Circular Dichroism Studies on Turnip Rosette Virus

Transcription

1 J. gen. Virol. (I978), 4 r, Printed in Great Britain 77 Circular Dichroism Studies on Turnip Rosette Virus ABIMBOLA O. DENLOYE,* R. B. HOMER,* AND R. HULLt * Department of Chemical Sciences, University of East Anglia, Norwich, U.K., and t John Innes Institute, Colney Lane, Norwich, U.K. (Accepted 5 May 1978) SUMMARY Circular dichroism studies show that the RNA of turnip rosette virus (TRosV), both within the particles and free in solution, has considerable base pairing. The protein of TRosV is characterized by a relatively high proportion of/?-structure and low proportion of a-helix. Swelling of the virus particles by raising the ph and removing divalent cations causes slight changes in the conformation of the RNA and some changes in the protein conformation. INTRODUCTION The particles of turnip rosette virus (TRosV) are isometric and probably consist of I8o identical protein subunits (mol. wt. 3I 30o) surrounding a single nucleic acid species of about 1.4 IO (Hull, I977a; R. Hull, unpublished observations). The virus particles appear to be held together by three types of bond, one involving divalent cations, one being ph dependent and one a salt link between protein and RNA (Hull, I977b ). If either the ph of the virus suspension is raised above neutrality, or the divalent cations are removed at acid ph, the virus particles remain salt stable. However, when the divalent cations are chelated at alkaline ph values the particles swell and, being held together by just the protein-rna links, become salt labile. Recently, Hull (i978) has shown that calcium is the divalent cation involved. Circular dichroism (CD) has been used to study the conformation of coat proteins and nucleic acids of viruses with isometric particles in only a few cases (e.g. Schubert, I969; Isenberg et al. 197I; Boulanger & Loucheux, i972; Incardona et al. I973; Piazzolla et al. I977). In this paper we describe observations on the conformation of TRosV particles and of protein subunits and RNA in the presence and absence of divalent cations at both acid and alkaline ph. METHODS TRosV was purified from Brassica rapa cv. Snowball plants, which had been infected for 12 to 20 days, using the method of Hull (I977 b) modified in that 0.2 M-sodium acetate buffer, ph 5"0, was used in the initial blending, o't M-acetate buffer, ph 5"0, was used to resuspend pellets and the ascorbic acid, K2HPO 4 and acidification to ph 4"8 were omitted. To prepare coat protein subunits and RNA, the virus was precipitated using polyethylene glycol 6ooo, the precipitate was collected by centrifugation (8ooo rev/min, io min, t Author to whom reprint requests should be addressed.

2 78 A.O. DENLOYE, R. B. HOMER AND R. HULL. (a)! (b) i! n mmlw i S 7 o X 120 i L (c) / l! (d) 120 I! I! VCavelength (nm) I t I I I Fig. I. CD spectra of TRosV protein subunits under different conditions. CD is expressed as mean residue ellipticity [0]. --, Measured values;..., values calculated from the analyses of secondary structure (Table I) using the reference spectra of Chen et al. (1974). (a) ph 5"o+Ca2+; (b) ph 5.o- Ca2+; (c) ph 8'25+Ca2+; (d) ph 8'25- Ca 2+. Sorval SS34 rotor) and resuspended in o.oi M-tris, 0-I M-NaC1, ph 8"25 (TN buffer). Na EDTA (ph 8"25) was added to give IO mm and the virus kept at room temperature for 3 min. Then an equal vol. of4m-lic1 (ph 8.25) was added and the solutionplaced at - 20 C for 16 h. After thawing, the precipitated RNA was collected by centrifugation (8o00 rev/min, IO min, Sorval SS34 rotor), washed twice in 2 M-LiC1, resuspended in TN buffer, precipitated using 2"5 vol. ethanol, resuspended in TN buffer, and dialysed as required. The protein-containing supernatant after adding LiC1 was dialysed extensively against o.oi i-tris, I'o M-NaC1, ph 8"25, and then dialysed as required. The LiC1 method gives protein and RNA with u.v. absorption spectra indicating that they are reasonably well separated (R. Hull, in preparation).

3 CD of turnip rosette virus 79 Table 1. Analyses of secondary structure of TRosV protein Integralt 208* method method c ---~-.... Conditions %c~ %c~ %fl %R Total% Protein subunits ph 5+Ca 2+ I4 Io I2o ph 5 -- Ca2+ 7 I I 2o ph 8.25+Ca z+ II IO Io9 ph Ca ~+ I2 I3 I Intact virus- RNA~ ph 5+Ca ~ ph 5-Ca o ph 8'25 + Ca 2+ ph 8"25- Ca I3 33 I I * Greenfield & Fasman (I969) method involving mean residue ellipticity at 2o8 nm. t Baker & Isenberg (1976) method employing an integration range of 2Ol to 243 nm. c = c~-helix, fl =/7-structure, R -- random or remainder structure. :~ From CD spectra of virus after subtracting contribution due to RNA. Samples of virus and nucleic acid were dialysed against four buffer systems: (a) o.oi M- sodium acetate, o-i M-NaC1, Io mm-cac12, ph 5"0, (ph 5+Ca2+); (b) o.oi M-sodium acetate, o. I M-NaC1, ph 5"o (ph 5- Ca2+); (c) TN buffer + Io mu-cac12 (ph 8"25 + Ca 2 ); (d) TN buffer (ph 8"25- Ca2+), Samples of protein were dialysed against the same buffer but using I.O M-NaC1 to prevent observable protein precipitation. Dialyses were performed at 4 C using four to six changes of buffer (each IOO to 5oo times the vol. of the sample) over a period of 48 to 72 h. Circular dichroism (CD) spectra were measured on a Cary 6I dichrograph calibrated with D-camphor-Io-sulphonic acid in accordance with the instrument manual. Virus and RNA were used at A2~0 = I and protein at A~ = I. The pathlength of the cells was varied according to the wavelength being measured. A mean residue weight of 1 I3 was used to calculate the mean residue ellipticity of the protein. In the analysis of the spectra, integration was performed by Simpson's rule at 3 nm intervals over the range 2oi to 243 nm using an I.C.L I9o 3 computer. RESULTS CD spectra of coat protein The CD spectra of TRosV coat protein subunits at ph 5.0 and 8-25 in the presence and absence of lo mm-cac12 are shown in Fig. I. It was not possible to measure the spectra of coat protein subunits with any reliability below 200 nm because of the I.o M-NaC1 necessary to prevent observable protein precipitation. Small Cotton effects ([0] ~< 5 o ) were observed between 250 to 30o nm. These could possibly be associated with near u.v. transitions of the aromatic amino acids; however they were not examined in detail. All the spectra exhibit only negative ellipticity and share the common features of a negative maximum at 200 to 2o2 nm and shoulder at 215 to 218 nm. The ph 5 spectra also have an additional pronounced shoulder at 230 nm. The short wavelength of the maximum suggests that the protein has a low helical content and a substantial amount of random or remainder structure; the 215 to 218 nm shoulder indicates some fl-sheet structure. The percentage of the a-helical, fl-sheet and remainder structures (Table I) were determined by the integral method of Baker & Isenberg 0976) 6 VIR 41

4 8o A. O. DENLOYE~ R. B. HOMER AND R. HULL i i i i i i I I I I.~ - ---~,....~._~ =~ '~ = ~ ~ ~ & I I! I I I I I I I Wavelength (nm) Fig. 2. CD spectra of TRosV particles and TRosV RNA at ph 5"o+Ca 2+. CD is expressed as specific ellipticity [~]. To obtain a direct comparison of the CD of the RNA and virus (2o ~ RNA) the spectra are plotted on different scales. Note also the different scales in the left and right hand portions of the spectra. --, virus;..., RNA. using reference spectra derived from proteins of known structure (Chen et al. I974). This method has recently been shown to be equivalent to an unconstrained least squares fitting procedure (Hammonds, I977). A comparison of the measured and calculated spectra in Fig. I shows that the position and ellipticity of the maxima are fairly well reproduced, whereas the other features are fitted less well. The results of this analysis (Table I) show that in all but one case the calculated totals depart significantly from Ioo %; this has also been found for several proteins by the originators of the method and will be discussed later in this paper. The c~-helix content, which is probably the most reliable parameter estimated by this method, is in moderate agreement with that calculated from the ellipticity at 2o8 nm by the method of Greenfield & Fasman 0969; Table I). In general the protein is characterized by a low s-helix content and a high proportion of remainder structure. It is evident from the spectra in Fig. I that changing either ph or calcium concentration perturbs the spectrum. The analysis (Table I) shows that the most substantial change accompanying the removal of calcium is the halving of the fl-structure content; this occurs at both ph values. Change in ph does not produce a consistent change in the proportions of any of the structural components but raising the ph is seen to eliminate the shoulder at 230 nm by increasing the negative ellipticity at this wavelength. CD spectra of RNA The CD spectrum of isolated TRosV RNA at ph 5 in the presence of calcium is shown in Fig. 2. No significant changes were produced by removing calcium ions or by changing ph. The wavelength of the positive CD maximum, 265 nm, is typical of an RNA with substantial base pairing (about 7o % ; Gratzer & Richards, I97~ ).

5 CD of turnip rosette virus 8I /.1 i * i i i i,! i i (a), o x 7 o,5 (c)! & (d) I I I I I I I I I I Wavelength (nm) Fig. 3" CD spectra of TRosV under various conditions. CD is expressed as specific ellipticity. (a) ph 5+Ca~+; (b) ph 5-Ca~+; (c) ph 8.25+Ca2+; (d) ph 8-25-Ca ~+. CD spectra of virus As the diam. of TRosV particles, about 30 nm (Horne et al. I977), is small compared with the wavelength of radiation used for CD spectra measurements, the spectra are relatively free from the distortions associated with light scattering (Gordon, I972) such as have been observed for viruses with rod-shaped particles (Vogel & Jaenicke, I974; Homer 6-2

6 82 A.O. DENLOYE, R. B. HOMER AND R. HULL I! i i! (a) I i!! (b) g r- 3 I00 20 (c) (d) 60 I! I I I I I! ~9 ~_0 2 Wavelength (nm) Fig. 4. CD spectra ( ) of TRosV protein in virus particles under various conditions compared with the calculated spectra (-... ). Calculations as in Fig. I. (a) ph 5+Ca2+; (b) ph 5-CaZ+; (c) ph Ca2+; (d) ph 8" 25-Ca ~+. & Goodman, I975). The CD spectra of the virus (Fig. 2 and 3) can be seen to be dominated by the RNA contribution (20 % by weight) in the 25o to 3oo nm region and by the protein contribution at shorter wavelength, I95 to 225 nm. In particles in the fully stable configuration (ph 5 + Ca2+) [Fig. 2 and 3 (a)] the intensity of the maximum of the RNA is nearly equal to that of free RNA, reflecting the small protein contribution, but the maximum is shifted by about 2 nm to the red. This probably indicates a small loss of base pairing (Gratzer & Richards, I970 on incorporation into the virion. Changing ph or removal of calcium does not cause any shift in the wavelength of the RNA maximum (Fig. 3) but removal of calcium does cause a reduction (about io%) in the specific euipticity at the maximum; this effect is slightly more marked at ph 8"25 than at ph 5. Thus, on the removal of calcium there is some reduction in the order (basestacking; Brahms et al. I967) of the RNA but not in the base-pairing. The similarity of the long wavelength spectra of the RNA in solution and in the virus validates the subtraction of the small RNA contribution in the I9o to 24o nm region to obtain the CD spectrum of the coat protein in the intact virus. The resulting spectra (Fig. 4) resemble those of the protein subunits shown in Fig. L but there is more variation in the position of the negative maximum (20o to 2o6 nm) and its ellipticity. Furthermore the 215 to 218 nm shoulder is not so prominent except at ph 5+Ca 2+ but the 230 nm shoulder

7 CD of turnip rosette virus 83 is present as a pronounced negative minimum under all conditions except ph 8"2 5 --Ca 2+. The analysis of the spectra by the Baker & Isenberg 0976) method again gives totals which differ significantly from loo % (Table I). The estimate for the a-helical content (Greenfield & Fasman, x969) shows the poorest agreement with the integral method for the ph 8"25- Ca 2+ spectrum. This spectrum, obtained under conditions where the virus is swollen (Hull, i977 b) has the negative maximum at the longest wavelength observed (206 nm); it shows the poorest visual fit with the calculated spectra. The reason for these spectral perturbations is not known. DISCUSSION The CD spectrum of the particles of TRosV in their most stable configuration shows properties which resemble those found by CD and other methods for several other viruses with isometric particles. The RNA in the virion has much secondary structure (basepairing), as has the RNA of brome mosaic virus (Incardona et al. 1973), chicory yellow mottle virus (CYMV; Piazzolla et al I977), bacteriophage U2 (Isenberg et al 1971) and bacteriophage MS2 (Thomas et al 1976). Considerable base-pairing has also been suggested (Schlessinger, I96O) from hyperchromicity data of southern bean mosaic virus which is in the same virus group as TRosV (Hull, 1977a ). CYMV is similar to TRosV in that its RNA shows a small degree of base unpairing which may facilitate protein-rna interactions on incorporation into the virion (Piazzolla et al 1977). Base-paired secondary structure of the RNA is not found in rod-shaped viruses such as tobacco mosaic virus (Tikchonenko, ~969) and potato virus X (Homer & Goodman, 1975). The protein in TRosV virions has a relatively low proportion of~-helix and a moderately high proportion of fl-structure. Other viruses with isometric particles also have proteins with low proportions of c~-helix and moderate to high proportions of fl-structure (Isenberg et al 1971; Thomas et al r976; Turano et al 1976; Piazzolla et al. 1977). In contrast, measurements on the proteins of viruses with rod-shaped particles indicate they have a relatively high proportion of c~-helix and low proportion of fl-structure (Schubert, 1969; Homer & Goodman, I975; Thomas & Murphy, 1975); the filamentous phage ft is an extreme example with approximately IOO % helix in the protein subunits (Marvin & Wachtel, 1975). It is interesting to speculate that these differences in proportions of basic structures might, in some way, be associated with the different shapes of the protein subunit needed to form rod-shaped or isometric particles. The removal of calcium from TRosV is characterized by some reduction in the base stacking of the RNA, such a change was not found in free RNA. The reduction in base stacking was greater at ph 8"25, under which conditions the virus particles became swollen. However, it is surprising that swelling of the virus particles, which involves an increase in hydrodynamic diam. of more than IO%, affects the CD spectrum of the RNA to such a limited extent. This suggests that although the RNA has considerable base pairing, there is enough flexibility in the remaining unpaired strands of RNA to allow the particles to expand. The data on the protein are more difficult to interpret mainly because of the difficulties in obtaining good spectra on isolated protein subunits and in evaluating the results. This is a problem common to all analyses of the CD of proteins. Whereas the CD spectra of the c~-helix as measured for polyamino acids and calculated from protein spectra are in reasonable agreement, there is greater divergence for the fl- and unordered structures. This is probably because there is more variability in the latter structures in proteins (Chen et al. 1974; Brahms et al. 1977).

8 84 A.O. DENLOYE, R. B. HOMER AND R. HULL Even though the CD spectra of TRosV protein were taken in high salt conditions, which in itself presented difficulties, it is still uncertain that aggregation of the subunits did not occur during the experiments. Among the reproducible features were the reduction in the proportion of p-structure on removal of calcium from free protein subunits, but this was not nearly so marked in the protein within the virions; also protein within the virions appears to have less a-helix than isolated protein subunits. A further feature of the protein spectra is the positive shoulder at 23o nm. This feature resembles one reported by Piazzolla et al. (I977) for CYMV and was interpreted as arising from the side chain of a tyrosine residue. Although this is a perfectly feasible assignment the shoulder is rather well reproduced by the calculated spectrum in Fig. 4(c), suggesting that it could originate from the peptide backbone itself. However the shoulder is ultimately assigned, Fig. I shows that in the protein subunits the ellipticity at 230 nm is a reflection of a conformational change associated with hydrogen ions but not calcium ion binding. In contrast, the changes in this feature in the protein moiety of the virus particles (Fig. 3) only occur under swelling conditions (ph 8.25-Ca2+); this suggests that the incorporation of protein subunits into virus particles partially locks their conformations. We thank Mrs Dawn B. Aldous for preparation of the virus and the Science Research Council for an equipment grant. REFERENCES BAKER, C. C. & ISENBERG, I. (I976). On the analysis of circular dichroic spectra of proteins. Biochemistry I5, BOULANGER, P. A. & LOUCHEUX, M. H. (I972). Conformational study of adenovirus type 2 hexon and fiber antigens. Biochemical & Biophysical Research Communications 47, I94-2oi. BRAHMS, J., MAURIZOT, J. C. & MICHELSON, A. M. (I967). Conformational stability of dinucleotides in solution Journal of Molecular Biology 25, BRAHMS, S., BRAHMS, J., SPACH, G. & BRACK, A. (I977). Identification of ~,/?-turns and unordered conformations in polypeptide chains by vacuum ultraviolet circular dichroism. Proceedings of the NationalAcademy of Sciences of the United States of America 74, CHE~, Y. H., YANk, J. T. & CHAN, K. T. (1974). Determination of the helix and/~ form of proteins in aqueous solution by circular dichroism. Biochemistry x6, 335o GORDON, D. T. 0972). Mie scattering by optically active particles. Biochemistry ix, o. ~RATZER, W. B. & VaCHARDS, r. O. (1971) Evaluation of RNA conformation from circular dichroism and optical rotary dispersion data. Biopolymers io, 26o GREENFIELD, N. & FASMAN, G. D. (I969). Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry 8, 4xo8-4I 16. HAMMONDS, R. G. (I977). Least-squares analysis of circular dichroic spectra of proteins. European Journal of Biochemistry 74, HOMER, R. a. & COODMAN, R. M. (I975). Circular dichroism and fluorescence studies on potato virus X and its structural components. Biochimica et Biophysica Acta 378, 296-3o4. HORNE, R. W., HARNDEN, J. M. & HULL, R. (1977). The in vitro crystalline formations of turnip rosette virus. I. Electron microscopy of two- and three-dimensional arrays. Virology 82, I5o-I62. mjll, R. (t977a). The grouping of small spherical plant viruses with single RNA components. Journal of General Virology 36, HULL, R. (I977b). The stabilization of the particles of turnip rosette virus and of other members of the southern bean mosaic virus group. Virology 79, HULL, R. (1978). The stabilization of the particles of turnip rosette virus. III. Divalent cations. Virology (in the press). INCARDONA, N. L., McKEE, S. & FLANAGAN, J. B. (I973). Non-covalent interactions in viruses: characterization of their role in the ph and thermally induced conformational changes in bromegrass mosaic virus. Virology 53, ISENBERG, H., COTTER, R. I. & GRATZER, W. B. (197 I). Secondary structure and interaction of RNA and protein in a bacteriophage. Biochimica et Biophysica Acta 232, I. MARVIN, D. A. & WACHTEL, r. J. (I975). Structure and assembly of filamentous bacterial viruses. Nature 253, I9-23.

9 CD of turnip rosette virus 85 PIAZZOLLA, P., GUANTIERI, V., VOVLAS, C. & TAMBURRO, A. M. (I977). Circular dichroism studies of chicory yellow mottle virus. Journal of General Virology 37, SCHL~SlNGER, D. (I960). Hypochromicity in ribosomes of Escherichia coli. Journal of Molecular Biology 2, SCHUBERT, D. (I969). Conformational changes during reversible depolymerization of the protein coat from bacteriophage ft. Biochimica et Biophysica Acta i88, I47-I54. THOMAS, G. J. & MURPHY, P. (I975). Structure of coat proteins of Pft and fd virions by Laser-Raman spectroscopy. Science i88, 12o5-12o7. THOMAS, G. I., PRESCOTT, B., MCDONALD-ORDZIE, P. E. & HARTMAN', K. A. (I976). Studies of virus structure by Laser-Raman spectroscopy. 2. MS2 phage, MS2 capsids and MS2 RNA in aqueous solutions. Journal of Molecular Biology lo2, Io3- I24. TmCHONENKO, J. L (t969). Conformation of viral nucleic acids in situ. Advances in Virus Research x5, 2ot-29o. TURANO, T. A., HARTMAN, K. A. & THOMAS, G. J. (I976). Studies of virus structure by Laser-Raman spectroscopy. 3. Turnip yellow mosaic virus. Journal of Physical Chemistry, I63. VOGEL, n. & JAEMCKE, R. (1974). Conformational changes and proton uptake in the reversible aggregation of tobacco mosaic virus protein. European Journal of Biochemistry 4I, 6o (Received 6 March t978 )