Proteins of cowpea chlorotic mottle, broad bean mottle, and brome mosaic viruses

VIROLOGY

47, 8-20 (1972)

Proteins

of Cowpea

Chlorotic Brome

HARI

Mottle,

Mosaic

0. ,4GRAWAL

Research Station,

AND

Canada. Agriculture, Awepted

Broad

Bean Mottle,

and

Viruses’ .J. H. TREK4INE Jhncouver,

B.C., Canada.

August 23, 1971

Molecular weights of virus protein subunits of cowpea chlorotic mottle virus (CCMV), broad bean mottle virus (BBhlV), and brome mosaic virus (BMV) were determined by means of polyacrylamide gel electrophoresis. Values of 19,200 for CCMV, 16,400 for BBMV, and 16,200 for BhlV were obtained. Smino acid compositions of the three viruses have been determined and compared with values reported earlier. Based on t,hese data CCMV, BBMV, and BMV were found to possess 179, 152, and 150 amino acid residues per protein subunit, respectively. The CCMV subunit on trypsin treatment gave one component with a molecular weight of 16,400 and 154 residues. Unlike CCMV in which only certain lysyl and arginyl bonds in the N-terminal portion of the protein are hydrolyzed by trypsin, BBMV did not show any selective response and some of the virus particles were completely digested under t.he same conditions. Thus the BBMV protein subunits are much more susceptible to tryptic action than those of CCMYV. Chymotrypsin t,reatment of BBMV hydrolyzed some virus particles completely while t.he protein of a small fraction was only partially hydrolyzed, suggesting the presence of peptide bonds susceptible to attack by enzyme in eit,her t.erminal of the molecule. Trypsin treatment of BMV produced at least four components. A stable spherical particle of 16 nm diameter (36 S) with an amino acid composition different from that of the virus and containing Sy;, R.NA was isolated after gel chromatography and density gradient centrifugation. However, a precipitate that formed during the digest.ion was found to cont,ain three components with molecular weights of 11,000, 9700, and 8800. Chymotrypsin treatment under similar condit,ions had no effect on this virus. INTRODUCTION

similar conditions BBMV gave a heterogeneous population sedimenting at 25 S and revealed disorganized material under the electron microscope (Bancroft et al., 1969). All three viruses were reported t.o be serologically distinct (Bancroft et al., 196s). A relationship among them has, however, recent,lg been found by H. A. Scott (personal communication). The addition of kypsin to CCRIV at pH 7.4 resulted in the complete hydrolysis of certain Iysyl and argingl bonds in the N-terminal portion of t,he protein molecule releasing 37 amino acid residues and was accompanied by disassembly of the virus (Chidlow and Tremaine, 1971). The remaining 80 %) of the protein molecule aggregated at pH 5.0 to form viruslike particles containing 8 cb R.NA. Chymotrypsin

Extensive studies have been made on the disassembly and renssemblv of cow-pea chlorotic mottle virus (CUM?), broad bean mottle virus (BBRIV), and brome mosaic virus (BlUV) (Hiebert et a.l., 1968; Wagner and Bancroft,, 19635). The three virusrs have similar disassembly and reassembly properties, and their coat, protein subunits can be mixed in various combinations to produce viruslike particles with mixed coat, prot#eins. BMV and CCXIV proteins aggregated at pH 5.0 in 0.2 31 NaCl to form a uniform 52 S pseudo-top (PT) component,, whereas under 1 Contribution No. 225, Research Station, Canada Department of Agriculture, 6660 N.W. Marine Drive, Vancouver 8, British Columbia.

8 Copyright

@ 1972 by Academic

Press,

Inr.

PROTEINS

OF CCMV,

under similar conditions cleaved 18 amino acids from the N-terminal of the CCMV protein. This cleavage did not alter the morphology of the virus particle, but the RNA content was decreased from 25 to 20%. The particles formed by the action of chymotrypsin were serologically identical with the virus. Similarities in the reassembly of CC&IV, BBMV, and BRW prompted us to determine their protein subunit molecular weights by polyacrylamide gel electrophoresis and to st,udy t,he effect of trypsin and chymotrypsin on BBMV and B&IV. The CCMV used here was the same isolate used by Chidlow and Tremaine (1971). The BBRIV and BMV isolates used showed similar properties as described by previous workers except that there were slight differences in the amino acid composition and the sediment,ation coefficients. MATERIALS

AND

METHODS

The BBRIV isolate was obtained from Dr. H. ,4. Scott, University of Arkansas, and the BMV isolate from Dr. J. T. Slykhuis, C.D.A., Ottawa. The two isolates were mul-

BBMV,

AND

BMV

9

t’iplied in plants of broad bean (T’icia fuba L. var. Exhibition Long Pod) and barley (Hordeurn vulgare L. var. Atlas), respectively. Infected plants were harvested about’ 10 days aft,er inoculation. The viruses were purified using sodium acetate buffer at, pH 5.0 as reported earlier (Chidlow and Tremaine, 1971), except that some of the material of low molecular weight was removed from the virus by chromatography on Sephadex G-200 equilibrated in 0.1 M acetate buffer, pH 5.0. The virus eluted from the column of Sephadex G-200 was pelleted, if necessary, by high-speed centrifugation and taken up in the acetate buffer at the required concentration. The virus was stored at 4” with 0.1 % chlorobutanol (1 , 1 ,1-t#richloro-2-methyl-2-propanol) added and was used as soon as possible after preparation. Virus concentrat,ion was det,ermined by absorbance at 260 nm using an E: “,y of 5.40 for BBMV and 5.08 for BMV, respectively. The average yield of virus was 400 mg/kg of leaves. Preparations at a concentration of 10 mg/ml showed a single symmetrical peak in the analyt.ical ultracentrifuge. Occasionally a trace of a second component sedimenting

FIG. 1. Electrophoresis of CCMV, BBM.V, and BMV proteins with marker proteins in 5’3, polyacrylamide gels in 0.1 df PO4 , pH 7.2 containing 0.1% SDS and O.lyO 2-mercaptoethanol. The gels in A were run for 105 min whereas in B they were run for 1‘20 min. Bands are from the top to the bottom of the gel: 1, bovine serum albumin (BSA), ovalbumin (OA), and TMV; 2, BSA, OA, and lysozyme (LYS) ; 8, CCMV 4, BBMV; 6, BMV; 6, CCMV, BBMV, and BMV; 7, CCMV; 8, CCMV after trypsin treatment; 9, CCM\ before and after trypsin treatment; 10, CCMV and BBMV; 11, CCMV and BMV; II, CCMV after trypsin treatment and BBMV.

10

AGRAWAL

AND

faster than the virus was found. Bockstahler and Kaesberg (1962) found a similar component with BRIV and assumed it to be a virus dimer. Polyacrylanlide gel electrophoresis in sodiunz dodecyl. sulfate (SDS). Gel electrophoresis in the SDS system was performed using essentially the techniques of Shapiro et aE. (1967) and Weber and Osborn (1969) with some modifications. Gels (5’%, 9 cm

TREMAINE

long) were prepared in glass tubes 10.5 cm long and 5 mm in diameter from the following solutions: Solution A: 10 g acrylamide and 0.3 g N , N’methylenebisacrylamide (Bis) dissolved in 100 ml of distilled water Solution B : 100 ml of 0.2 M sodium phosphate buffer pH 7.2 containing 0.2 g SDS and 0.2 ml N , N , N’ , N’-betramethylethylenediamine (TERIED)

TABLE AMINO

ACID COMPOSITION MOTTLE VIRUS

OF COWPEI CHLOROTIC (BBMV) PROTEIN .IND

1

MOTTLE VIRUS (CCMV) PROTEIN, BRO.ID BROME MOS\IC VIRUS (B&IV) PROTEIN

CChIV Amino acid

Chidlow and lYremaim (1971)”

Trypsin 5 S componente

BBbIV jancroft el al. (1968)

NR-

BhlV

9R i< 0.097’

Subunit molecular weight

12 2 7 10 16 15 17 i 9 20 2 19 1 i 16 5 -l 4/i

9 2 2 8 13 15 15 G 7 22 2 17 1 i 15 5 4 4k

12 2 9 11 17 16 16 i 10 25 2 19 1 i 16 5 4 4

123 16 98 109 80 144 145 i0 92 li9

12 2 10 10 8 15 14 7 8’ 18 2 18 2 6 14 3 5

12 2 10 11 8 14 14 i

9 17 2” 15 2 5 14 3 5

lil 17 55 147 30 50

11 2 9 11 8 1-l 14 i 8 18 2 18 2 ci 15 3 6

-

-

-

-

--

-

179 19,20(

154 lG,-iO(

1530 183 19,6oCI

152 lfi,4oC

154 16,GOCI

154 16,60(

Stubbs and FIaesbergh ww

IiRU

NR x 0.065’

152 37 159 125 132 153 232 79 135 405 --

10 2 10 8 9 10 15 5 9 26 1’ 14 2 6 13 4 4 2k

10 3 11 8 9 10 14 5 8 26 1 14 2 7 12 4 4 2

150 16, KM

150 16,200

I-

LYS His Arg Asp Thrb Serb Glu Pro Glyc Ala CYS Vald Met Ile Leu ‘W Phe Trp

BE.IN

213 32 9B 196 56 61 -i 263

-

a NR = nanomoles recovered. Averaged from two analyses of each of 24- and 72-hr hydrolyzates. b Extrapolat.ed to zero hydrolysis time. c Only 24-hr hydrolysis analyses included. d Only 72-hr hydrolysis analyses included. e Data reported by Chidlow and Tremaine (1971) in nanomoles X O.Oi99, figures rounded to integer value. Values for trypsin 6 S component were multiplied by 0.0643 and figures rounded. ’ Data reported by the authors in molar percentages X 1.58 and figures rounded to integer value. @Data reported by the authors in micromoles X 19.2 and figures rounded to integer value. * Data reported by the authors in molar percentage X 1.44 and figures rounded to integer value. i Figures rounded to integer value. i -, Not determined. k Assumed value.

PROTEINS

OF CCMV, BBMV,

Solution C: 1 g ammonium persulfate in 100 ml 0.1 :11 sodium phosphate buffer pH 7.2 Ten milliliters of Solution A were mixed with 10 ml of Solution B, and then 1 ml of Sol&on C was added to t,he mixture. The gel solution was mixed well, gently poured into the glass t#ubes, overlaid with about 0.2 ml of distilled water, and allowed to polymerize in presence of light at room temperature for 15-20 min. The tray buffer was 0.1 31 sodium phosphate, pH 7.2, containing 0.1% SDS and 0.1% 2-mercaptoethanol. Viral proteins were prepared by boiling the virus for 1 min in 0.1 41 sodium phosphate buffer, pH 7.2, containing 4 11 urea, 1% SDS, and 1% 2-mercaptoethanol. Glycerol was then added t#o50 %. The marker proteins were also prepared in a similar manner. Ten microliters of the above sample solution containing 1.5 mg/ml were layered under the electrode buffer. Electrophoresis was carried out at 6 mA per gel for 105 min at room temperature. Gels were stained overnight with Coomassie Brilliant Blue R250 (0.25 %) dissolved in 10 % trichloroacetic acid. Destaining was done by several changes in 10 % acetic acid over a period of 48 hr. Mobilities were usually compared for sets of 8 tubes run simultaneously with several proteins singly as well as in combination. The proteins used as markers were: bovine serum albumin (BSA, MW = 68,000), ovalbumin (OA, RIW = 43,000), tobacco mosaic virus protein (TRIV, MW = 17,500), and lysozyme (LYS, MW = 14,300). Enzymatic digestion of virus. Solutions of BBRIV and B&IV, at 10 mg/ml in 0.1 M sodium acetate buffer pH 5.0, were adjusted to pH 7.4 by overnight dialysis at 4” against 0.02 Ji TrisoHCl, pH 7.4, containing 0.1 31 NaCl and 1 X 10” M Cleland’s reagent (or 0.1% 2-mercaptoethanol). The required amount of freshly dissolved enzyme was added in solution (1.0 mg/ml in the dialysis buffer) and the reaction was followed by measuring the absorbance of the solution at 320 nm. Enzymatic reactions were at room temperature of approximately 23’.

AND B&IV

11

Chromatography on Sephadex G-200. A column of Sephadex G-200 (90 X 2.5 cm diameter) was equilibrat,ed in 0.1 M sodium acetate buffer, pH 5.0. The sample (20-40 ml) was applied to the column using an LKB peristaltic pump having a flow rate of 15 ml;!hr. The column n-as elut,ed wit#h t,he same buffer at the same flow rate. The

FIG. 2. Schlieren patterns of BBMV solut,ions before and after various treatments. (A) BBhWin 0.02 JI Tris.HCl pH 7.4 containing 0.1 M NaCl and 1 x 10-a .I1 Cleland’s reagent, before incubat.ion wit,h trypsin; virus concent,ration is 10 mg/ml. (B, top) Same as (-4) after incubation wit.h 0.2”, trypsin for 149 min. (B, bottom) Same as (B, top) after adjusting to pH 5.0. Sedimentation is from left. t.o right, pict.ure t.aken 16 min after reaching 35,609 rpm; bar angle 60”.

12

AGRAWBL

AND

effluent was monitored at 254 nm and collected in 15-ml fractions. Chrovnatography on Sephadex G-50. The digest mixture (10 ml, 10 mg/ml) was loaded on to a Sephadex G-50 column (40 X 2.5 cm diameter) using an LKB peristaltic pump. The column was then eluted with 0.1 J1 sodium acet,at,e buffer pH 5.0 at 15 ml/hr and the effluent monitored for absorbance at 254 nm by an LK3 Uvicord ultraviolet adsorptiometer. Elution wit,h distilled water (Chidlow and Tremaine, 1971) resulted in the adsorption of BBMV and BRIV. Fractions of 7.5 ml were collected, from which 0.5-ml aliquots were removed and mixed with 0.5 ml of Spinco amino acid analysis ninhydrin reagent in best tubes of 9 X 1 cm diameter. The mixture was heated in a boiling mater bath for 15 min and cooled for 10 min in a cold water bath. A mixture of 2.5 ml of equal parts of ethanol and water (v/v) was then added and absorbance at 570 nm was measured on a Beckman DU spectrophotometer. The nucleic acid content of the fractions was estimated from the ratio of absorbance at 230 nm and 260 nm. Electron ~vzicroscopy. Specimens were stained with 2% uranyl acetate at8 pH 5.0 or 1% potassium phosphotungstate at pH 7.0, and observed in a Philips ERI 200 electron microscope. Amino acid analysis. Analyses were performed on undegraded nucleoprotein and on the Sephadex G-50 isolated fractions. In both cases the solutions were made 6 N with respect, t.o HCI and sliquots were pipetted into 2 hydrolysis tubes. The tubes were evacuated, sealed, and hydrolyzed in an oven at, 107” for 24 and 72 hr. When nucleic acid was present there was usually some black insoluble material formed, which was removed by filtering with a fine sinteredglass filter before analysis. The hydrolyzates were generally light yellow. Excess HCl was removed by evapornt,ion i?a ~wz~o and t.he samples were dissolved in 5-10 ml of citrate buffer, pH 2.2. -4liquots of the hydrolyzates of from l-2 ml containing approximately 0.4 mg of amino acids were analyzed wi-ith a Spinco amino acid analyzer. The recoveries of amino acids were determined with the use of Spinco calibration mixture.

TREMAINE RESULTS

Protein Subunit Molecular Weight and A,vnino Acid Composition A comparison of molecular weights of protein subunits of CCMV, BBMV, and BMV in polyacrylamide gel elect,rophoresis gave values of 19,200, 16,400, and 16,200, respectively. The CCRIV-trypsin 6 S component run under similar conditions gave a value of 16,400. The relative movement of these proteins singly and in combination, and of the marker proteins is shown in Fig. 1. CCMV protein moved more slowly than the BBMV and BMV proteins. There was only a slight difference in t,he movement of BBMV and BRSV proteins, and when run toget,her they formed a wider band. The amino acid compositions of CCRZV, BBMV, and BRIV, and the CCMV-trypsin 6 S component are given in Table 1. The number of amino acid residues is based on the molecular weight determinations, and data reported by ot’her workers were recalculated on the same basis for comparison (Table 1). It is evident from the t#able that CCRIV has 179 residues while the 6 S component obtained aft,er the trypsin treatment has lTj4 residues. BBJIV was calculated to 2.01.6l.Bt.4 t *

1.2-

s L

1.w

2

0.6-

/

: 0.60.40.2-

FIG. 3. Absorbance at, 570 nm after reaction with ninhydrin, of fractions obtained from a trypsin digest. of BBMV, pH 5.0, by chromatography on Sephadex G-50 equilibrated in 0.1 111 acet.ate buffer, pH 5.0.

PROTEINS

OF CCMV,

BBMV,

AND

BMV

rapid initial decreasein absorbance followed by a more gradual decrease on addition of trypsin to CCMV (Chidlow and Tremaine, The Action of Trypsin on BBAW 1971) could be due to decreasein pH. Titration of the digest to pH 5.0 did not The addition of trypsin at a concentration of 0.2 % (w/w) of the virus concentration to alter the sedimentation rate of the virus solutions of BBMV in 0.02 M Tris*HCl, pH (75 S) ; however, before the addition of 7.4, cont#aining 0.1 111NaCl and 1 X 10” 211 trypsin the virus sedimented 5 S faster at Cleland’s reagent caused a decrease of opal- pH 5.0 than at pH 7.4. The quantity of a escenceindicating disassembly of some of the slower-sedimenting, heterogeneous componvirus particles. The reaction was followed by ent was greater at pH 7.4 than at pH 5.0 observing the decrease in absorbance at 320 (Fig. 2). Decrease in this material was acnm. The decrease was fast in t)he first few companied by an increase in absorbance at minutes but slowed as t’he pH of the digest 320 nm. The digest was chromatographed on decreased to 7.15. On adjustment of the pH a Sephadex G-50 column in 0.1 M acetate to 7.4 by the addition of 1.0 M TriseHCl, pH buffer pH 5.0. The ultraviolet absorption 7.5, the decrease in absorbance accelerated profile showed 3 peaks: the first, eluted in again and dropped to 66 cb of the original fraction 9 contained viruslike particles; the value in 2.5 hr. At this time the reaction mix- second and third contained RNA. After reture was adjusted to pH 5.0 by the addition action with ninhydrin (Fig. 3) 2 major peaks of 1.0 AI sodium acetate buffer pH 4.8. The were detected at 570 nm. The ultraviolet have 152 amino acid residues and BMV gave 150 residues.

TABLE

2

AMINO ACID COMPOSITION OF BRO.ID BE-IN MOTTLE VIRUS (BBMV) PROTEIN AND OF FRACTIONS OBT.UNED WHEN BBbIV W.YS DIGESTED WITH TRYPSIN AND CHYMOTRYPSIN AT pH 7.4 BBhIV

BBMV

-amino -kid

Molar yp

LyS His Al-g

8.04 1.05 (3.41 7.12 5.23 9.41 9.48 4.58 6.01 11.70

143 18 113 124 95 16B 166 80 103 205 -

11.18 0.98 3.59 9.Bl 1.96 3.66 __. 100.00

Asp Thrb Serb Glu Pro GIY’ Ala cys Vald Met Ile Leu Tyr Phe

+ trypsin

Fraction 9 NRa

Molar To

Fractions

BBMV 15-24

+ chymotqpsin

Fraction 9

Fractions

1623

NRa

hiolar y0

NRQ

Molar To

NRa

Molar To

8.10 1.02 G.40 7.03 5.38 9.41 9.40 4.53 5.83 11.62 -

199 23 154 166 128 218 212 106 127 279 -

8.58 0.99 6.64 7.16 5.52 9.40 9.14 4.57 5.48 12.03 -

188 24 147 lG0 112 213 217 102 132 260 -

8.2G 1.05 6.46 7.03 4.92 9.36 9.53 4.48 5.80 11.42 -

95 4 80 75 96 115 77 33 79 152 -

8.67 0.36 7.30 6.84 8.75 10.49 7.02 3.01 7.21 13.87 -

203 14 64 171 35 65 __~

11.50 O.i9 3.63 9.69 1.98 3.68

2i3 20 77 219 37 81 __

11.77 0.87 3.32 9.44 1.60 3.49 ~

266 20 83 219 46 87 -~

11.68 0.88 3.65 9.62 2.02 3.82

85 3 34 108 23 37

7.76 0.27 3.10 9.85 2.09 3.38

1765

100.00

2319

100.00

2276

100.00

1096

100.00

D NR = nanomoles recovered. Averaged from two analyses * Extrapolated to zero hydrolysis time. c Only 24-hr hydrolysis analyses included. d Only 72-hr hydrolysis analyses included. e Calculat.ed from Table 1.

of each of 24- and 72-hr hydrolyaates.

14

AGRAWAL

AND TREMAINE

and 570 nm profiles were similar to those obtained by Chidlow and Tremaine (1971) with CCRIV tryptic digest.s. Aliquots of fractions 15-24, comprising the second 570 nm peak were bulked and dried by flash evaporation. Amino acid analyses of these aliquots, the untreated virus, and fraction 9 showed similar compositions (Table 2). These results indicate that fract,ion 9 was composed mainly of unhydrolyzed virus. The presence of the small amount of slower sedimenting material (Fig. 2) was not detectable b,: amino acid analyses. Fractions 15-24 consisted of peptides from the complete hydrolysis of most, lysyl and arginyl bonds in the protein molecules of some of t,he virus particles. These result,s are different from those of Chidlow and Tremaine (1971) with CCRIV when addit#ion of trypsin resulted in the cornplebe hydrolysis of only certain lgsyl and arginyl bonds in the N-terminal portion of the protein molecule. The compositions: of BBMV reported by Yamazaki and Iiaesberg (1963) and by Miki and Knight (1963) are similar to the composition of our st,rain (Table 1).

3.W 2.62.62.42.22.0-

0.6. 0.60.40.2 L-4

Lyy 2

FRYmON14

L 2'2 ' 26

'1

Nlkll3ER

FIG. 4. Absorbance at 570 nm after reaction with ninhydrin of fractions obtained by chromaThe effect of chymotrypsin on BBMV was tography of a chymot,rypsin digest of BBMV, pH investigated under condit,ions similar t#o 5.0, on Sephadex G-50 equilibrated in 0.1 kf acetate t,hose described for t’rypsin. Chvmotrypsin at buffer pH 5.0.

The Actiotl oj Chytuotrypsin on BBMV

0.3%., caused a 15Yb decrease in absorbance at 320 nm in the first 4 min. Ahhough the pH remained constant the absorbance increased in 3 hr to 120 ‘ii. of the original value. Upon tit&on to pH 5.0 t.he digest became turbid and was chromatographed on Sephadex G-50 equilibrated in 0.1 JI acetat#e buffer pH 5.0. The elution profiles at 254 nm and at. 570 nm after react,ion with ninhxdrin (Fig. 4) were similar t.o t,hose obt,ained with trypsin. The amino acid analyses of fract,ion 9 and fractions l&23 (Table 2) show that t,he mola,r percent,ages of amino acids for fract,ion 9 are quitme similar to t.hose of the corresponding fraction of t,he t#rypsin digest and the untreated virus. The analvsis of t#he peptide fractions 16-23 showed significant differences from fract,ion 9 and the untreated virus. It did not’ resemble eit,her a limited digest,ion as in the case of CCRIV (Chidlow and Tremaine, 1971) or a complet,e digestion as wit,h trypsin (Table 2).

Examination in the analytical ultracentrifuge of the unreacted BBMV, and the chymotryptic digest at pH 7.4 and at 5.0 gave results similar to those with trypsin (Fig. 2). From G-50 chromatography of the tryptic digest in 0.1 ilf acetate pH 5.0, fraction 9 contained only virus sedimenting at SO S (Fig. 5) but, from the chymotryptic digest fraction 9 contained virus (80 S) and a small quantity of a 66 S component. The Action of Trypsin on BMV The addition of trypsin t’o solutions of BMV under conditions similar to those described for BBMV also caused a decrease in opalescence indicating disassembly of some of the virus particles. The decrease in absorbance at 320 nm was fast in the first few minutes and then slowed down slight,13 with a decrease in pH. On adjusting the pH

PROTEINS

OF CCMV, BBMV,

AND BMV

15

FIG. 5. Schlieren pattern of BBMV fraction 9 obtained after incubation with chymotrypsin (top) and trypsin (bottom) and chromatography on Sephadex G-50 column equilibrated with 0.1 Af acetate buffer pH 5.0. Sedimentation is from left to right, picture taken 1Gmin after reaching 35,600 rpm; bar angle 60”. FIG. 6. Schlieren pattern of BMV solutions before and after trypsin treatment. Top: BMV in 0.02 df Tris.HCl pH 7.4 containing 0.1 III NaCl and 1 X 10-Sdl Cleland’s reagent after incubation with trypsin for 83 min and then adjustment to pH 5.0. Bottom: BMV at pH 7.4 just, before adding trypsin (dilution l/3, 5.5 mg/ml). Sedimentation is from left to right, picture taken 1F min after reaching 35,606 rpm; bar angle 60”.

to 7.4 the decrease in absorbance accelerat,ed again and dropped to 76 ‘% of the original value in 83 min. At t,his time the reaction mixt,ure was adjusted t,o pH 5.0 by the addition of 1.0 III sodium acetate buffer pH 4.8. Examination of t,his digest in the analytical ultracentrifuge showed 3 peaks (Fig. 6). The fastest component sediment.ed at t,he same rat.e as the untreated virus; the others at 57 S and 36 S. A precipitate appeared and was removed by a low speed centrifugation of 15 min at 10,000 rpm in a Spinco Model L, then the supernatant was chromatographed on a Sephadex G-50 column in 0.1 M acetat,e buffer, pH 5.0. Elution profiles at 260 nm resembled the BBiIIV tryptic digest. Two peaks, fraction 12 and fractions 23-26, mere found at 570 nm after reaction with ninhydrin (Fig .7). Fractions 23-26 containing the pept,ides were pooled and dried by flash evaporation. Amino acid analyses of aliquots of fractions 23-26 (Table 3) indicated that the composition of the peptides differed greatly from the unt,reat,ed virus. Examinat,ion of fraction 12 in the elect,ron microscope and the

analytical ultracentrifuge showed particles of 25 nm and 16 nm diameter and 2 components sediment,ing at 78 S and 36 S (Fig. 8). After concentrat,ing these components by centrifugation at 38,000 rpm for 3 hr, they were separated by density gradient centrifugation. Amino acid analyses after dialysis demonst#rated that the 78 S particles (20% RNA) were unreact#ed virus but the molar percentages of arginine, threonine, and methionine in the analyses of the 36 S component (6 % RNA) were much lower than those in the virus analysis (Table 3). The molar ratios of arginine, threonine, alanine, and met,hionine were higher in the peptide fraction than in the virus (Table 3). A portion of t,he pelleted precipitate obtained from the digest above was dissolved in 2 ml of 12 N HCl. Aliquots were hydrolyzed for amino acid analyses. The amino acid composition of the precipitate was different, from t.hat of the untreat,ed virus, the peptides, or the 36 S component. Another portion of this precipibate was dissolved by boiling for 1 min in 0.1 d1 sodium phosphate

16

AGRAWAL

3-o2.62.e 2.4. 2.22.0. u

1.B

ii <

16

2

I.6

AND TREMAINE

BRIV at pH 7.4 caused no significant. decrease in absorbance at 320 nm. No peptides were recovered after chromatography on Sephadex G-50. Examination of B&IV at pH 7.4 (Fig. 9, bottom) and pH 5.0 (Fig. 9, top) aft#er the addition of chymotrypsin showed homogeneous virus components sedimenting at 74 and 78 S, respectively. Sedimentation rates of BMV at pH 7.4 and pH 5.0 before the addition of chymotrypsin were similar. These resulbs demon&rated that. BMV was not altered by chymotrypsin. DISCUSSION

%

A comparison of the properties of CCRIV, BBRLV, and BMV (Table 4) shows bhat in l.Otheir physical and chemical characteristics 0.6the three viruses have many similarities, but, their amino acid compositions (Table 1)) 0.6. isoelectric points, and molecular weights of their subunits are different. However, these 04 different subunits can combine with RNA 0.2to form infectious viruslike particles wilth mixed protein coats (Wagner and Bancroft, 2 6 L.-.,---l.--T ... & 10 I 1441. '18 22, ' 26I b--T-30 196s). The three viruses have recently been FRACTION NUMBER found to be serologically related (H. A. Fro. 7. Absorbance at, 570 nm after reaction Scott,, personal communication). These with ninhydrin, of fractions obtained by chromafindings imply a certain degree of homology tography of a trypsin digest of BMV adjusted to in t,heir subunits ahhough the degree of pH 5.0, on Sephadex G-50 equilibrated in 0.1 1cf similarity necessary for the formation of acetate buffer pH 5.0. mixed prot,ein coats is not known. The molecular weight of the protein subbuffer, pH 7.2 containing 1% SDS, 4 III urea units, based on amino acid composition and and 1% 2-mercaptoethanol, then subjected to electrophoresis as before. Three com- fingerprinting were 19,600 for CCRIV, ponents wilth molecular weight,s of 11,000, 20,500 and 20,900 for BBRV, and 20,000 9700, and 8800 were found. When digests for BiUV (Table 4). Employing the polyacrylamide gel electrophoresis technique were maintained at pH 7.4 for 23 hr, larger values of 19,200, 16,400, and 16,200 were quantities of precipitate were formed; found for the protein subunit,s of CCRIV, neither 36 S component nor residual virus BBMV, and BMV, respectively. The rewere detectable in t.he analytical ultraliability of this technique has been estabcentrifuge. lished (Dunker and Rueckert, 1969; Weber When several other digests were examined in the analytical ultracentrifuge at, pH 7.4 and Osborn, 1969; Lesnaw and Reichmann, and after adjustment t’o pH 5 t#here were 1970). Lesnaw and Reichmann (1970) relarger quantities of the 36 S component at port,ed a value of 18,000 based on gel pH 5 t,han at pH 7.4 but larger quant,ities of elect,rophoresis for BMV coat protein subslowly sedimenting material at pH 7.4 t,han unit,. The molecular weights obtained in our at pH 5. These results indicated an assembly stud? are supported by the results of CCRIV of 36 S component from the slowly sediment,- trypsm treatment. The CCMV subunit of 19,200 with 179 residues, on t,rypsin t,reating material upon pH 5 adjustment. merit gave a protein with a molecular weight TIM Bct,ion of Chymotrypsin on BMV The addition of chymotrypsin (0.2%) to of 16,400 (154 residues). Thus t.rypsin Yo2

PROTEINS

OF CCMV, TABLE

BBMV,

AND

17

BMV

3

AMINO ACID COMPOSITION OF BROME ~~0s.i~ VIRUS (BMV) PROTEIN AND OF FRXTIONS WHEN BMV W-is DIGESTED WITH TRYPSIN IT pH 7.4 BMV Amino acid

BMV + Molar yoe

36 S component of Fraction 12 NR%

Molar To

-

trypsin Precipitate

Fractions 23-26 NR’

OBTMNED

Molar yo

NR*

Molar ‘%

Lys His .4rg .4sp Thrb Serb Glu Pro Gly” Ala cys

6.72 1.64 7.03 5.52 5.83 6.76 10.25 3.49 5.97 17.90 -

149 41 66 131 94 165 231 74 143 355 --

7.15 1.96 3.19 6.30 4.55 7.92 11.14 3.56 6.89 17.09 -

44 0 413 61 127 36 63 0 42 273 -

3.78 0 35.51 5.24 10.91 3.10 5.42 0 3.61 23.47 -

62 ‘23 53 74 59 72 118 48 65 191 -

5.41 2.01 4.62 6.45 5.14 6.28 10.29 4.19 5.67 16.65 -

Vald Met Be Leu TS’ Phe

9.41 1.41 4.24 8.66 2.47 2.70

189 17 90 199 69 64

9.09 0.82 4.34 9.57 3.29 3.08

13 51 20 10 6 4

1.12 4.38 1.72 0.86 0.51 0.3-l __

126 5 41 126 40 45

10.99 0.44 3.57 10.99 3.49 3.92 -~

100.00

1163

100.00

1148

100.00

100.00

207:

(1NK = nanomoles recovered. Averaged from two analyses b Extrapolated to zero hydrolysis time. c Only 24-hr hydrolysis analyses included. d Only 72-hr hydrolysis analyses included. c Calculated from Table 1.

cleaved 25 residues from the protein subunit, and we have verified this value (unpublished results) by a quantitative recovery and amino acid analysis of CCMV tryptic peptides. The estimation of 37 residues (Chidlom and Tremaine, 1971) released upon addition of trypsin to CCMV is therefore incorrect. The number of amino acid residues in the subunits of CC&IV, BBRLV, BRIV, and trgpsin-treated CCIJIV were calculated (Table 1) t,o correspond with our molecular weight determinations. The results of analvses of two strains of BRIV are similar and ‘indicate only 3 amino acid exchanges. Similarly! the three strains of BBlUV show 3 to 4 amino acid exchanges. Comparison of BBMV, BRIV, and trypsin-treated CCRIV indicate 16 to 1s amino acid exchanges.

--

of each of 24 and 72-hr hydrolyzates.

We attempted to establish regions of homology between the proteins of CCRIV, BBRIV, and BMV by treating these viruses with t,rypsin and chymotrypsin. Our studies indicated that trypsin either completely hydrolyzed t,he protein of some BBMV particles or did not alter them. Chymotrypsin also completely hydrolyzed some of the virus particles, but t,here was an indication of the formation of a small amount of an intermediat,e which was less sensitive to chymotryptic action and also a small amount of precipitate formed during the digestion. The quantities of the intermediate were too small for further study. Although chymotrypsin did not hydrolyze BlUV, treatment with trypsin yielded many products. Three components sedimenting at 36 S, 57 S, and 78 S were observed in varying amounts in different digests. In addition a

AGRAWAL

AND TREMAINE

FIG. 8. Schlieren pattern of BMV fraction 12, obtained after incubation with trypsin. The virus was incubated with 0.2yfi (w/w) trypsin for 83 min, and the digest after adjustment to pH 5.0 and a low speed centrifugation, was chromatographed on Sephadex G-50 column in 0.1 M acetate buffer pH 5.0. Sedimentation is fromleft to right, picture taken 12 min after reaching 35,600 rpm; bar angle 60’. FIG. 9. Schlieren pattern of BMT’ solutions after chymotrypsin treatment,. Top: B&K in 0.02 M Tris.HCl, pH 7.4, containing0.1 IIf NaCl and 1 X lO+ M Cleland’s reagent after incubation with chymotrypsin for 5 hr and then adjustment to pH 5.0. Bottom: BMV at pH 7.4 before titration to pH 5.0. Sedimentation is from left to right, picture taken 10 min after reaching 35,600 rpm; bar angle 60”. large amount of precipitate, containing components with molecular weights of 11,000, 9700, and 8800 formed during digestion. The amino acid composition of this precipitate was different from t#he untreated virus (Table 3). Similarly, the amino acid compositions of the 36 S component and the peptides (fractions 23-26, Table 3) were also different from the untreated virus and from each other. The peptides were apparently produced not only by the formation of the 36 S component but also by the production of the three components of the precipitate mentioned above. Hence this technique for locating sequences similar to CClUV is complicated by the number of products formed. Stubbs and Kaesberg (1964) reported the formation of a precipitate upon titration of a tryptic digest of urea denatured BMV protein to pH 3.3. The amino acid composition of this precipitate had lower proportions of lysine, arginine and isoleucine and higher proportions of valine, leucine, tyrosine and phenylalanine than the virus. The composition of the precipitate obtained upon digestion of whole virus in our study re-

sembles that obtained by Stubbs and Kaesberg. after the release of 25 residues from CCMV, the remaining protein has about the same subunit molecular weight as that of the other two viruses. It is possible that these three viruses originated from a common ancestor either simultaneously or independently by a deletion of a part of the protein chain. Hence a part of the coat protein genome could have been deleted in the course of evolution. The addition of approximately 75 nucleotides to the coat prot,ein genome of a common ancestor is also possible. Lane and Kaesberg (1971) found that all 3 RNA molecules x&h molecular weights of 1.09 X 106, 0.99 X 106, and 0.75 X lo6 are essential for infectivity of BMV. These RKAs are found in 3 types of virions and the smallest RNA contains the coat protein gene. If the three viruses are derived from a common ancestor, CCMV and BBMV should also have multiple genetic components allowing the viruses t,o adapt t,o environmental variation by selecting components from a pool. A complete amino acid sequence of the

PROTEINS

OF CCMV, BBMV,

19

AND BMV

TABLE 4 PROPERTIES OF COWPEA CHLOROTICMOTTLE VIRUS (CCMV), BROAD BEAN MOTTLE VIRUS (BBMV) AND BROME hiowc VIRUS (BMV) BBMV

BMV

260 bi (wit,h a central hole 110 .< in diameter)

228 5.400

260 Kc (with a central cavity 80 d in diameter) 86b 87m (at pH 6); 78” (at pH 5.0) 79” (at pH 7) ; 74n (at pH 7.4) 21.46 5.08h

1.7p 4.6 X lo6 (normal)O 4.4 X 10” (swollen)a 1.1 x 106Q

2.1g; 2.op 5.20 X 1O’Q

1.76; 1.8~ 4.6 x loch

1.1 x 1068

1.0 x 1066

3; 23, 18, and 13 Sa

2; 17 and 10 S”

3; 27, 22, and 14 Sd

19,600”; 19,200”

20,500”; 20,900’; 16,400”

20,OOOb.c; 16,200”

l&3-, lslj,

194’, 1906, 152”

189”, 150”

180’”

180”

18Ob

1.382”

1.363”

1.36la

3.6Q Tyrosinea’ i Probably acetylatedp

5.5’ rllanineBB f None6 (probably acetylated)

7.96 -4rgc -

Virus

CCRIV

Particle size, average diamet,et

~20(1 m&ml)

87”~ j (at pH 5.0)

Percentage RNA absorbancy index (OD 260 cm2/md OD 260:280 ratio Molecular weight, virus Molecular weight, RNA/ virus particle No. of pieces of RNA and their sedimentation rate Molecular weight, protein subunit Total number of amino acid residues per subunit Number of structural protein subunits/particle Average density of virus particles (g/ml) Isoelectric point I* = 0.1 C-terminal amino acid N-terminal amino acid

77asj (at pH 7.07.3) 24a 6.00

179”

a Bancroft et al. (1968). b Bockstahler and Kaesberg (1962). c Stubbs and Kaesberg (1964). d Bockstahler and Kaesberg (1965). e Yamazaki and Kaesberg (1963). f Miki and Knight (1965). QTamazaki et al. (1961). * Kodama and Bancroft (1964).

t,hree viruses would establish whether the 25 amino acids mere lost or gained from eit,her end of the protein molecule or from different regions during evolution. It is also possible that some amino acid residues were cleaved from the protein subunits of BBMV and BJIV by proteolytic enzymes in ho. ACKNOWLEDGMENTS Our thanks are due to our Director, Dr. R. E. Fitzpatrick, for his interest in the problem, and to the National Research Council of Canada for

849 80n (at pH 5.0) 75” (at pH 7.4)

i Finch and Klug (1967). j Chidlow and Tremaine (1971). k Bancroft et al. (1967). 1 Semancik (1966). m Incardona and Kaesberg (1964). * This work (cysteine and tryptophan determined). p Our unpublished resu1t.s.

were not

financial support to one of us (H. 0. Agrawal). The authors gratefully acknowledge the technical assistance of Mr. A. Valcic. Nofe added in proof:

Recently

Scott

and

Slack (1971) have reported a serological relationship between BRV and CCMV. According to t,hem BMV and CCMV reacted with BRIV antiserum but BBMV did not. ilntisera produced for CC&IV and BBMV reacted only with the homologous virus antigen. Scott, H. A., and Slack, S. A. (1971). T’iroloqy,

47,49049?.

20

AGRAWAL

AND TREMAINE

REFERENCES BANCROFT, J. B., HILLS, G. J., and M.~RKHAM, R.

pH-induced st.ructural change in bromegrass mosaic virus. Biophys. J. 4,11-21. KODAM~, T., and BANCROFT, J. B. (1964). Some properties of infectious ribonucleic acid from broad bean mottle virus. Virology 22,23-32. LtlNE, L. C., and KAESBERG, P. (1971). Multiple genetic components in bromegrass mosaic virus.

(1967). A study of the self-assembly process in a small spherical virus. Formation of organized structures from protein subunits in vitro. ViroZogy 31, 354379. B.~NCROFT, J. B., HIEBERT, E., REES, M. W., and lvature (London) 232, 40-43. hfaRgHaM, R. (1968). Properties of cowpea LESN.I~, J. A., and REICHMANN, M. E. (1970). chlorotic mottle virus, its protein and nucleic Determination of molecular weights of plant acid. T’irology 34, 224239. viral protein subunits by polyacrylamide gel BbNCROFT, J. B., BRACKER, C. E., and WAGNER, electrophoresis. Virology 42,724731. G. W. (1969). Structures derived from cowpea MIKI, T., and KNIGHT, C. A. (1965). Preparation of chlorotic mottle and brome mosaic virus probroad bean mott.le virus protein. T’irology 25, tein. Virology 38, 324-335. 478-481. BOCKSTAHLER, L. E., and KAESBERG, P. (1962). SEMBNCX, J. S. (1966). Studies on electrophoretic The molecular weight and other biophysical heterogeneity in isometric plant viruses. ViroZproperties of bromegrass mosaic virus. Biophys. ogy 30, 698-704. J. 2. 1-9. SHAPIRO, A. L., VIBUEL.~, E., and MAIZEL, J. v., BOCKSTAHLER, L. E., and KAESBERG, P. (1965). JR. (1967). Molecular weight estimation of polyIsolat.ion and properties of RNA from bromepept,ide chains by electrophoresis in SDS-polygrass mosaic virus. J. IVOZ.Biol. 13,127-137. acrylamide gels. Biochem. Biophys. Res. ComCHIDLO~, J., and TREMAINE, J. H. (1971). Limited mun. 28, 815-820. hydrolysis of cowpea chlorotic mottle virus by STUBBS, J. D., and KAESBERG, P. (1964). A protein trypsin and chymotrypsin. Virology 43,267-278. subunit of bromegrass mosaic virus. J. AfoZ. DUNKER, A. K., and KUECKERT, R. R. (1969). BioZ. 8, 314-323. Observations on molecular weight determinaWAGNER, G. W., and BANCROFT,J. B. (1968). The self-assembly of spherical viruses with mixed tions on polyacrylamide gel. J. BioZ. Chem. 244, coat proteins. T’irology 34,748-756. 5074-5080. WEBER, K., and OSBORN,M. (1969). The reliability FINCH, J. T., and KLUG, A. (1967). Structure of of molecular weight determinations by dodecyl broad bean mottle virus. I. Analysis of electron sulfate-polyacrylamide gel electrophoresis. J. micrographs and comparison with turnip yellow Bid. Chem. 244,4406-4412. mosaic virus and it.s top component. J. iliol. YA~z.~~ECI, H., and KAESBERG, P. (1963). Isolation BioZ. 24, 289-302. and characterization of a protein subunit of HIEBERT, E., BINCROFT, J. B., and BRACKER, C. broad bean mottle virus. J. Mol. Biol. 6, +65E. (1968). The assembly in. vitro of some small 473. spherical viruses, hybrid viruses, and ot.her T.~MAZ.XI, H., B.~NCROFT, J., and K.~ESBERG, P. nucleo-proteins. Virology 34, 492608. (1961). Biophysical studies of broad bean mottle virus. Proc. Sat. Acad. Sci. U. S. 47,979-983. INCARDONA, N. L., and K.~ESRERG, P. (1964). A