Studies on the amino and carboxyl terminal amino acid sequences of reovirus capsid polypeptides

62, 174-186 (1973)

VIROLOGY

Studies

on the Amino Sequences

DONALD

M. PETT,

Department

and

of Reovirus

THOMAS

of Microbiology

Carboxyl

Terminal

Capsid

C. VANAMAN,

Amino

Acid

Polypeptides AND

WOLFGANG

and Immunology, Duke University Durham, North Carolina 27710

Medical

K. JOKLIK Center,

Accepted November 28, 1972 Techniques are described for isolating several reovirus capsid polypeptides in amounts sufficient for determination of amino and carboxyl terminal amino acid sequences and fingerprinting. Among them are chromatography on CM- and DEAESephadex in the presence of urea, and gel filtration on agarose A-15 m in the presence of sodium dodecyl sulfate. Using a combination of these procedures, and starting with either virions or cores, polypeptides ~3, ~2, and ~2 have been obtained essentially pure. Polypeptides Xl and x2 have been obtained as a mixture which has so far not been resolved. All reovirus capsid polypeptides except ~2 possess blocked amino terminal amino acid residues. The amino terminal amino acid sequence of polypeptide p2 is HJX-Pro-Gly-Gly-Val-Pro-. This suggests that polypeptide ~2 is derived from its precursor, polypeptide ~1, by cleavage of the amino terminal portion of the polypeptide chain. The carboxyl terminal regions of at least three of the five major reovirus capsid polypeptides are different. Polypeptide 03 ends in -(val,val,leu)-COOH; polypeptide ~2 in -leu-(arg, tyr, tyr)-Arg-COOH; and either one or both of the two polypeptides Xl and h2 terminate(s) in -Arg-COOH, the adjacent amino acid sequence being different from that of ~2. INTRODUCTION

possesses two capsid shells which are composed of seven polypeptides which fall into three size classes, designated X, p and u (Smith et al., 1969). The outer shell is composed of three polypeptides, polypeptides 112(MW 72,000), al (MW 43,000), and a3 (MW 34,000) ; the inner shell, the core, is composed of four polypeptides, namely polypeptide Xl (MW 155,000), X2 (MW 140,000), ~1 (MW SO,OOO),and ~2 (MW 36,000). The approximate number of molecules of each of these polypeptides per virion is a3, 850; ~2, 550; ~2, 200; Xl, 100; X2, 100; ul, 25; ~1, 25 (Smith et al., 1969). All these polypeptides are primary gene products except ~2, which is derived by cleavage from ~1 (Smith et al., 1969; Zweerink and Joklik, 1970; Zwcerink et al., 1971; Joklik, 1972). WC present in this paper the results of Reovirus

further studies on the characterization of reovirus capsid polypeptides. In particular, procedures have been devised for the isolation and purification of several of them in milligram amounts; their amino acid composition has been determined; and the nature of their amino and carboxyl terminal amino acid residues has been investigated. The results of these studies indicate that all reovirus capsid polypeptides except ~2 possess blocked amino terminals, and that at least 3 of the 5 major capsid polypeptides have different carboxyl terminal amino acid sequences. These conclusions are at variance with thost: reached by Roy et al. (1972), whose work suggested that the amino and carboxyl terminal regions of several reovirus capsid polypept’ides, even those of different size classes, may be identical. 174

Copyright All rights

@ 1973 by Academic Press, of reproduction in any form

Inc. reserved.

REOVIRUS MATERiALS

AND

CAPSID

METHODS

Cells and virus. Mouse L cells were grown in suspension culture in Eagle’s MEM (Joklik’s modification, Grand Island Biological Company) containing 57, fetal ,calf serum. Three-liter batches of cells, at a density of 1 X lo6 cells/ml, were’infected at a mult’iplicity of 2000 virus particles per cell with reovirus strain Dearing. After 1 hr, onethird volume of fresh prewarmed medium was added. Cells were harvested after 24 hr at 34”, and virus was purified as described previously (Smith et al., 1969). Virus was always banded twice in CsCl. Preparation of reovirus cores. Cores were prepared as described by Skehel and Joklik (1969), using virus at a conceptration of 5 mg/ml in SSCl and chymotrypsin at 50 pg/ml. After 90 min at 37” cores were pelleted and banded in preformed CsCl density gradients (1.28-1.48 g/ml in SSC, 2 hr at 25,000 rpm). The core band at 1.43 g/ml was d’la 1yzed against 10 mM Tris1 mM EDTA, pH 8.0. SDS-Polyacrylamide gel electrophoresis. Seven and one-half percent gels (0.4 cm X 12 cm) were made with 7.5y0 acrylamide, 0.1 M phosphate buffer, pH 7.2, 0.185% bisacrylamide, 6 M urea, 0.02 M EDTA, 0.1% SDS, 0.1% TEMED, and 0.08% ammonium persulfate to catalyze polymerization. Polypeptides were dissolved in 0.2 ml of 5 M urea-lojo SDS-l% 2-ME, heated at 100” for 2 min, and applied to gels after the addition of 0.05 ml of 0.05yo bromophenol blue. J$lectrophoresis buffer consist,ed of 0.1 M sodium phosphate, pH 7.2, 0.1% SDS, arid 0.02 M EDTA (Zweerink et al., 1971). Glectrophoresis was carried out at 5 mA p ?r gel for 18 hr. Gels were stained for 30 min at room temperature with 0.2yo Coomassie Brilliant’ Blue R250 in 50?& 1 The following abbreviations are used: SSC, 0.15 M sodium chloride-O.015 Jf sodium citrate, pH 7.4; EDTA, ethylenediaminetetracetic acid; Tris, tris(hydroxymethyl)aminomethane; SDS, sodium dodecyl sulfate; TEMED, N,N,N’,N’t’etramethyleneethylenediamine; 2-ME, 2-mercaptoethanol; NEMO, 4-ethylmorpholine; DFP, diisopropylfluorophosphate; PITC, phenylisothiocyanate; PTC, phenyl thiocarbamyl.

POLYPEPTIDES

175

methanol-10yO acetic acid and destained in a Hoeffer ‘gel destainer with 7.5yo acet)ic acid-5 % methanol. Preparation of ion-exchange and gel jiltration resins. DEAE-Sephadex and CM-Sephadex were swollen for 24 hr in 6 M urea in 0.1 M sodium acetate pH 6.5, 1 mM EDTA, 0.1% 2-ME, 0.01 M glycine. The resins were degassed’ under vacuum for 5 min and adjusted to pH 6.5 with HCl. Columns were poured and operated with a maximum hydroststtic head of 30 cm. Preparation of polypeptides for ion-exchange chromatography. RNA tias removed from protein by phenol extraction. Virus at a concentration of 5 mg/ml in SSC was shaken for 5 min with an equal volume of 70y0 phenol in water. The phenol and aqueous phases were separated by centrifugation and the polypeptides precipitated from the phenol phase by addition of 5 volumes of icecold acetone: 1 N HCl (4O:l). The precipitate was washed 3 times with acetoneHCl, dried under a stream of nitrogen, and redissolved in 6 M guanidine . HCl in 0.1 M sodium acetate, pH 6.5, I mM EDTA, 0.01 M glycine and 0.1% 2-ME. Prior to application to ion-exchange columns, the protein solution was dialyzed against two lots of 50 volumes of 6 M urea in 0.1 M sodium acetate, pH 6.5, 1 mM EDTA, 0.01 M glycine and 0.1% 2-ME. During this dialysis the polypepbide concentration had to be kept below 0.5 mg/ml so as to prevent aggregation which causes irreproducible elution profiles. Recovery of polypeptides after chrom.atograph,y on CM- and DEAE-Sephadex. Pooled fractions corresponding to peaks were dialyzed successively against 1% acetic acid and water, and lyophilized. Preparation of polypeptides for gel Jiltration. Virions or cores were dissolved in 10 mM Tris . HCl, pH 8, 1 mM EDTA, 2% 2-ME and 2% SDS. Removal of SDS from polypeptides. After chromatography on agarose A-15m, SDS was removed from polypeptides as described by Weber and Kuter (1971). PolypeptideSDS samples were lyophilized and dissolved in 10 M urea in 0.5% acetic acid and 0.1% 2-NE, and applied to 0.9 X 5 cm columns

176

PETT,

VANAMAN,

of BioRad AG l-X8, 200-400 mesh, acetate form, in the same solvent. The columns were washed with 2 column volumes of solvent, and the eluates dialyzed a.gainst 100 vol of 1% acetic acid, and two lots of 100 volumes of water. The samples were then lyophilized. Determination of amino acid compositions. Protein samples were hydrolyzed in 6 N HCI at 110” in sealed evacuated Pyrex ignition tubes. A crystal of phenol was added to each tube to improve tyrosine yields. Time course hydrolyzates were analyzed so as to establish accurate amino acid compositions. Hydrolyzates were analyzed with a Beckman Model 121 Automatic Amino Acid Analyzer equipped with high-sensitivity cuvettes and an electronically expanded recorder range. L-Norleucine and a-amino-&guanidinopropionic acid (2 X 10m5M) were included in sample dilutor buffers to serve as internal indicators of sample injection reproducibility (variation f0.5%). Tryptophan was determined on maleylated polypeptides by the method of Scoffone et al. (1968) after reduction and alkylation. Half-cystine was determined as cysteic acid after performic acid oxidation (Hirs, 1967). The amino acid composition of each of the purified reovirus polypeptides was calculated using the molecular weights previously determined by Smith et al. (1969). Alkylation of polypeptides. Polypeptides were reduced by incubation in 6 M guanidine.HCl, 0.1 M NEMOeHCl, 0.01 M 2ME, pH 8.5, at 37” for 2 hr and t’hen alkylated by the addition of a 2-fold molar excess of iodoacetic acid. The resulting reaction mixture was incubated for 15 min at 25” while the pH was maintained at 8.5 by the addition of 1 N sodium hydroxide. A IO-fold molar excess of 2-ME was then added, and the reaction was allowed to proceed at pH 8.5 until no further base addition was required. Reaction mixtures were dialyzed at 4’ against 4 lots of 100 volumes of deionized water, shell-frozen, and lyophilized. Determination of the amino acids at the amino terminus. Amino-terminal determinations were performed using the dansyl technique essentially as described by Weiner et

AND JOKLIK

al. (1972). A sample of polypeptide (lo-20 nmoles) was dansylated in 1% SDS and 0.5 M NaHC03, pH 9.6, by adding 10 ~1 of 10% dansyl chloride in acetone per milliliter of reaction mixture. After 30 min at 37”, protein was recovered by precipitation with 5 volumes of acetone. The precipitate was centrifuged down, washed once with acetone and then with 1 N HCl to remove dansic acid (Gros and Labouesse, 1969), and finally dried under a stream of nitrogen. Aliquots of dansylated proteins were hydrolyzed at 105” for both 4 hr and 18 hr in sealed tubes in the dark. The hydrolyzates were evaporated to dryness, redissolved in 0.5 ml of water, brought to pH 3.5 with HCl, and extracted three times with 0.5!ml of et)hyl ether. The ether extracts were evaporated to dryness under a stream of nitrogen, redissolved in a small volume of 5Oyc pyridine, and spotted on one side of 7.5 X 7.5 cm double-layered polyamide sheets. One nanomole each of dansyl-proline, dansyl-glycine, dansyl-glutamic acid, dansyl-serine, and dansyl-isoleutine were spotted on the reference side. The dansyl amino acids were then separated by two-dimensional chromatography as described by Hartley (1970), using 1.5% formic acid in the first dimension and benzene-acetic acid (9:l) in the second. After development, plates were viewed under a Mineralight UVS-12 and coincidence between unknowns and standards noted. Plates were photographed under TJV-light using a Polaroid MP-3 camera and Polaroid 107 black and whit,e film. The pH 3.5 aqueous phase from each sample was analyzed for 0-dansyl-tyrosine, cr-dansyl-arginine, a-dansyl-histidine and [dansyl-lysine by means of 2-dimensional chroma,tography on double-layered polyamide sheets using solvents II and IV of Gros and La,bouesse (1969). An aliquot of the 1% hr hydrolyzate was always analyzed for total a,mino acids, so that, a quantitative estimat,e could be made of the dansylamino acid yield. Determination of amino terminal amino acid sequences. Amino terminal sequence determinations were performed using the modified dansyl-Edman procedure described by Weiner et al. (1972) with minor changes.

REOVIRUS

CAPSID

Purified polypeptides were dissolved in 0.5 ml 2% SDS-O.5 M sodium phosphate, pH 9.6. An aliquot was removed and subjected to dansyl analysis as described in the preceding paragraph. The remainder was subjected to one cycle of Edman degradation. Coupling with PITC was initiated by the addition of 25 ~1 of the reagent; the solution was mixed vigorously and incubated at 50” in an atmosphere of nitrogen for 15 min. Another 25 ~1 of PITC was then added with further mixing followed by incubation for another 15 min as just described. The PTC-protein derivatives were then precipitated with acetone containing 1 mW 2ME, recovered by centrifugation, washed once with acetone, and dried in vacua for 30 min at 60”. Cyclization and cleavage were then accomplished by incubation with 500 ~1 of anhydrous trifluoroacetic acid at 50” for 5 min; 1 mg of SDS was added, trifluoroacetic acid was removed under a stream of I\Tz , and the sample was dried for 30 min at 60” in vucuo. The sample was then redissolved in coupling buffer for another cycle, an aliquot again being removed for dansyl analysis (5 nmoles per cycle). Determination of the amino acids at the carboxyl terminus. The carboxyl terminal amino acid residues of purified reovirus polypeptides were determined by carboxypeptidase digestion of polypeptides maleylated with a 100-fold excess of maleic anhydride in 6 M guanidine . HCl and 0.1 M T\TEMO.HCI, pH 9.0. The maleylated polypeptides were dialyzed against, water, shell frozen and lyophilized. Digestion of maleylated polypeptides with carboxypeptidase A and B was carried out, in 0.25 iV potassium phosphate, pH 7.65, using an enzyme to substrate ratio of 1:50. Samples were removed at various times, adjusted to pH 2 with 1 N HCl, and stored frozen until analyzed. DFP-treated carboxypeptidase A (Worthington, crystalline suspension, 50 units/mg) was centrifuged at 2000 (/ for 5 min before use; the pellet was resuspended in water, recentrifuged and dissolved in 10% LiCl to give a stock solution containing 1 mg/ml. DFP-treated carboxypept’idase B (Worthington, 10 mg/ml, 95 units/mg) was used as supplied. Both enzymes had negligible tryptic and chymotryptic activities.

177

POLYPEPTIDES

Digestions were carried out adding either carboxypeptidase A or B first, as described below. Released amino acids were quantitated by amino acid analysis. The concentration of the polypeptide being degraded was determined by amino acid analysis of a 24-hr acid hydrolyzate of an aliquot of the reaction mixture. Materials. Sequenal grade dansyl chloride (10% solution in acetone), dansyl amino acid standards, PITC, n-propanol, SDS, trifluoroacetic acid, L-norleucine, a-amino-& guanidinopropionic acid hydrochloride, ninhydrin, and stannous chloride monohydrate were products of the Pierce Chemical Company. Benzene, ethyl acetate, and n-butyl chloride were used as supplied by Burdich and Jackson Laboratories. Pyridine (spectral grade), 4-ethylmorpholine (practical), iodoacetic acid, acrylamide and N , N’-methylenebisacrylamide were products of Eastman Organic Chemicals; the last two were recrystallized from chloroform and acetone, respectively. Pyridine and 4-ethylmorpholine were redistilled following reaction with ninhydrin under reflux conditions. Urea solutions were deionized by passage through Amberlite MB-3 immediately prior to use. Other chemicals were obtained as reagent grade and used without further purification. The standard amino acid calibration mixture was obtained from the Beckman Instruments Company. Cheng-chin doublelayered polyamide sheets were obtained from Gallard-Schlesinger. RESULTS

Fractionation of Reovirus Polypeptides CM-Sephadex and DEAE-Sephadex

on

Phenol-extracted reovirus polypeptides were chromatographed on columns of CMSephadex in urea-acetate, which separated polypeptide ~2, which was unretarded, from polypeptides Xl, X2, and ~2, which eluted together at 0.15-0.2 M LiCl, and from polypeptide ~3, which eluted at 0.34.35 M LiCl (Figs 1 and 2). Absolute separation of polypeptides ~2 and Xl + X2 was often not achieved with the first column (as shown in Fig. 2), but, could be accomplished by rechromatography. On DEAE-Sephadex only polypeptide ~2

178

PETT,

VANAMAN,

//’

E c 0

2 .c 0’

, /’ g

0.2 :-x’

,I’

022

I’ /’ I’

I 0

II

y’ 20

40

’

AND JOKLIK

ten species of viral double-stranded RNA but no viral polypeptides (Fig. 6). Peak II cont’ained polypeptides Xl and X2; peak III, polypeptides ~1 and ~2; and peak IV, polypeptides ul, 02, and ~3. Peak V contained no material capable of being st’ained with Coomassic Brilliant Blue; it possessed a high 260 nm:280 nm ratio and presumably

ci 3

1’ II

III

,

I

I

60 Fraction

El0

100

120

FIG. 1. Chromatography of reovirus polypeptides on CM-Sephadex. Polypeptides (100 mg) were prepared for chromatography as described in Materials and Methods. After dialysis against 6 M urea, 0.1 M sodium acetate, pH 6.5, 1 rnn/l EDTA, 0.01 M glycine, and 0.1% Z-ME (ureaacetate), the preparation was centrifuged at 5000 rpm to remove aggregated material; the supernatant was then applied to a 2.5 X 25 cm column of CM-Sephadex in urea-acetate and washed into the column with several bed volumes of ureaacetate. Polypeptides were eluted with a 250-ml linear gradient of 0 to 0.4 M LiCl in urea-acetate. Fractions of 2.5 ml were collected. The optical density at 280 nm of every other fraction was measured, and the molarity of LiCl of every tenth fraction was determined by measuring conductivity.

was retarded; it could be eluted in 1 M LiCl (Figs. 3 and 4). The elution profiles of reovirus polypeptides on CM- and DEAE-cellulose were very similar to those on CM- and DEAESephadex. Recovery in all cases was between 20 and 30yo of the material applied to columns. CM- and DEAE-Sephadex were chosen for routine use owing to t’heir greater ease of preparation and handling. Gel Filtration

of Reovirus Pclypeptides

in

Reovirus polypeptides fall into three size classes with molecular weights of 34,00042,000, 72,000--80,000 and 140,000-155!000, respectively (Smith et al., 1969). These size classes could be separated by gel filtration on Agarose A-15m in the presence of SDS. Six distinct peaks were eluted (Fig. 5). Peak I eluted in the void volume; it contained all

Fro. 2. SDS-polyacrylamide gel electrophoresis of polypeptides in pooled fractions comprising peaks I, II, and III resulting from CM-Sephadex chromatography (see Fig. 1). Samples containing about 50 pg polypeptide were made l\yO with respect to SDS and electrophoresed on polyacrylamide gels. Electrophoresis was from bottom to top. In the first gel (V) whole virion polypeptides were electropboresed.

REOVIRUS

CAPSID

POLYPEPTIDES

179

columns used here was 50 mg. Recovery was in excess of 90%. Attempts to rechromatograph peaks II, III, and IV on CM-Sephadex or DEAE-Sephadex were unsuccessful, probably owing to small and variable amounts of SDS not removed by passage through AG l-X8 (see Materials and Methods). In summary, polypcptide ~2 (together with 3-8oj, polypcptide ~1 from which it is derived) could bc prepe.red either by chromatography on CX-Scphadex or DEAESephadex, or preferably on agarose; poly-

0.E

E p,, N g

0.4

L I.OM LiCl 0.3

I ICI

I 20

I 30 Fraction

I

40

I 50

60

FIG. 3. Chromatography of reovirus polypeptides on DEAE-Sephadex. The sample was prepared as described in the legend to Fig. 1. A step elution was performed with buffer containing 1M LiCl in urea-acetate. Column dimension and fraction size as for Fig. 1.

contained the reovirus oligonucleotides (Nichols et al., 1972). Peak VI corresponded t.o 2-ME which emerged when one bed volume of eluent had passed through the column. It was reported previously that polypeptide ~2 is present in virions in the form of a disulfide-bonded dimer (Smith et al., 1969). Additional evidence for this observation was obtained by chromatographing reovirus dissociated with SDS in the absence of 2-ME on Agarose A-15m. The elution profile then lacked peak III and exhibited a correspondingly increased peak II, which upon analysis in SDS-polyacrylamide gels after reduction with 2-ME proved to contain not only polypeptides Xl and X2, but. also polypeptides ~1 and ~2. The maximum amount of polypeptide that could be resolved on the 2.5 X 90 cm

FIG. 4. SDS-polyacrylamide gel electrophoresis of polypeptides in pooled fractions comprising peaks I and II eluted from DEAE-Sephadex (see Fig. 3). For further details see legend to Fig. 2. Electrophoresis was from bottom to top.

PETT,

VANAMAN,

AND JOKLIK

in proline and lysine and poor in phenylalanine and arginine; the ratio 1ysine:arginine was about 1.5. It contained 5 half-cystine residues per molecule, in line with the ob-

I so

Fraction

I 100

I IS0

FIG. 5. Gel filtration of reovirus polypeptic lee on sgarose A-15m in SDS. Reovirus was suspended in 2 ml 0.01 M Tris+HCl, pH 8, 1 mM EDTA and 2% 2-ME, and solubilized by the addition of SDS to 2%, followed by heating at 100’ for 1 min. Sucrose was then added to 200% and the sample underlaid onto a 2.5 X 90 cm column of agarose A-15m, 20@400 mesh, in 0.01 Tris.HCI, pH 8, 0.5% SDS, 1 mM EDTA, and 0.1% 2-ME. The column was developed with this solvent at a flow rate of 15 ml/hr under a maximum hydrostatic head of 30 cm, and fractions of 1.75 ml were collected.

peptide u2 was prepared by chromatographing the polypeptides of reovirus cores on sgarose, or by agarose chromatography of peak II eluted from CM-Sephadex columns; while polypeptide u3 was obtained directly as peak III eluted from CMSephadex. No method of column chromatography, either ion exchange or gel filtration, was capable of separating polypeptides Xl and h2, which were obtained as peak II eluted from agarose columns. Amino Acid Composition of Reovirus Polypeptides The amino acid composition of polypeptides ~3, ~2, ~2, and Xl + X2 are shown in Table 1. None of the compositions presented highly unusual features. Polypeptide a3 was relatively rich in histidine and tryptophan and poor in threonine; the ratio lysine/ arginine was close to 1. Polypeptide a2 was relatively rich in phenylalanine and poor in lysine; the ratio 1ysine:arginine was only about 0.2. Polypeptide ~2 was relatively rich

FIG. 6. SDS-polyacrylamide gel electrophoresis of material in pooled fractions comprising peaks I, II, III, and IV resulting from agarose-SDS gel filtration (see Fig. 5). For further details see legend to Fig. 2. All gels were stained for both polypeptides (with Coomassie Brilliant Blue) and RNA (with Methylene Blue). In gel V solubilked whole virus was electrophoresed. Electrophoresis was from bottom to top. The extremely tight bands in the first two gels are due to doublestranded RNA, The bottom band comprises the 3 L species, the next band the 3 M species, and the next 3 bands the 4 S species (species S3 and S4 are not resolved).

REOVIRUS

CAPSID TABLE

I

181

POLYPEPTIDES 1

AMINO ACID COMPOSITION OF SEVERAL REOVIRUS CAPSID POLYPEPTIDESO 703 02 4

Residue

Moles/100 r noles amino acid

rI-

--

Moles/100 loles amine acid

Residues/ mole of protein

Moles/100 r noles amino acid

Moles/l00 rnoles amino acid -

R;Zgf protein

_2.8 1.7 5.3 11.8 7.4 7.9 9.4 5.4 6.2 7.8 7.1 3.4 6.0 9.3 4.1 4.2 ND ND

LYS His -4% ASP Thr* Serb Glu Pro GUY Ala ValC Met Ilec LX Tyr

Phe Cysd (half) Trpe

5.4 0.7 3.8 9.5 7.2 8.8 9.9 6.9 6.2 9.6 7.3 2.4 5.6 7.8 2.9 2.5 0.7 2.4

36 5 25 62 48 59 66 46 41 64 49 16 37 52 19 17 5 16

1.4 1.7 6.6 10.7 7.2 8.0 10.7 4.3 8.3 9.5 6.0 2.0 4.0 10.7 3.7 5.2 ND ND

663

-

5 6 23 37 25 28 37 15 29 33 21 7 14 37 13 18 ND ND -

-

348 -

that it exists in virions as disulfide-bonded dimers. Polypeptides Xl + X2 presented no unusual features; the ratio lysine : arginine was about 0.5. The Amino Terminal Amino Acids of Reovirus Polypeptides For the identification of the amino terminal amino acids the dansylation procedure was employed. Two types of experiments were carried out. In the first, reovirus polypeptides were allowed to react with dansyl chloride, then hydrolyzed and the dansylated amino acid residues identified. Analysis of a portion of the reaction mixture by SDS-polyacrylamide gel electrophoresis showed that all polypeptides had reacted since fluorescent bands corextensively, responding to each of the major polypeptide bands were readily distinguished under ultra-

317 -

a Averages of at least two determinations. * Corrected for desbructive losses during hydrolysis. c From 120 hr hydrolyzates. d Determined as cysteic acid. 6 Trp was determined on carboxymethylated and maleylated servation

16 12 18 38 15 21 29 17 26 19 27 9 11 27 9 11 2 10

5.0 3.7 5.7 12.0 4.7 6.6 9.1 5.4 8.2 6.0 8.5 2.8 3.5 8.5 2.8 3.5 0.6 3.2

& and ~3.

violet light. The only dansylated amino acids present in the hydrolyzate were dansylproline and [-dansyl-lysine. Dansyl-proline was present in an amount sufhcient to represent the aminoterminal residue of any one of the major polypeptides Xl, X2, ~2, ~2, and ~3, but not of all of them. Although the amount of virus analyzed was sufficient for amino terminal dansyl derivatives from the minor polypeptides ~1 and al to have been detected, none were observed. The fact that proline was the only amino-terminal amino acid residue suggested that several of the reovirus capsid polypeptides possessed blocked amino terminals. In order to determine which of the polypeptides contained amino-terminal proline, 2.5 mg of virus was dansylated in the same manner, and the polypeptides were separated by SDS-polyacrylamide gel elect’ro-

182

PETT,

VANAMAN,

AND

JOKLIK

FIG. 7. Identification of the amino terminal dansyl derivative of polypeptide ~2. Two-dimensional thin-layer chromatography on double-sided polyamide sheets was performed on an aliquot of the dansyl derivative. The origin, in the lower left-hand corner, is indicated by the bright spot of dansamide applied after chromatography. Chromatography in the first dimension was in 1.5% formic acid and in the second in benzene:acetic acid (9:l). (A) Standards (1 nmole each); (B), (C) and (D) the dansyl derivatives of the amino terminal amino acids of polypeptide ~2, Xl + ~2, and 03, respectively.

phoresis (250 pg/gel). The fluorescent bands were then cut out, the polypeptides eluted, precipitated with acetone, hydrolyzed, and analyzed as described in Materials and Methods. Polypeptides Xl and X2 were not readily distinguished by fluorescence and were therefore eluted together. Likewise polypeptides ~1 and $2 were analyzed together since ~1 migrated too close to ~2, which in any case was present in large excess. Polypeptides a2 and a3 were well separated and analyzed individually; and the region just behind a2 was examined for (~1, which, however, did not fluoresce sufficiently to be detected. The amount of polypeptide in each hydrolyzed sample was quantitated

by assaying an aliquot in the amino acid analyzer; they were: polypeptides Xl + X2 (in approximately equal amounts) 6 nmoles; ~1 + ~2 (more than 90% ~2), 2.2 nmoles; ul, 0.20 nmole; ~2, 1.6 nmoles; and ~3, 2.5 nmoles. The only region which yielded an amino terminal residue was that containing polypeptides ~2 and ~1; this residue was proline (Fig. 7). This was confirmed by dansylating the polypeptides in peaks II, III, and IV after elution from an agarose-SDS column (which contained polypeptides Xl + X2, ~1 + ~2, and al + u:! + ~3, respectively, see Figs. 5 and 6) and analyzing their hydrolyzates. Again no dansylated amino

REOVIRUS

CAPSID

POLYPEPTIDES

183

terminal amino acids were detected apart from dansyl-proline in peak III. The conclusion drawn from these experiments was that all reovirus capsid polypeptides had blocked amino terminal amino acid residues except polypeptide ~2 (and possibly Al), the amino terminal amino acid of which was proline. The Amino Terminal Amino Acicl Sequence 0-f Polypeptide $2 ” .._Employing the dansyl-Edman procedure as described in Materials and Methods, the 0 60 120 160 240 first 5 amino acids at the amino terminus of Minutes polypeptide ~2 were sequenced. The seFIG. 8. Digestion of maleylated polypeptide quence determined was H&Pro-Gly-GlyA and B. Maleylated, Val-Pro-. The first four residues of this a3 with carboxypeptidase sequence are the same as those found by lyophilized polypeptide a3 (3.5 mg) was dissolved in 2.5 ml of 0.25 fif potassium phosphate buffer, Roy et al. (1972) in all size classes of reovirus B (100 rg) was added, polypeptides. Attempts at further se- pH 7.65. Carboxypeptidase and the mixture incubated at 37’ for 30 min. A quencing were unsuccessful, owing to the sample (0.4 ml) was then withdrawn and added low yields obtained after encountering pro- to 1 ml 0.01 N HCl; after centrifugation, the superline at residue 5. natant, was analyzed in the amino acid analyzer. The Carboxyl Terminal Amino Acid quences of Reovirus Polypeptides

Se-

All reovirus capsid polypeptides were found to be extremely insoluble in the absence of guanidine.HCl, urea, or SDS. Since these reagents are incompatible with the use of carboxypeptidase, reovirus polypeptides were first rendered soluble by maleylation. Polypeptide US. Approximatelv 100 nmoles of polypeptide a3 (3.5 mg), purified by CMSephadex chromatography, was maleylated, dialyzed, lyophilized, and digested first with carboxypeptidase B, then with carboxypeptidase A. Released amino acids were quantitated in the amino acid analyzer and a portion of the reaction mixture prior to addition of the enzyme hydrolyzed to determine the total amount of polypeptide present. A control containing no polypeptide a3 indicated that neither carboxypeptidase autolyzed detectably. The results are shown in Fig. 8. No amino acids were released by carboxypeptidase B. Carboxypeptidase A released valine and leucine in a ratio of about 2 : 1. Methionine, arginine, isoleucine, tyrosine, and phenylalanine were all released in roughly equal

Carboxypeptidase A (50 fig) was then added to the remaining reaction mixture and incubation continued for 4 hr. Samples were withdrawn at the times indicated and analyzed in the same manner. Parallel analyses were performed on a reaction mixture containing no substrate.

although smaller amount’s. The carboxyl terminal sequence of polypeptide a3 was therefore most likely -val-leu-val-COOH, -leu-val-val-COOH or -val-val-leu-COOH. Polypeptide ~2. The carboxyl terminal amino acid sequence of polypeptide ~2 was determined similarly using maleylated material purified by agarose gel filtration in SDS. Carboxypeptidase A released no amino acids in 120 min. Aft,er addition of carboxypeptidase B, arginine and tyrosine were released rapidly and leucine, alanine, phenylalanine, isoleucine, and valine more slowly (Fig. 9). By 4 hr about 2.5 moles of tyrosine, 2 moles of arginine, and 1.5 moles of leucine had been released per mole of polypeptide. The most likely sequence suggested by these data is -leu-(arg ,tyr ,tyr)-Arg-COOH. Polypeptides Xl + X2. The carboxyl terminal sequences of unfractionated maleylated polypeptides Xl + X2 (7 mg) was determined in the same manner (Fig. 10).

PETT,

VANAMAN,

AND

JOKLIK ‘r

TY~

7 0

60

120 180 Minutes

240

300 CPA

FIG. 9. Digestion of maleylated polypeptide ~2 with carboxypeptidase A and B. Maleylated lyophilieed polypeptide ~2 (7 mg) was dissolved in 4 ml of 0.25 M potassium phosphate, pH 7.65. Carboxypeptidase A (50 pg) was added for 2 hr at room temperature. At this time 0.4 ml was withdrawn and analyzed as described in the legend to Fig. 8. Carboxypeptidase B (50 fig) was then added and incubation continued for 3 hr. Samples were withdrawn and analyzed at the times indicated.

Carboxypeptidase A released no amino acids in 120 min. After addition of carboxypeptidase B tyrosine was released extremely rapidly, followed by leucine, phenylalanine, alanine, valine, arginine, isoleucine, and serine, in that order. These data indicate that arginine is the carboxyl-terminal residue of Xl or X2, or of both Xl and X2; and that the amino acid sequence adjacent to this carboxyl terminus is rich in aromatic and hydrophobic amino acids. If arginine is the carboxyl terminal residue of only one of these two polypeptides, that of the other may be proline, glutamic acid, aspartic acid, or maleylated lysine, none of which would have been released in detectable amounts by either carboxypeptidase A or B under the conditions employed.

/ 120

IS0

240

MINUTES

FIG. 10. Digestion of maleylated polypeptides Xl + X2 with carboxypeptidase A and B. Unfractionated, maleylated, and lyophilized polypeptides Xl + X2 (7 mg) were used. For further details see legend to Fig. 9. DISCUSSION

The isolation of individual reovirus capsid polypeptides in amounts large enough for determination of terminal amino acid sequences and fingerprinting studies may be achieved using a variety of column chromatographic systems. First, in the form of their SDS-complexes, they could be separated by gel filtration in agarose into three size classes which comprise polypeptide Xl + X2, ~1 + ~2, and al + a2 + a3, respect,ively. Second, in the presence of 6 M urea, they could be separated into polypeptide ~2 (and perhaps ~1) which was not retarded, a mixture of three polypeptides (Xl, X2, and ~2) which was eluted with 0.15-0.20 M LiCl, and polypeptide ~3, which was eluted with 0.30-0.35 M LiCl. It was remarkable that, polypeptides Xl, X2, and ~2, which together comprise about 95y0 of the inner capsid

REOVIRUS

CAPSID

shell (Smith et aZ., 1969), were eluted together from CM-Sephadex; this may imply some degree of interaction among them even in the presence of 6 M urea. By contrast polypeptides ~2 and ~3, which together constitute about 98% of the outer capsid shell, were easily separated. Finally, in the presence of 6 &! urea polypeptide ~2 could be separated from t’he rest by chromatography on DEAE-Sephadex, since it alone was retarded. Using these three systems, and also taking advantage of the availability of reovirus cores, which may be prepared from virions by treatment with chymotrypsin (Shatkin and Sipe, 1968; Skehel and Joklik, 1969), the following capsid polypeptides could be prepared in large amounts: ~3, ~2, ~2 + ~1, and Xl + X2. For sequencing studies, contamination of ~2 by ~1 is of little consequence since the amount of ~1 does not exceed 5% of that of ~2, and since ~2 is in any case derived from ~1 by cleavage of no more than 10% of its amino acids. Separation of Xl and X2, however, presents a serious problem which has not yet been solved in any system tested. Although these two polypeptides are coded by different genome RNA segments, they appear to possess very similar physicochemical properties. All reovirus polypeptides are extremely insoluble in the absence of denaturing agents such as guanidineaHC1, urea, or SDS. This greatly complicates amino acid sequence analyses using enzymes, such as the determination of amino acid sequences at the carboxyl termini using carboxypeptidases, or the preparation of tryptic peptide maps. This problem can be overcome in part by maleylation, which renders t’he polypept,ides more soluble. Our results indicate that all reovirus capsid polypeptides except ~2 possess blocked amino terminal residues, the nature of which is not yet’ known. It is unlikely that the amino groups are formylated, since formyl groups would have been removed under some of the conditions used. However, they could be acetylated, or the amino terminal residues could be cyclized glutamine residues, that is, pyrrolidone carboxylic acid (PCA). There is ample precedent for either

POLYPEPTIDES

185

alternative: acetylation of amino terminal amino groups can proceed with high efficiency in mammalian cells and even in cellfree extracts (Strous et al., 1972), and many mammalian proteins start with PCA (Narita, 1970). The only reovirus polypeptide which does not have a block amino-terminal amino group is polypeptide ~2, the amino-terminal amino acid sequence of which is HZN-ProGly-Gly-Val-Pro-. This implies that when polypeptide ~1 is cleaved to polypeptide ~2, its amino terminal portion is removed; however, it is also conceivable that polypeptide ~1 is the only primary gene product. among the reovirus capsid polppeptides which does not have a blocked amino terminal a~mino group. These results are at variance with those of Roy et al. (1972), who reported that the amino terminal amino acid sequence of at least one reovirus polypeptide each of size class is HzN-Pro-Gly-Gly-Val-. These results are most readily explained, against the background of our findings, on the basis of contamination of the X and u size classes with derivatives of polypeptide ~2. Incomplete reduction of ~2 will result, in the presence of t’races of its dimer, which coelectrophoreses with Xl and X2; and aging of reovirus is often accompanied by cleavage of ~2 to polypept’ides (nonessential noncapsid polypeptides, Zweerink et al., 1971; Joklik, 1972) which electrophorese near the g family of polypeptides. As for the carboxyl terminals, we find that polypeptide u3 terminates with (val, va1, leu)-COOH and polypeptide ~2 with -leu(arg, tyr, tyr)-Arg-COOH; and tha.t either Xl or X2, or both, also terminate(s) wit’h -Arg-COOH. However the adjacent amino acid sequence(s) of Xl and/or X2 must differ markedly from that of ~2, since the kinetics of individual amino acid release is quite different (compare Figs. 9 and 10). These results suggest that the major species of reovirus capsid polypeptides terminate in unique amino acid sequences at their carboxyl terminal ends. Roy et al. (1972), on the other hand, found that valine, isoleucine, and leucine were released in equimolar amounts from all three size classes of re-

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ovirus polypeptides. This may have been due to the fact that they identified released amino acids by the dansyl technique, with which Arg and Tyr are not easily identified and quantitated. The availability of techniques for preparing several reovirus polypeptides in large amounts permits attempts at localizing lesions of reovirus ts mutant in specific polypeptides, using tryptic peptide map analysis. Such studies are currently underway. ACKNOWLEDGMENTS This work was supported by Research Grant No. AI-08909 and Health Sciences Advancement Grant No. 5 SO4 RR 06148 from the United States Public Health Service. Donald M. Pett was the recipient of a USPHS Postdoctoral Fellowship. REFERENCES GROS, C., and LABOUESSE, B. (1969). Study of the dansylation reaction of amino acids, peptides and proteins. Eur. J. Biochem. 7, 463-470. HARTLEY, B. S. (1970). Strategy and tactics in protein chemistry. Biochem. J. 119, 805-822. HIRS, C. H. W. (1967). Performic acid oxidation. Methods Enzymol. 11, 197-199. JOKLIK, W. K. (1972). Studies on the effect of chymotrypsin on reovirions. Virology 49, 700715. NARITA, K. (1970). End group determination. In “Protein Sequence Determinations” (S. B. Needham, ed.), pp. 25-90. Springer-Verlag, Berlin and New York. NICHOLS, J. L., BELLAMY, A. R., and JOKLIK, W. K. (1972). Identification of the nucleotide sequences of the oligonucleotides present in reovirions. Virology 48, 562-572.

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ROY, D., GRAZIADEI, W. D., III, LENGYEL, P., and KONIGSBERG, W. (1972). Amino terminal sequences of several reovirus type 3 capsid proteins are identical. Biochem. Biophys. Res. Commun . 46, 1066-1071. SCOFFONE, E., FONT.IR.A, A., and ROCCHI, R. (1968). Sulfenyl halides as modifying reagents for polypeptides and proteins. I. Modification of tryptophan residues. Biochemistry 7,971-979. SHATKIN, A. J., and SIPE, J. D. (1968). RNA polymerase activity in purified reoviruses. Proc. Nat. Acad. Sci. U.S. 61, 1462-1469. SKEHEL, J. J., and JOICLIK, W. K. (1969). Studies on the in vitro transcription of reovirus RNA catalyzed by reovirus cores. Virology 39, 822831. SMITH, R. E., ZWEERINK, H. J., and JOKLIK, W. K. (1969). Polypeptide components of virions, top component, and cores of reovirus type 3. Virology 39, 791-810. STROUS, G. J. A. M., BERNS, T. J. M., v.4~ WESTKEENEN, H., and BLOEMENDAL, H. (1972). Synthesis of lens protein in uivo. Role of methionyl-tRNAs in the synthesis of calf lens 01crystallin. Eur. J. Biochem. 30, 48-52. WXBEX, K., and KUTER, D. J. (1971). Reversible denaturation of enzymes by sodium dodecyl sulfate. J. Biol. Chem. 246, 45044509. WEINER, A. M., PLATT, T., and WEBER, K. (1972). Amino-terminal sequence analysis of proteins purified on a nanomole scale by gel electrophoresis. J. Biol. Chem. 247, 3242-3251. ZWEERINK, H. J., and JOKLIK, W. K. (1970). Studies on the intracellular synthesis of reovirus-specified proteins. Virology 41, 501-518. ZWEERINK, H. J., MCDOWELL, M. J., and JOKLIK, W. K. (1971). Essential and nonessential noncapsid reovirus proteins. Virology 45, 716723.