 1) show no change in the yields of virus upon addition of Mg 2 + or spermine. The present reassembly therefore shows several clear differences from that previously described in not requiring high pH, low temperature, low ionic strength or the presence of divalent cations. (c) Properties of the reassembled particles The physical properties of reassembled CCMV are similar to those of native virus. The molecular mass of the reassembled virus, determined by sedimentation equilibrium in the analytical ultracentrifuge, was 4.5 • 106 daltons, the same, within
350
K . W . A D O L P H AND P. J. G. B U T L E R
experimental error, as native CCMV. The sedimentation coefficient, 82o.w, of reassembled CCMV was found to be around 80 S at low ionic strength (Tables 1 and 2), which is again similar to the sedimentation coefficient of native CCMV in the same conditions (Adolph, 1975b). I n preliminary experiments (Adolph & Butler, 1975) the reassembled product was less stable t h a n native CCMV in CsC1, and we had suggested t h a t this might be due to the lack of some annea]ing process. This has now been overcome b y using freshly isolated protein (see Materials and Methods) so t h a t the virus particles are reassembled within two to four hours of disassembly of the virus to isolate the protein. The b u o y a n t density of reassembled CCl~V in CsC1 was measured and found to be the same (to ~4thin 0.004 g/cm 3) as t h a t of native CGMV. The measurement of the b u o y a n t density was made b y centrifuging reassembled CGM-V and native virus to equilibrium in separate cells, but under identical conditions. Since the buoyant density depends upon the relative proportions of protein and R N A contained in the virus particles, the similarity of the b u o y a n t densities confirms t h a t the proportions of protein and R N A are the same in the reassembled and native virus particles. TABLE 2
Specificity of assembly with CCM V protein Final solution conditions pH 6, I ~-- 0"2 Time for assembly 0.5 h 20 h Encapsidation Encapsidation a~o.~(S) (%) ~o..(S) (%) CCMV RlqA BSV RNA TCV RNA TYMV RhTA TMV RNA poly(A) poly(C) 9poly(U)
75 76 76 67 105 77 69 65 70
60 64 56 41 25 25 70 51 62
81 80 80 85 114 87 62 70 74
57 60 53 45 21 32 72 44 48
pH 7"5, I = 0.2 20 h Encapsidation S~o.w(S) (%) 77 79 80 77 -76 65 54 67
23 27 14 27 -32 71 21 42
When examined in the electron microscope, reassembled CCMV shows the same general appearance and diameter as native CCMV, and also displays the characteristic 2-fold, 3-fold, 5-fold and local 2-fold views of the T ---- 3 surface lattice of native CCMV, as previously demonstrated (Adolph & Butler, 1975). l~urthermore, preparations of reassembled virus are substantially homogeneous, with few particles revealing an abnormal morphology. The infectivity of reassembled CCMV was compared to those of native virus and of the purified R N A in a dilution assay on cowpea plants. Both reassembled and native CCM~ produced systemic infections when applied at a concentration of 0.1 mg]ml, but not at 0.02 mg/ml. However, the purified RNA, which had been used in the reassembly, did not produce an infection when inoculated at 0.02 mg/ml (equivalent to virus at 0.1 mg]ml). Although these results should be considered preliminary, t h e y again confirm the similarity of the reassembled virus to the native CCM~.
REASSEMBLY
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351
(d) Stability of reassembledGGMV at high TH Particles of CCI~V undergo an abrupt structural transition as the pH of a virus solution is raised above pH 6.5 (Bancroft, 1970; Adolph, 1975b). The transition is observed at low ionic strength (ionic strength 0.2) as a decrease in the sedimentation coefficient, s20.w, of the virus particles from 83 S to 73 S. This behaviour of CCMu has been described as "swelling", since it is associated with an increase in the radius of the virus particles (Bancroft, 1970). Since the isolated protein shell disaggregates above pH 5.5 (Adolph & Butler, 1974), the swelling of CCMW only at a higher pH must reflect the stabilizing interaction of RNA with the protein subunits of the capsid. The sedimentation behaviour of reassembled CCMV at high pH therefore provides a sensitive test for the quality of the reassembly into the virus structure, though this is not essentially related to the specific CCM-V RNA (see below).
\
"~ o 85
\
g ~g g .--
\
75
7O
I
5
I
\
I
6
7
I
8
pH FIG. 2. Stability of reassembled CCM-V a t high pH, ionic s t r e n g t h 0.2. The points are plotted for the m a j o r b a n d of optical density (260 nm) which sediments near the rate for virus.
Samples of reassembled CCM-V were dialyzed against buffers in the range of pH from 5 to 8, ionic strength 0.2, 5~ for 3 hours. The sedimentation coefficients, s20.w, of the reassembled material were then measured, and are shown as a function of pH in Figure 2. Unlike the RNA-free capsid, reassembled virus does not disaggregate at higher pH values, but sediments at the rates appropriate for virus over the range of pH. Indeed, a decrease in the sedimentation coefficient of the reassembled virus is found, which is similar to that due to the "swelling" behaviour of native virus, and is also centered at pH 6.5, as it is with native CCMW. The product of this reassembly is therefore, not simply an RNA molecule trapped inside the protein shell, but rather interacting with the protein subunits similarly to the RNA in the native virus.
(e) The low specificity of reassembly with COMV protein The specificity of encapsidation with CCMV protein was tested by reassembly with various natural RNAs and synthetic polynucleotides. The natural RNAs were those of three other spherical plant viruses: tomato bushy stunt virus, turnip crinl~le virus and turnip yellow mosaic virus, and the rod-shaped tobacco mosaic virus. Three
352
K. W. ADOLPH AND P. J. G. B U T L E R
synthetic polynucleotides were studied: polyadenylic acid (poly(A)), polycytidylic acid (poly(C)) and polyuridylic acid (poly(U)). Reassembly was carried out with the final solution conditions pH 6, I -~ 0.2, 25~ where almost 70% of CCMV RNA is encapsidated (Fig. 1), and also with p H 7.5, I ~ 0.2, 25~ where 30% of CCMV RNA sediments as virus following reassembly. At the lower pH, the percentage encapsidation and sedimentation coefficient (S2o.w) of the reassembled material was determined at 0.5 hour after mixing weakly buffered protein at p H 7.5 with strongly buffered polynucleotide at pH 6, and at 20 hours after dialyzing to p H 6 a mixture of the protein and polynucleotide at p H 7-5. In each case the weight ratio of protein to RNA was 4 : 1. The results of performing the reassembly ~dth CCM~7 protein and the various natm'al and synthetic polynucleotides are summarized in Table 2. In these experiments CCMV protein does not show a strong specificity for CCMV RNA, all the natural RNAs and synthetic polynucleotides were encapsidated to a substantial extent and the percentages of the various encapsidated polynucleotides sedimenting at a similar rate to virus were comparable to the percentage of CCi~V RNA, particularly with a final solution pH of p H 6. For example, after 0.5 hour at pH 6, I ---- 0.2. 25~ 60~ of CCMV RNA was encapsidated, 64% of BSV RNA, 50% of TMV RNA and 62% of poly(U). There was little difference in the eneapsidation of the RNAs and synthetic polynucleotides after reassembly for 0-5 hour or for 20 hours. The reassembly reaction seems to be completed within 0.5 hour and may be even much sooner. Raising the final pH had a similar effect on the percentage encapsidation of heterologous RNAs and synthetic polynucleotides (except for poly(A)) to that found with CCMV RNA (Table 2 and Fig. 1). The sedimentation coefficients, S2o.w, of the products of assembly of the natural RNAs with CCMV protein (20 h, pH 6, I ---- 0.2) are close to that of native or reassembled CCMV. With TMV RNA an additional component sedimenting at about l l 0 S can frequently be resolved as previously reported by Verduin & Bancroft (1969). The preparations assembled with poly(A)), poly(C) and poly(U) have lower sedimentation coefficients, but these are still greater than that of the RNA-free capsid (52 S). With all the plant virus RNAs and synthetic polynucleotides, the reassembled material was found by electron microscopy to be in the form of spherical particles similar to CCMV. Hence in this reassembly with CCMV protein virus-like material is produced ~4th all of the polynucleotides. The different times allowed for reassembly at pH 6, I ---- 0.2 (0.5 or 20 h) made no qualitative difference to the results. However, the sedimentation coefficients with all the RNAs and synthetic polynucleotides after the shorter reassembly time averaged 70/0 less than after the longer time. With a final p H of 7.5 and after 20 hours reassembly, the sedimentation coefficients averaged about 90% of those found after 20 hours reassembly at p H 6, with the notable exception of that for the poly(A) nucleoprotein, although no reason is known for this anomaly. The RNA prepared from CCMV, BSV, TCV, TYMV and TMV for use in the assembly system is mainly intact, as judged by the sedimentation coefficients and sedimentation profiles. Since these heterologous RNAs are significantly larger than CCMV RNA (BSV RNA has a molecular weight of 1.65 x 106, while TMV RNA is 2.2 • l0 s) more of these heterologous RNAs must be encapsidated in each particle during the reassembly. Although there is no evidence for the location of this extra RNA, packing it into the shell will present no problem (see Discussion). The molecular weights of the synthetic polynucleotides were calculated from their sedimentation
REASSEMBLY OF CCMV U N D E R MILD CONDITIONS
353
coefficients using the formula s20.w ---- 1 • 10 -2 M~ (Mahler & Cordes, 1966). Thus the sedimentation coefficients, 82o.~, for poly(A), poly(C), and poly(U) of 9.3 S, 3"5 S and 5.3 S, correspond to molecular weights of 2.5 • l0 s, 4.3 • 10 a and 9.1 • 104, respectively. Hence 4 molecules of poly(A), 25 molecules of poly(C) or 12 molecules of poly(U) would need to be encapsidated in each particle to equal the 1.1 • 10 s daltons of RNA that is contained in a native CCMV particle. However, the lower sedimentation coefficients for the synthetic polynucleotide-containing particles than for those with CCMV RNA or the other plant virus RNAs suggest that less poly(A), poly(C) or poly(U) is contained in these reassembled particles. The sedimentation profiles of the products of assembly show that when the assembly is performed rapidly, by mixing weakly buffered protein with strongly buffered R N A at the final pH, the resulting material is somewhat more heterogeneous than for a slow assembly. The material was examined in the analytical ultracentrifuge within 0"5 hour, but the actual reaction time is probably much shorter. It thus appears that a better product is formed with a slower reassembly. The sedimentation patterns also reveal another difference between the rapid and slow assembly with CCMV protein. With the rapid assembly, more of the non-encapsidated material sediments faster than the virus, compared to the slow assembly where more of the material has a smaller s20.w value than virus. (f) Subunit composition of the 3 S protein aggregate Above p H 5.5 the purified protein of CCMV exists in small aggregates sedimenting at 2.7 S, which for convenience we designate the "3 S" aggregate (Adolph & Butler, 1974). Below this critical p H value, the 3 S protein assembled into a shell that was identified as the capsid. Since no protein aggregates of a size intermediate between the 3 S form and the capsid were found, it seems probable that the 3 S protein is important ill the association reaction. The subunit composition of the 3S protein aggregate is therefore of interest. Our previous sedimentation equilibrium experiments (Adolph & Butler, 1974) showed a weight average of 1.5 to 1.8 subunits of the protein monomer (of molecular mass 19,400 daltons; Bancroft et al., 1971) in the 3 S aggregate. To define the nature of the 3 S protein more precisely, we have determined the number, weight and z-average molecular weights over a range of protein concentrations by high-speed equilibrium. These experiments were carried out at two characteristic p H values and at ionic strength 0.2 and 5~ pH 6.0, which is just above the threshold of p H for appreciable assembly of the 3 S protein into capsid and p H 7.5, where more than 90% of the protein is in the 3 S form (Adolph & Butler, 1974). The number and weight-average molecular weights are shown for the two p H values (Fig. 3) (the z-averages were not included since they are less reliable as data for any numerical analysis). At any given protein concentration the number, weight and z-averages went in descending order (the opposite of the expected behaviour for an aggregating system), suggesting that the aggregate was essentially monodisperse and that non-ideality was a bigger effect than aggregation. An attempt was therefore made to fit the data with the non-ideality equations: l[~]n,ap p =
1/~..o + (B/2)c
l/Y/w,ap~ = 1/5/w.o + Be,
354
K.W. 3"0
A N D P . J . G. B U T L E I %
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(b)
(c)
(d)
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2"0
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0-0
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I
I
0.2
0.4
0.6
0.0
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I
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0.2
0.4
0.6
Concentration (mglml) FIo. 3. Molecular weight averages for the 3 S protein aggregate a t p H 6.0 a n d 7.5, ionic s t r e n g t h 0.2 a n d 5~ a t different concentrations. Replicate experimental curves are shown as broken lines, while theoretical curves fitted to these b y a least-squares analysis are shown as solid lines (for details see text). (a) p H 6.0, ll~n; (b) p H 7.5, ~ n ; (c) p H 6.0, ll~w; (d) p H 7.5, dl~,.
where B is the second virial coefficient and c is the protein concentration. This procedure yielded the calculated curves shown as the solid lines in Figure 3, with the following values for -~/o and B (which were the same for both the number and weight averages): p H 6.0: 1~/o = 2.00 (=I=0.04), B = 0.34 (• p H 7-5: ~ o = 2.08 (-~0.03), B = 0"48 (• These calculated curves closely fit the data, in agreement with the hypothesis that the 3 S protein is essentially dimeric and the spread of molecular weights is due to non-ideality. Although the error in the z-averages makes these more difficult to fit numerically, their values are consistent with this interpretation. Since the 3 S aggregate remains a dimer as the p H of a protein solution is decreased from p H 7.5 to p H 6-0, where assembly into capsid is beginning to be appreciable, the low molecular weight protein aggregate which assembles into the shell is likely to be the dimer. 4. D i s c u s s i o n In this paper we discuss mild conditions for the direct reassociation of the isolated protein and RNA components of a simple, spherical virus, cowpea chlorotie mottle virus. Such physiologically plausible conditions include a p H near neutrality, ionic
REASSEMBLY
OF CCMV UNDER
MILD
CONDITIONS
355
strength 0.1 to 0.2, and a temperature around 25~ We believe that the identification of these conditions is worthwhile since only under such mild conditions can aspects of normal virus assembly be studied. Any reassembly requiring unphysiological temperatures, changes of ionic strength, or the presence of denaturing agents, clearly departs from the normal assembly of the virus at least in this respect and may not even follow the same pathway. The method for reassembly of CCMV described here differs in its properties from that described previously (Bancroft & Hiebert, 1967; Hiebert & Bancroft, 1969; Bancroft, 1970). This required relatively high pH, low ionic strength and the presence of divalent cations. Moreover, to obtain particles that resembled native CCM-~, this earlier technique was usually carried out at around 4~ whereas virus growth (and the reassembly described in this paper) occurs at temperatures of 20 or 25~ Other differences are that CCMV can now be reassembled at pH 5 or 6 to yield the properties of native virus, whereas previously at pH 6 an abnormal nueleoprotein was found which sedimented 10 S slower than the virus and was not very infective. The earlier method of reassembly had only yielded virus by dialysis from a salt concentration of 0.5 ~ or higher to a very low salt concentration (0.01 M-KCI, 0.01 MTris (pH 7.5), 5• -3 ~-MgC12). However we now find that reconstitution occurs normally at physiological ionic strengths of 0.1 or 0.2, although particles can still be formed at ionic strengths of 0.5 or 1.0. Finally, Mg2+ (or another polyvalent cation) was previously required, otherwise only a slightly infective, slowly sedimenting heterogeneous aggregate was formed. But with the present reassembly technique, no requirement is seen for the presence of Mg2+ or other such cations. Hence the direct reassembly under mild conditions that we describe is not merely a variant on the earlier reconstitution procedure but rather shows different properties which may reflect its distinct and probably more physiological nature. ~rom our previous study of the states of aggregation of CCMV protein (Adolph & Butler, 1974) we had speculated upon possible conditions for direct reassociation of the protein with CCM-~ RNA, which were at least plausible for the in vivo reaction. In particular, the observation of hexagonal arrays of CCMV protein subunits on electron microscope grids, without any species corresponding to such arrays in the free solution, led us to propose that there might be a nucleation barrier for protein aggregation which could be overcome by interaction of the disaggregated protein with the RNA to form virus particles. This hypothesis is supported by the hysteresis in going from the disaggregated state of the protein to the capsid (Jaerot, 1975). By supplying the protein in a non-equilibrium form (operationally, by reducing the pH of the protein solution upon interacting with the RNA) we were able to obtain an efficient RNA-dependent reassembly. Protein that is supplied to the RNA in this way may possibly be in a state closer to that of the freshly synthesized coat protein in a natural infection. The experiments described in this paper have not revealed any strong specificity of CCI~V protein for its own RNA. Indeed, a number of other plant virus RNAs and synthetic polynucleotides were encapsidated with similar efficiency to CCM-V RNA. While the ability to package the synthetic polynucleotides within the protein shell is hardly surprising, since they are all much smaller than C ~ I V RNA, the detail of the packaging of the larger heterologous RNAs is less immediately obvious. Part of the RNA of a spherical virus is intercalated into the protein shell from the inside and has the icesahedral symmetry of that shell, as seen in TYMV (Klug eta/., 1966) and,
356
K. W. A D O L P H AND P. J. G. B U T L E R
at higher resolution, in BSV (Harrison & Jack, 1975). The remainder of the R N A is less clearly ol~anized, but is packed in a highly hydrated state within the protein shell. Low-angle X - r a y scattering studies of brome mosaic virus, which is very closely related to CCMV, showed that this less organized R N A occurred mainly as a shell between radii of 40 A and 95 A (Anderegg et al., 1963). These measurements have since been confirmed by low-angle neutron scattering and the extent of the R N A penetration into the protein shell determined (Jacrot et al., 1976). Interestingly, the urn'elated wild cucumber mosaic virus has a protein shell of similar dimensions to t h a t of BMV, but there is no central hole in the R N A core (Anderegg et al., 1971), and such a progressive filling of the central hole is found with increasing I~NA sizes for a range of viruses (Fischbach & Anderegg, 1976). Moreover, the I~NA of wild cucumber mosaic virus is about 2-4 • 106 in molecular weight, compared to 1.0 • 106 for BMW or CCMV, and therefore larger than any of the heterologous RNAs employed in this study. Hence although the amount of R N A in intimate contact with the protein shell is probably constant in either the homologous or the heterologous RNA-containing particles, and possibly has a similar stabilizing effect on these particles, the excess R N A in the heterologous ones can readily be accommodated within the regular shell. This low specificity of CCMV reassembly is in marked contrast to the other virus assembly system t h a t has been investigated in detail, t h a t of tobacco mosaic virus. I n the case of TMV a strong specificity is sho~,~ for the R N A b y the "disk" of TMV protein and this specificity resides in a specific region of the R N A sequence (Butler & Klug, 1971 ; Zimmern, 1976). Another difference in the initial events of reassembly of CCMV and TMV is the absence of an aggregate of intermediate size, corresponding to the TMV protein "disk", with CCMV protein (Adolph & Butler, 1974). I n the absence of a strong specificity for CCMV, other local factors in the plant cell m u s t ensm'e t h a t the CCM-V protein subunits encapsidate mainly the proper I~NA molecules and these have yet to be described. We thank Dr B. Jacrot of the Institut Max yon Laue-Paul Langevin, Grenoble, France, for helpful discttssion. One of the authors (K. W. A.) was supported by a postdoctoral fellowship from the American Cancer Society. REFERENCES Adolph, I~. W. (1975a). J. Gen. Virol. 28, 137-145. Adolph, K. W. (1975b). J. Gen. Virol. 28, 147-154. Adolph, K. W. & Butler, P. J. G. (1974). J. Mol. Biol. 88, 327-341. Adolph, K. W. & Butler, P. J. G. (1975). Nature (London), 255, 737-738. Anderegg, J. W., Geil, P. H., Beeman, W. W. & Kaesberg, P. (1961}. Biophys. J . 1, 657-667. Anderegg, J. W., Wright, M. & Kaesberg, P. (1963). Biophys. J. 3, 175-181. Bancroft, J. B. (1970). Advan. Virus. Res. 16, 99-134. Bancroft, J. B. & Hiebert, E. (1967). Virology, 32, 354-356. Bancroft, J. B., Hiebert, E., Rees, M. W. & Markham, R. (1968). Virology, 34, 224-239. Bancroft, J. B., McLean, G. D., Rees, M. W. & Short, M. N. (1971). Yirology, 45, 707-715. Butler, P. J. G. & Klug, A. (1971). Nature New Biol. 229, 47-50. Durham, A. C. H. & Klug, A. (1971). Nature N e w Biol. 229, 42-46. Durham, A. C. I~., Finch, J. T. & Klug, A. (1971). N a t u r e N e w Biol. 229, 37-42. Fischbach, F. A. & Anderegg, J. W. (1976). Biochim. Biophys. Acta, 432, 404-408. FraenkeLConrat, H. & Williams, R. C. (1955). Proc. Nat. Acad. Sci., U.S.A. 41, 690-698. Harrison, S. C. & Jack, A. (1975). J. Mol. Biol. 97, 173-191. Hiebert, E. & Bancroft, J. B. (1969). Virology, 39, 296-311.
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I4iebert, E., Bancroft, J. B. & Bracker, C. E. (1968). Virology, 34, 492-508. Hung, P. P. & Ovcrby, L. R. (1969). Biochemistry, 8, 820-828. Jacrot, B. (1975). J. Mol. Biol. 95, 433-446. Jacrot, B., Pfeiffer, P. & Witz, J. (1976}. Phil. Trans. Roy. Soc. set. B, 276, 109-112. Kaper, J. lYl. & Geelen, J. L. M. C. (1971). J. Mol. Biol. 56, 277-294. Klug, A., Longley, W. & Leberman, R. (1966). J. Mol. Biol. 15, 315-343. Mahler, H. R. & Cordes, E. H. (1966). Biological Chemistry, H a r p e r a n d Row, New York and London. Roberts, J. W. & Steitz, J. E. A. (1967}. Proc. Nat. Acad. Sci., U.S.A. 58, 1416-1421. Verduin, B. J. M. & Bancroft, J. B. (1969). Virology, 37, 501-506. Zimmern, D. (1976}. Phil. Trans. Roy. Soc. set. B, 276, 189-204.
Studies on the Assembly o f a Spherical Plant Virus H I . t Reassembly o f Infectious Virus U n d e r Mild Conditions KENNETH W. ADOLPH~ AND P. J. G. BUTLER
Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge CB2 2QH, England (Received 2 July 1976) Mild solution conditions are described for the direct reassembly of a simple spherical vimm, cowpea chlorotic mottle virus (CCMV), from its isolated protein and RNA components. Previously the reconstitution of spherical viruses has only been accomplished in conditions which are clearly non-physiological. The reassembly proceeds efficiently near neutral pH, at ionic strength 0.1 or 0.2 and 25~ and requires the protein to be in a non-equilibrium state. Addition of Mg 2+ is not required. At pH 6, about 70% of the RNA is eneapsidated as infectious virus. The 3 S protein aggregate {which assembles into the capsid) is monodisperse and consists of dimers of the subunit. The material is indistinguishable from native CCMV by physicochemical and structural characterization and also shows similar infectivity to native CCMV in a dilution assay on cowpea plants. However, the specificity of the protein for its homologous RNA is not strong and a number of plant virus RNAs and synthetic pol)~mcleotides can be encapsidated with effieiencies comparable to that found for the CCMV RNA. 1. Introduction Many simple viruses have been reconstituted from their isolated protein and nucleic acid components, but the reassembly of spherical viruses has previously only been accomplished under conditions which are clearly non-physiological. For example, reassembly of infectious bacteriophage R17, containing the A-protein, requires the components to be mixed under denaturing conditions and renatured from 5"7 M-urea (Roberts & Steitz, 1967). Similarly, the reconstitution of bacteriophage Qfl was accomplished by renaturation from 8 M-guanidine hydrochloride (Hung & Overby, 1969). The previously reported reassembly of cowpea chlorotic mottle virus involved dialysis from a high salt concentration, and had to be carried out at a low temperature (Bancroft & Hiebert, 1967; Hiebert & Bancroft, 1969). Cucumber mosaic virus can be reversibly dissociated with lithium chloride (Kaper & Geelen, 1971). The reassembly of simple spherical viruses thus differs considerably from t h a t of the rod-shaped tobacco mosaic virus, which was the first virus to be shown to selfassemble from its isolated protein and nucleic acid components (Fraenkel-Conrat & Williams, 1955). Investigations of the states of aggregation of tobacco mosaic virus -~Paper I I in this series is Jeerer (1975). :~Present address: Department of Biochemical Sciences, Princeton University, Princeton) N.J. 08540, U.S.A. 23* 345
346
K. W. A D O L P H A N D P. J. G. B U T L E R
protein and of the control of polymerization into the protein helix (Durham et aL, 1971; Durham & Klug, 1971) have led to an understanding of the physiologically plausible conditions for the reproducible and rapid formation of complete virions (Butler & Klug, 1971). Physiologically significant assembly processes are likely to occur at p H values near neutrality, ionic strengths of 0.1 or 0.2, and temperatures of 20 to 37~ A n y reassembly which requires the presence and subsequent removal of denaturing agents or of high concentrations of salt at low temperature clearly departs from the normal assembly of the virus. I n this paper we discuss the reassembly of CCMVt under mild conditions of pH, ionic strength and temperature, which are plausible for the in vivo assembly. A preliminary account of these results has been presented (Adolph & Butler, 1975). This procedure for reassembly shows clear differences in its properties from the earlier procedure of Bancroft and his co-workers (Bancroft & Hiebert, 1967; Hiebert & Bancroft, 1969). 2. M a t e r i a l s a n d M e t h o d s
(a) Virus growth and purification Cowpea ehlorotic mottle virus was grown in cowpea plants (Vigna unguiculata (L.) Walp. var. Blackeye), and purified from the sap by polyethylene glycol precipitation followed by differential centrifugation. The purified virus was stored in sodium acetate, pH 5, ionic strength 0.1 at 4~ Concentrations of CCMV were estimated ushlg an extinction coefficient E~ ~ = 5.87 (Bancroft et al., 1968). (b) Preparation of protein Two methods were used to isolate CCMV protein from the purified virus. One procedure, which we had previously used in investigating the states of aggregation of the protein (Adolph & Butler, 1974), involving dialyzing CCM-V against 1 M-calcium chloride at pH 8 to disassemble the virus and precipitate its RNA. In this procedure, any residual, degraded RNA was removed by ion exchange chromatography using a small cohmm of DEAE-eel]ulose. A second method allowed the protein to be isolated more rapidly, by avoiding lengthy dialyses to eliminate the Ca 2+. Here the virus was dialyzed against 1 M-KC1, 0.01 ~-Tris, 10 -3 M-dithiothreitol (pH 7.5) for 2 h at 5~ and then the RNA was removed by sedimentation (Hiebert & Bancroft, 1967). This method was used to provide the very fresh protein which, as previously reported by Hiebert and Bancroft, yielded better reassembly of virus. Concentrations of CCMV protein were estimated spectrophotometrically using an extinction coefficient E~ ,~ = 1.2 (Hiebert et al., 1968). (c) Preparation of R N A CCMV RNA is only extracted in low yield with phenol, and so a procedure was used which involved dissociating the virus in SDS and then separating the RNA from the protein by sedimentation into a sucrose gradient (Adolph, 1975a). The virus was dissociated at a concentration of about 5 mg/ml in sodium acetate, pI-I 5"5, ionic strength 0.1 with 2% SDS for 3 to 5 rain at 50~ The mixture was layered on a 5% to 200/o sucrose gradient in 0.1 M-sodium acetate, 0.01 M-Tris (pI-I 7.5), 1% SDS, and centrifuged for 18 h at 20,000 revs/min and 20~ in a Beckman SW27 rotor. Fractions could either be collected to isolate the individual RNA species of the multieomponent CCMV genome, or, more frequently, the entire central portion of the gradient removed to recover the mixed RI~IA. The mixture t Abbreviations used: CCMV, cowpea chlorotio mottle virus; SDS, sodium dodecyl sulphate; BM'V, brome mosaic virus; BSV, tomato bushy stunt virus; TCV, turnip crinkle virus; TYM~r turnip.yellow mosaic virus; TMV, tobacco mosaic virus.
REASSEMBLY
OF CCMV UNDER
MILD
CONDITIONS
347
of the RNA species was precipitated with 2.5 vol. ethanol at --20~ for 18 h and resuspended in 0.1 m-sodium acetate, 0.01 M-EDTA, 0.01 ~-Tris, pH 7.5. The ethanol precipitation was repeated once or twice to remove residual sucrose, and the RNA was then taken up in an appropriate buffer and stored at -- 20~ The other plant virus RNAs were purified by extraction with phenol. For each virus, bentonite was added to the virus solution (5 mg/10 mg of virus), which was then made 2~/o in SDS. The mixture was shaken at room temperature for 20 rain with an equal volume of phenol (saturated with 0.01 ~-EDTA, 0.01 ~-Tris, pH 7-5), following which the aqueous, RNA-containing phase was separated from the phenol phase by centrifugation. After twice repeating the extraction procedure, the RNA was precipitated with ethanol and stored in an appropriate buffer at --20~ The CCMV RNA concentration was determined from an extinction coefficient E~~ ---- 33.84 (Bancroft et aL, 1968), while the extinction coefficients of the other RNAs were taken to be E~ % -~ 25-0. (d) Buffers The buffers used to obtain the required pH values were" formate from pH 3.5 to 3.75, acetate from pH 4-0 to 6"0, phosphate from p H 6.25 to 7.5, and Tris at p i t 8.0. Buffers were prepared to an ionic strength of 0.1 and made up to higher ionic strengths by the addition of 3 ~-KC1.
(e) Analytical ultracent~rlfugation Sedimentation coefficients, buoyant densities and molecular weights were measured with an MSE analytical ultracentrifuge mark II, equipped with an ultraviolet scanning system. A least-squares program was used to calculate the sedimentation coefficients from values of ln(r) against t, and the sedimentation coefficients were corrected to give values of S2o.w. The sedimentation coefficients were generally determined at 15,000 revs/min at 25~ The percentage encapsidation was estimated from the ratio of the absorbance (at 260 nm) of material sedimenting at the rate of virus to the total absorbance by the sample at the beginning of the centrifugation, with allowance made for radial dilution. (f) Electron microscopy A Philips EM300 electron microscope was used to examine samples of the reassembled virus. Grids were prepared by adding a drop of reassembled virus to a "holey" carbon grid, washing with several drops of water after adsorption for about 5 min, and staining with a drop of 1~/o (w/v) uranyl acetate in water. The particles that were photographed were in a film of stain over holes in the carbon.
3. Results (a) Reassembly of infectious virus Reassembly of CCM-V involves the co-aggregation of the protein and the R•A and can occur under mild conditions which are plausible for the in rive assembly. A high yield of reassembled virus can be obtained, for example, b y mixing a weakly buffered protein solution (at p H 7.5, ionic strength 0.2) with a strongly buffered R N A solution (at p i t 6-0, ionic strength 0.2) to give a final p i t near 6. An alternative procedure is to mix the protein and R N A at p H 7.5 and then dialyze to p i t 6. I n both cases, with a 4:1 weight ratio of protein-to-RNA, nearly 70% of the R N A is encapsidated as infectious CCMV. (The solution conditions for reassembly arc discussed in greater detail in the following section.) Under these conditions for the reassembly, the isolated CCMV protein exists at equilibrium primarily (over 90%) as a low molecular weight aggregate (Adolph & Butler, 1974), which is found to be a dimer (see below), while the remaining protein
348
K.W.
ADOLPH
A N D P . J . G. B U T L E R
(under 10~o at the total protein concentration employed) occurs as capsid. Recent potentiometric titration experiments (Jaerot, 1975) have demonstrated hysteresis through the pH region where the dimer aggregates into the eapsid. Direct mixing of the equilibrium protein and RNA led primarily to the formation of small amounts of material sedimenting at the rate of capsid and not of reassembled virus. A high yield of virus could be obtained, however, by taking advantage of the hysteresis and supplying the protein in the non-equilibrium form, obtained by rapidly reducing the pH. (b) Effect of conditions on reassembly The effect of varying the final pH of the reassembly solution at ionic strength 0.2 was studied. CCMV was reassembled with a 5 : 1 weight ratio of protein-to-RNA for 16 hours at 5~ Three classes of material were found: reassembled virus sedimenting at about 80 S, non-encapsidated RNA, and heterogeneous material which sediments faster than reassembled virus, and their proportions varied with pH (Fig. 1). At pH 6.0 almost 70~o of the material sediments at the rate of virus, and at pH 6.5 50% of the RNA was encapsidated. For lower pH values, even greater percentages of
90 O
~
~
E
,&. . . . . .
C
o
9
50 "\ /
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A"
\X \
2O I
;':
9 9
5"0
-
I
5"5
I
9 _J
6'0
"
9 I
6"5
_.__
b
I
7.0
7.5
pH
FIG. 1. Effect of varying the final p H of the reassembly solution upon the percentage of reassembled virus at ionic strength 0.2. --O--O--, Reassembled virus ( ~ 8 0 S); - - A - - A - - , non-encapsidated R N A (<<80 S); "" I " " I " ", rapidly sedimenting material (>> 80 S).
the optical density (at 260 nm) is found as virus-like material; by pH 5-0, nearly 90% of the material is in this form. But the interpretation of the results at pH 5.5 and below is complicated by the fact that the isolated protein itself aggregates into a shell below pH 5.5 (Adolph & Butler, 1974), and so in a reassembly at these low pH values the RNA may be being trapped by the aggregating protein rather than causing the assembly. At pH values of the final reassembly solution above pH 6.5, the percentage of reassembled virus plateaus at about 30~/o. The non-eneapsidated RNA varies from undetectable amounts at pH 5.0 to about 60~/o at pH 7.0 and above, while throughout the range of pH, approximately 13% of the material is in a form which sediments much faster than virus. The overall picture from these experiments
R E A S S E M B L Y OF CCMV U N D E R MILD C O N D I T I O N S
349
is that near neutral pH, at ionic strength 0.2, substantial co-aggregation of CCMV R N A with the protein occurs to form virus. Reassembly, as previously described, requh'ed low temperatm'es (4~ low ionic strengths (0.01 M-KCI, 0"01 M-Tris), and the presence of divalent cations (normally as 5 • 10 -3 M-MgC12) (Bancroft & Hiebert, 1967; Hiebert & Bancroft, 1969; Bancroft, 1970). The effect of these parameters was therefore tested under the present reassembly conditions. Reassembly at p H 6.0 with ionic strengths other than 0.2 was investigated and the results are shown in Table 1. Over the range from 0.1 to 1"0, ionic strength has little effect on the reassembly. The main difference is the slight decrease in the sedimentation coefficient, s20.w, with increasing ionic strength. A similar effect was found with native CCMV at p H 4.5 (Adolph, 1975b).
TABLE 1
Effect of solution conditions on reassembly of CCM V
pH 6, I = 0.1 pH 6, I = 0.5 pI-I 6, I = 1.0 pH 6, I = 0.2 25~ pH 6, I = 0.2 +0.01 ~-Mg2+ pH 5'75, I = 0.2 + 0"01 ~1-spermine
su0.w(S) of product
Percentage eneapsidation of RNA
81 76 72 76
53 58 57 60
7(.)
56
82
51
Reassembly was performed at 5~ (unless otherwise indicated) for 20 h. After reassembly at 25~ (pH 6.0, ionic strength 0.2), the percentage encapsidation and sedimentation coefficient, Seo.w, of the reassembled virus were the same as those for reassembly at 5~ (Table 1). A low, and unphysiological temperature is therefore not required for this reassembly. Although divalent cations need not be added to the reassembly solutions, any effect they might have was tested by adding 0-01 M-MgC12 or 0.01 M-spermine (which acted in a manner similar to Mg 2+ in reassembly, Hiebert & Bancroft, 1969) to the reassembly solutions. The results (Table 1) show no change in the yields of virus upon addition of Mg 2 + or spermine. The present reassembly therefore shows several clear differences from that previously described in not requiring high pH, low temperature, low ionic strength or the presence of divalent cations. (c) Properties of the reassembled particles The physical properties of reassembled CCMV are similar to those of native virus. The molecular mass of the reassembled virus, determined by sedimentation equilibrium in the analytical ultracentrifuge, was 4.5 • 106 daltons, the same, within
350
K . W . A D O L P H AND P. J. G. B U T L E R
experimental error, as native CCMV. The sedimentation coefficient, 82o.w, of reassembled CCMV was found to be around 80 S at low ionic strength (Tables 1 and 2), which is again similar to the sedimentation coefficient of native CCMV in the same conditions (Adolph, 1975b). I n preliminary experiments (Adolph & Butler, 1975) the reassembled product was less stable t h a n native CCMV in CsC1, and we had suggested t h a t this might be due to the lack of some annea]ing process. This has now been overcome b y using freshly isolated protein (see Materials and Methods) so t h a t the virus particles are reassembled within two to four hours of disassembly of the virus to isolate the protein. The b u o y a n t density of reassembled CCl~V in CsC1 was measured and found to be the same (to ~4thin 0.004 g/cm 3) as t h a t of native CGMV. The measurement of the b u o y a n t density was made b y centrifuging reassembled CGM-V and native virus to equilibrium in separate cells, but under identical conditions. Since the buoyant density depends upon the relative proportions of protein and R N A contained in the virus particles, the similarity of the b u o y a n t densities confirms t h a t the proportions of protein and R N A are the same in the reassembled and native virus particles. TABLE 2
Specificity of assembly with CCM V protein Final solution conditions pH 6, I ~-- 0"2 Time for assembly 0.5 h 20 h Encapsidation Encapsidation a~o.~(S) (%) ~o..(S) (%) CCMV RlqA BSV RNA TCV RNA TYMV RhTA TMV RNA poly(A) poly(C) 9poly(U)
75 76 76 67 105 77 69 65 70
60 64 56 41 25 25 70 51 62
81 80 80 85 114 87 62 70 74
57 60 53 45 21 32 72 44 48
pH 7"5, I = 0.2 20 h Encapsidation S~o.w(S) (%) 77 79 80 77 -76 65 54 67
23 27 14 27 -32 71 21 42
When examined in the electron microscope, reassembled CCMV shows the same general appearance and diameter as native CCMV, and also displays the characteristic 2-fold, 3-fold, 5-fold and local 2-fold views of the T ---- 3 surface lattice of native CCMV, as previously demonstrated (Adolph & Butler, 1975). l~urthermore, preparations of reassembled virus are substantially homogeneous, with few particles revealing an abnormal morphology. The infectivity of reassembled CCMV was compared to those of native virus and of the purified R N A in a dilution assay on cowpea plants. Both reassembled and native CCM~ produced systemic infections when applied at a concentration of 0.1 mg]ml, but not at 0.02 mg/ml. However, the purified RNA, which had been used in the reassembly, did not produce an infection when inoculated at 0.02 mg/ml (equivalent to virus at 0.1 mg]ml). Although these results should be considered preliminary, t h e y again confirm the similarity of the reassembled virus to the native CCM~.
REASSEMBLY
OF CCMV UNDER
I~IILD C O N D I T I O N S
351
(d) Stability of reassembledGGMV at high TH Particles of CCI~V undergo an abrupt structural transition as the pH of a virus solution is raised above pH 6.5 (Bancroft, 1970; Adolph, 1975b). The transition is observed at low ionic strength (ionic strength 0.2) as a decrease in the sedimentation coefficient, s20.w, of the virus particles from 83 S to 73 S. This behaviour of CCMu has been described as "swelling", since it is associated with an increase in the radius of the virus particles (Bancroft, 1970). Since the isolated protein shell disaggregates above pH 5.5 (Adolph & Butler, 1974), the swelling of CCMW only at a higher pH must reflect the stabilizing interaction of RNA with the protein subunits of the capsid. The sedimentation behaviour of reassembled CCMV at high pH therefore provides a sensitive test for the quality of the reassembly into the virus structure, though this is not essentially related to the specific CCM-V RNA (see below).
\
"~ o 85
\
g ~g g .--
\
75
7O
I
5
I
\
I
6
7
I
8
pH FIG. 2. Stability of reassembled CCM-V a t high pH, ionic s t r e n g t h 0.2. The points are plotted for the m a j o r b a n d of optical density (260 nm) which sediments near the rate for virus.
Samples of reassembled CCM-V were dialyzed against buffers in the range of pH from 5 to 8, ionic strength 0.2, 5~ for 3 hours. The sedimentation coefficients, s20.w, of the reassembled material were then measured, and are shown as a function of pH in Figure 2. Unlike the RNA-free capsid, reassembled virus does not disaggregate at higher pH values, but sediments at the rates appropriate for virus over the range of pH. Indeed, a decrease in the sedimentation coefficient of the reassembled virus is found, which is similar to that due to the "swelling" behaviour of native virus, and is also centered at pH 6.5, as it is with native CCMW. The product of this reassembly is therefore, not simply an RNA molecule trapped inside the protein shell, but rather interacting with the protein subunits similarly to the RNA in the native virus.
(e) The low specificity of reassembly with COMV protein The specificity of encapsidation with CCMV protein was tested by reassembly with various natural RNAs and synthetic polynucleotides. The natural RNAs were those of three other spherical plant viruses: tomato bushy stunt virus, turnip crinl~le virus and turnip yellow mosaic virus, and the rod-shaped tobacco mosaic virus. Three
352
K. W. ADOLPH AND P. J. G. B U T L E R
synthetic polynucleotides were studied: polyadenylic acid (poly(A)), polycytidylic acid (poly(C)) and polyuridylic acid (poly(U)). Reassembly was carried out with the final solution conditions pH 6, I -~ 0.2, 25~ where almost 70% of CCMV RNA is encapsidated (Fig. 1), and also with p H 7.5, I ~ 0.2, 25~ where 30% of CCMV RNA sediments as virus following reassembly. At the lower pH, the percentage encapsidation and sedimentation coefficient (S2o.w) of the reassembled material was determined at 0.5 hour after mixing weakly buffered protein at p H 7.5 with strongly buffered polynucleotide at pH 6, and at 20 hours after dialyzing to p H 6 a mixture of the protein and polynucleotide at p H 7-5. In each case the weight ratio of protein to RNA was 4 : 1. The results of performing the reassembly ~dth CCM~7 protein and the various natm'al and synthetic polynucleotides are summarized in Table 2. In these experiments CCMV protein does not show a strong specificity for CCMV RNA, all the natural RNAs and synthetic polynucleotides were encapsidated to a substantial extent and the percentages of the various encapsidated polynucleotides sedimenting at a similar rate to virus were comparable to the percentage of CCi~V RNA, particularly with a final solution pH of p H 6. For example, after 0.5 hour at pH 6, I ---- 0.2. 25~ 60~ of CCMV RNA was encapsidated, 64% of BSV RNA, 50% of TMV RNA and 62% of poly(U). There was little difference in the eneapsidation of the RNAs and synthetic polynucleotides after reassembly for 0-5 hour or for 20 hours. The reassembly reaction seems to be completed within 0.5 hour and may be even much sooner. Raising the final pH had a similar effect on the percentage encapsidation of heterologous RNAs and synthetic polynucleotides (except for poly(A)) to that found with CCMV RNA (Table 2 and Fig. 1). The sedimentation coefficients, S2o.w, of the products of assembly of the natural RNAs with CCMV protein (20 h, pH 6, I ---- 0.2) are close to that of native or reassembled CCMV. With TMV RNA an additional component sedimenting at about l l 0 S can frequently be resolved as previously reported by Verduin & Bancroft (1969). The preparations assembled with poly(A)), poly(C) and poly(U) have lower sedimentation coefficients, but these are still greater than that of the RNA-free capsid (52 S). With all the plant virus RNAs and synthetic polynucleotides, the reassembled material was found by electron microscopy to be in the form of spherical particles similar to CCMV. Hence in this reassembly with CCMV protein virus-like material is produced ~4th all of the polynucleotides. The different times allowed for reassembly at pH 6, I ---- 0.2 (0.5 or 20 h) made no qualitative difference to the results. However, the sedimentation coefficients with all the RNAs and synthetic polynucleotides after the shorter reassembly time averaged 70/0 less than after the longer time. With a final p H of 7.5 and after 20 hours reassembly, the sedimentation coefficients averaged about 90% of those found after 20 hours reassembly at p H 6, with the notable exception of that for the poly(A) nucleoprotein, although no reason is known for this anomaly. The RNA prepared from CCMV, BSV, TCV, TYMV and TMV for use in the assembly system is mainly intact, as judged by the sedimentation coefficients and sedimentation profiles. Since these heterologous RNAs are significantly larger than CCMV RNA (BSV RNA has a molecular weight of 1.65 x 106, while TMV RNA is 2.2 • l0 s) more of these heterologous RNAs must be encapsidated in each particle during the reassembly. Although there is no evidence for the location of this extra RNA, packing it into the shell will present no problem (see Discussion). The molecular weights of the synthetic polynucleotides were calculated from their sedimentation
REASSEMBLY OF CCMV U N D E R MILD CONDITIONS
353
coefficients using the formula s20.w ---- 1 • 10 -2 M~ (Mahler & Cordes, 1966). Thus the sedimentation coefficients, 82o.~, for poly(A), poly(C), and poly(U) of 9.3 S, 3"5 S and 5.3 S, correspond to molecular weights of 2.5 • l0 s, 4.3 • 10 a and 9.1 • 104, respectively. Hence 4 molecules of poly(A), 25 molecules of poly(C) or 12 molecules of poly(U) would need to be encapsidated in each particle to equal the 1.1 • 10 s daltons of RNA that is contained in a native CCMV particle. However, the lower sedimentation coefficients for the synthetic polynucleotide-containing particles than for those with CCMV RNA or the other plant virus RNAs suggest that less poly(A), poly(C) or poly(U) is contained in these reassembled particles. The sedimentation profiles of the products of assembly show that when the assembly is performed rapidly, by mixing weakly buffered protein with strongly buffered R N A at the final pH, the resulting material is somewhat more heterogeneous than for a slow assembly. The material was examined in the analytical ultracentrifuge within 0"5 hour, but the actual reaction time is probably much shorter. It thus appears that a better product is formed with a slower reassembly. The sedimentation patterns also reveal another difference between the rapid and slow assembly with CCMV protein. With the rapid assembly, more of the non-encapsidated material sediments faster than the virus, compared to the slow assembly where more of the material has a smaller s20.w value than virus. (f) Subunit composition of the 3 S protein aggregate Above p H 5.5 the purified protein of CCMV exists in small aggregates sedimenting at 2.7 S, which for convenience we designate the "3 S" aggregate (Adolph & Butler, 1974). Below this critical p H value, the 3 S protein assembled into a shell that was identified as the capsid. Since no protein aggregates of a size intermediate between the 3 S form and the capsid were found, it seems probable that the 3 S protein is important ill the association reaction. The subunit composition of the 3S protein aggregate is therefore of interest. Our previous sedimentation equilibrium experiments (Adolph & Butler, 1974) showed a weight average of 1.5 to 1.8 subunits of the protein monomer (of molecular mass 19,400 daltons; Bancroft et al., 1971) in the 3 S aggregate. To define the nature of the 3 S protein more precisely, we have determined the number, weight and z-average molecular weights over a range of protein concentrations by high-speed equilibrium. These experiments were carried out at two characteristic p H values and at ionic strength 0.2 and 5~ pH 6.0, which is just above the threshold of p H for appreciable assembly of the 3 S protein into capsid and p H 7.5, where more than 90% of the protein is in the 3 S form (Adolph & Butler, 1974). The number and weight-average molecular weights are shown for the two p H values (Fig. 3) (the z-averages were not included since they are less reliable as data for any numerical analysis). At any given protein concentration the number, weight and z-averages went in descending order (the opposite of the expected behaviour for an aggregating system), suggesting that the aggregate was essentially monodisperse and that non-ideality was a bigger effect than aggregation. An attempt was therefore made to fit the data with the non-ideality equations: l[~]n,ap p =
1/~..o + (B/2)c
l/Y/w,ap~ = 1/5/w.o + Be,
354
K.W. 3"0
A N D P . J . G. B U T L E I %
- -
2"0
~
ADOLPH
(o)
(b)
(c)
(d)
7==::
1.0
-g o.o "~
3.0 -
b "6
2"0
1.0
0-0
I
I
I
0.2
0.4
0.6
0.0
I
I
I
0.2
0.4
0.6
Concentration (mglml) FIo. 3. Molecular weight averages for the 3 S protein aggregate a t p H 6.0 a n d 7.5, ionic s t r e n g t h 0.2 a n d 5~ a t different concentrations. Replicate experimental curves are shown as broken lines, while theoretical curves fitted to these b y a least-squares analysis are shown as solid lines (for details see text). (a) p H 6.0, ll~n; (b) p H 7.5, ~ n ; (c) p H 6.0, ll~w; (d) p H 7.5, dl~,.
where B is the second virial coefficient and c is the protein concentration. This procedure yielded the calculated curves shown as the solid lines in Figure 3, with the following values for -~/o and B (which were the same for both the number and weight averages): p H 6.0: 1~/o = 2.00 (=I=0.04), B = 0.34 (• p H 7-5: ~ o = 2.08 (-~0.03), B = 0"48 (• These calculated curves closely fit the data, in agreement with the hypothesis that the 3 S protein is essentially dimeric and the spread of molecular weights is due to non-ideality. Although the error in the z-averages makes these more difficult to fit numerically, their values are consistent with this interpretation. Since the 3 S aggregate remains a dimer as the p H of a protein solution is decreased from p H 7.5 to p H 6-0, where assembly into capsid is beginning to be appreciable, the low molecular weight protein aggregate which assembles into the shell is likely to be the dimer. 4. D i s c u s s i o n In this paper we discuss mild conditions for the direct reassociation of the isolated protein and RNA components of a simple, spherical virus, cowpea chlorotie mottle virus. Such physiologically plausible conditions include a p H near neutrality, ionic
REASSEMBLY
OF CCMV UNDER
MILD
CONDITIONS
355
strength 0.1 to 0.2, and a temperature around 25~ We believe that the identification of these conditions is worthwhile since only under such mild conditions can aspects of normal virus assembly be studied. Any reassembly requiring unphysiological temperatures, changes of ionic strength, or the presence of denaturing agents, clearly departs from the normal assembly of the virus at least in this respect and may not even follow the same pathway. The method for reassembly of CCMV described here differs in its properties from that described previously (Bancroft & Hiebert, 1967; Hiebert & Bancroft, 1969; Bancroft, 1970). This required relatively high pH, low ionic strength and the presence of divalent cations. Moreover, to obtain particles that resembled native CCM-~, this earlier technique was usually carried out at around 4~ whereas virus growth (and the reassembly described in this paper) occurs at temperatures of 20 or 25~ Other differences are that CCMV can now be reassembled at pH 5 or 6 to yield the properties of native virus, whereas previously at pH 6 an abnormal nueleoprotein was found which sedimented 10 S slower than the virus and was not very infective. The earlier method of reassembly had only yielded virus by dialysis from a salt concentration of 0.5 ~ or higher to a very low salt concentration (0.01 M-KCI, 0.01 MTris (pH 7.5), 5• -3 ~-MgC12). However we now find that reconstitution occurs normally at physiological ionic strengths of 0.1 or 0.2, although particles can still be formed at ionic strengths of 0.5 or 1.0. Finally, Mg2+ (or another polyvalent cation) was previously required, otherwise only a slightly infective, slowly sedimenting heterogeneous aggregate was formed. But with the present reassembly technique, no requirement is seen for the presence of Mg2+ or other such cations. Hence the direct reassembly under mild conditions that we describe is not merely a variant on the earlier reconstitution procedure but rather shows different properties which may reflect its distinct and probably more physiological nature. ~rom our previous study of the states of aggregation of CCMV protein (Adolph & Butler, 1974) we had speculated upon possible conditions for direct reassociation of the protein with CCM-~ RNA, which were at least plausible for the in vivo reaction. In particular, the observation of hexagonal arrays of CCMV protein subunits on electron microscope grids, without any species corresponding to such arrays in the free solution, led us to propose that there might be a nucleation barrier for protein aggregation which could be overcome by interaction of the disaggregated protein with the RNA to form virus particles. This hypothesis is supported by the hysteresis in going from the disaggregated state of the protein to the capsid (Jaerot, 1975). By supplying the protein in a non-equilibrium form (operationally, by reducing the pH of the protein solution upon interacting with the RNA) we were able to obtain an efficient RNA-dependent reassembly. Protein that is supplied to the RNA in this way may possibly be in a state closer to that of the freshly synthesized coat protein in a natural infection. The experiments described in this paper have not revealed any strong specificity of CCI~V protein for its own RNA. Indeed, a number of other plant virus RNAs and synthetic polynucleotides were encapsidated with similar efficiency to CCM-V RNA. While the ability to package the synthetic polynucleotides within the protein shell is hardly surprising, since they are all much smaller than C ~ I V RNA, the detail of the packaging of the larger heterologous RNAs is less immediately obvious. Part of the RNA of a spherical virus is intercalated into the protein shell from the inside and has the icesahedral symmetry of that shell, as seen in TYMV (Klug eta/., 1966) and,
356
K. W. A D O L P H AND P. J. G. B U T L E R
at higher resolution, in BSV (Harrison & Jack, 1975). The remainder of the R N A is less clearly ol~anized, but is packed in a highly hydrated state within the protein shell. Low-angle X - r a y scattering studies of brome mosaic virus, which is very closely related to CCMV, showed that this less organized R N A occurred mainly as a shell between radii of 40 A and 95 A (Anderegg et al., 1963). These measurements have since been confirmed by low-angle neutron scattering and the extent of the R N A penetration into the protein shell determined (Jacrot et al., 1976). Interestingly, the urn'elated wild cucumber mosaic virus has a protein shell of similar dimensions to t h a t of BMV, but there is no central hole in the R N A core (Anderegg et al., 1971), and such a progressive filling of the central hole is found with increasing I~NA sizes for a range of viruses (Fischbach & Anderegg, 1976). Moreover, the I~NA of wild cucumber mosaic virus is about 2-4 • 106 in molecular weight, compared to 1.0 • 106 for BMW or CCMV, and therefore larger than any of the heterologous RNAs employed in this study. Hence although the amount of R N A in intimate contact with the protein shell is probably constant in either the homologous or the heterologous RNA-containing particles, and possibly has a similar stabilizing effect on these particles, the excess R N A in the heterologous ones can readily be accommodated within the regular shell. This low specificity of CCMV reassembly is in marked contrast to the other virus assembly system t h a t has been investigated in detail, t h a t of tobacco mosaic virus. I n the case of TMV a strong specificity is sho~,~ for the R N A b y the "disk" of TMV protein and this specificity resides in a specific region of the R N A sequence (Butler & Klug, 1971 ; Zimmern, 1976). Another difference in the initial events of reassembly of CCMV and TMV is the absence of an aggregate of intermediate size, corresponding to the TMV protein "disk", with CCMV protein (Adolph & Butler, 1974). I n the absence of a strong specificity for CCMV, other local factors in the plant cell m u s t ensm'e t h a t the CCM-V protein subunits encapsidate mainly the proper I~NA molecules and these have yet to be described. We thank Dr B. Jacrot of the Institut Max yon Laue-Paul Langevin, Grenoble, France, for helpful discttssion. One of the authors (K. W. A.) was supported by a postdoctoral fellowship from the American Cancer Society. REFERENCES Adolph, I~. W. (1975a). J. Gen. Virol. 28, 137-145. Adolph, K. W. (1975b). J. Gen. Virol. 28, 147-154. Adolph, K. W. & Butler, P. J. G. (1974). J. Mol. Biol. 88, 327-341. Adolph, K. W. & Butler, P. J. G. (1975). Nature (London), 255, 737-738. Anderegg, J. W., Geil, P. H., Beeman, W. W. & Kaesberg, P. (1961}. Biophys. J . 1, 657-667. Anderegg, J. W., Wright, M. & Kaesberg, P. (1963). Biophys. J. 3, 175-181. Bancroft, J. B. (1970). Advan. Virus. Res. 16, 99-134. Bancroft, J. B. & Hiebert, E. (1967). Virology, 32, 354-356. Bancroft, J. B., Hiebert, E., Rees, M. W. & Markham, R. (1968). Virology, 34, 224-239. Bancroft, J. B., McLean, G. D., Rees, M. W. & Short, M. N. (1971). Yirology, 45, 707-715. Butler, P. J. G. & Klug, A. (1971). Nature New Biol. 229, 47-50. Durham, A. C. H. & Klug, A. (1971). Nature N e w Biol. 229, 42-46. Durham, A. C. I~., Finch, J. T. & Klug, A. (1971). N a t u r e N e w Biol. 229, 37-42. Fischbach, F. A. & Anderegg, J. W. (1976). Biochim. Biophys. Acta, 432, 404-408. FraenkeLConrat, H. & Williams, R. C. (1955). Proc. Nat. Acad. Sci., U.S.A. 41, 690-698. Harrison, S. C. & Jack, A. (1975). J. Mol. Biol. 97, 173-191. Hiebert, E. & Bancroft, J. B. (1969). Virology, 39, 296-311.
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I4iebert, E., Bancroft, J. B. & Bracker, C. E. (1968). Virology, 34, 492-508. Hung, P. P. & Ovcrby, L. R. (1969). Biochemistry, 8, 820-828. Jacrot, B. (1975). J. Mol. Biol. 95, 433-446. Jacrot, B., Pfeiffer, P. & Witz, J. (1976}. Phil. Trans. Roy. Soc. set. B, 276, 109-112. Kaper, J. lYl. & Geelen, J. L. M. C. (1971). J. Mol. Biol. 56, 277-294. Klug, A., Longley, W. & Leberman, R. (1966). J. Mol. Biol. 15, 315-343. Mahler, H. R. & Cordes, E. H. (1966). Biological Chemistry, H a r p e r a n d Row, New York and London. Roberts, J. W. & Steitz, J. E. A. (1967}. Proc. Nat. Acad. Sci., U.S.A. 58, 1416-1421. Verduin, B. J. M. & Bancroft, J. B. (1969). Virology, 37, 501-506. Zimmern, D. (1976}. Phil. Trans. Roy. Soc. set. B, 276, 189-204.