THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed m U.S.A. Vol. 255, No. 14, Issue of July 25, pp 6962-6965. 1980

Alignment of the Peptides Derived from Acid-catalyzed Cleavage of an Aspartylprolyl Bond in the Major Internal Structural Polypeptide of Avian Retroviruses*

(Received for publication, October I, 1979, and in revised form, April 4, 1980)

Ajit S. Bhown, J. Claude Bennett, and Eric Hunter From the Department of Microbiology a n d the Department of Medicine, University of Alabama in Birmingham, Birmingham, Alabama 35294

The major internal structural polypeptide (p27) of Rous sarcoma virus (RSV), and the analogous poly- peptide (~270) of Rous-associated virus-0 (RAV-0), an endogenous virus released spontaneously by some chicken cells) have been cleaved selectively at a single aspartylprolyl peptide bond to yield two fragments. The NHa- and COOH-terminal amino acid sequences of p27 and p2TO and their mild acid-cleavage fragments have been determined. These results show the existence of an identical cleavage site and a similar N H 2 - and COOH-terminal amino acid sequence in both the poly- peptides. Furthermore they indicate that the difference in the molecular weights of p27 and p2T0 results from an insertion of amino acids in the COOH-terminal pep- tide of ~ 2 7 ~ rather than a shift in the scission site of the precursor molecule.

Avian sarcoma leukosis viruses (ASV) possess a major internal structural polypeptide (p27) of 27,000 daltons, which forms the inner capsid shell of the virion. The form of this polypeptide is highly conserved in those exogenous oncovi- ruses that are horizontally transmitted among chickens. How- ever, the analogous polypeptide (p270) (1, 2) of Rous-associ- ated virus-0 (RAV-0), an endogenous virus released sponta- neously by some chicken cells, is somewhat larger, having a molecular mass of 29,000 (2). The isolation of the RAV-0’ p270 polypeptide in sufficient quantity from a recombinant Rous sarcoma virus, PR-E-95c, prompted us to undertake structural studies of these molecules in order to determine whether the difference in their molecular weights results from a change in the proteolytic cleavage site for p270 or from an insertion/deletion of amino acids in the precursor molecule (Pr76). This communication presents results on the partial amino acid sequence at the NH2 and COOH terminus of p27 and p2TO and their mild acid cleavage fragments which support the latter possibility.

MATERIALS AND METHODS

Chemicals-Sequencer grade chemicals were purchased from Spinco Division, Beckman Instruments (Palo Alto, CA). Methanol for high pressure liquid chromatography was the product of Mallinck- rodt. Carboxypeptidase A was obtained from Worthington. AU other

* This work was supported by Grants AM 03555, AI 09153, and VC 215A. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: RAV-0, Rous-associated virus-0; RSV, Rous sarcoma virus; ASV, avian sarcoma leukosis virus; PTH, phenylthiohydantoin; SDS, sodium dodecyl sulfate.

chemicals were of highest purity grade and were obtained from Pierce, or Fisher, or both.

Purification of Viral Proteins-The viral polypeptides internally labeled with [35S]methionine were purified from RSV-PR-C, or RSV- PR-E-95c grown in tissue culture. RSV-PR-E-95c was kindly provided by Dr. Maxine Linial, Hutchinson Cancer Center, Seattle, Wash. Details of its isolation and initial characterization have been published elsewhere (2, 3). Both viruses were recloned prior to large scale culture. RSV-PR-C was grown in chick-helper-factor negative C/E chicken embryo cells seeded in roller bottles. RSV-PR-E-95c was also grown in roller bottles; however, quail embryo cells were used for this E-subgroup virus. Both viruses were purified by a combination of discontinuous sucrose/tartrate and continuous sucrose density gra- dients as described (4). Virus was banded at a density of approxi- mately 1.16 g / d , and was assessed for purity by sodium dodecyl sulfate/polyacrylarnide gel electrophoresis prior to protein purifica- tion. The p27 and p270 polypeptides were separated from the other structural components of the virions by gel filtration on Sepharose- CL-GB with 6 M guanidine hydrochloride as solvent, as described previously (5). Viral polypeptides prepared in this way were better than 95% pure.

Polyacrylamide Gel Electrophoresis-The products of acid-cata- lyzed cleavage were separated on slab gels containing a gradient of acrylamide concentrations from 5 to 20%. A discontinuous buffer system, essentially as described by Laemmli (6), was used in these experiments. For analytical studies, gels 1.5 mm thick were employed since these gave superior resolution. Preparative gels from which polypeptides were eluted were 3 mm thick. Polypeptides were located by Coomassie blue staining or scanning the gels a t 280 nm on a scanning spectrophotometer (model SD 3000, Schoeffel Instruments Corp., Westwood, NJ). The molecular weights of viral polypeptides were determined by comparing their migration to that of protein standards electrophoresed in adjacent wells. The protein standards used in these experiments were phosphorylase-B (Mr = 94,000), bovine serum albumin (M, = 68,000), ovalbumin (Mr = 43,000), carbonic anhydrase (M, = 30,000), soybean trypsin inhibitor (M, = 21,000), and lysozyme (M, = 14,300) (Bio-Rad Inc., Richmond, CA).

Amino Acid Analysis-Amino acid analyses were performed on samples hydrolyzed with constant boiling HCI at llO°C for 20 h, using a Durmm D-500 amino acid analyzer (7).

Acid Digestion-Acid-catalyzed hydrolysis of aspartylprolyl bonds was performed essentially as described by Mole et al. (8). Lyophilized p27 and ~ 2 7 ~ polypeptides were dissolved in 50% formic acid (v/v) a t a concentration of 0.1 mg/100 pl. The tubes were sealed at atmos- pheric pressure, kept a t 37°C for 96 h, and the product lyophilized.

Electrodialysis-Acid-cleaved fragments were eluted from gel bands by electrodialysis (9) with an average yield of 90%. The gel slices containing the peptide were cut into small pieces with the help of a stainless steel spatula and the sample eluted using 0.1 M and 0.05 M N-ethylmorpholine acetate buffer, pH 8.5, using an ISCO (Model 1750) sample concentrator.

NH2-terminal Amino Acid Sequence Determination-Sequential Edman degradation of peptides eluted from the gels was achieved on a modified (10) Beckman 890C automated sequenator using 0.5 M Quadrol as a coupling buffer. Phenylthiohydantoins (PTH) were separated by high pressure liquid chromatography (HPLC) and iden- tified spectrophotometrically a t 254 nm (Waters Associates). The PTH-derivative of threonine was detected at 313 nm as the PTH- derivative of dehydrothreonine. Thin layer chromatography (11) was

6962

Acid-catalyzed Hydrolysis of Structural Polypeptide of Avian Retroviruses 6963

employed as a second method of PTH identification and to dif€eren- tiate the paire of PTH-derivatives of leucine-isoleucine and valine- methionine which co-elute in the high pressure liquid chromatogra- phy system used. The PTH-derivative of arginiie was identified by high pressure liquid chromatography after injecting 25 to 50% of the aqueous layer.

COOH-terminal Amino Acid Sequence Determination-The amino acid sequence at the carboxyl end was determined enzpat i - cally with carboxypeptidase A essentially as described by Oroszlan et al. (12). The digestion was carried out a t ambient temperature in 0.2 M N-ethylmorpholine acetate, pH 8.5, with 0.1% SDS and without SDS. Protein samples (1 of 2 nmol each) in four to five different tubes were dissolved in 50 pl of buffer containing SDS, heated for 10 min at 80°C, and cooled to mom temperature. Carboxypeptidase A, made up in the same buffer without SDS was added to the substrate to give an enzyme/protein ratio of 1:lO. Digestion was terminated at various time intervals by lowering the pH with 1 to 2 drops of acetic acid, freezing, and lyophilizing the mixture immediately. The lyophilized mixture was analyzed for the released amino acids on an amino acid analyzer. Since p27 was internally labeled with [%]methionine, in one set of experiments the release of radioactivity during the course of digestion was monitored to confum the presence of methionine a t the carboxyl end. In such an experiment the incubation of protein with enzyme was terminated at different time intervals by adding trichloroacetic acid to a final concentration of 10% and cooling to 0°C. The reaction mixture was centrifuged and the supernatant siphoned. out very carefully without disturbing the protein pellet. The clear solution was counted for release of radioactivity in an LKB liquid scintillation counter (1210 Ultrabeta) using an ACS liquid scintillant.

RESULTS

Amino Acid Analysis-The average values from three de- terminations of the amino acid composition of p27 and p270 are presented in Table I. No attempt was made to correct for losses of serine and threonine during acid hydrolysis. An examination of the number of individual residues per molecule of p270 indicates an overall increase in most amino acids compared to p27; however, the values for histidine, tyrosine, and phenylalanine are the same in both p27 and p270 poly- peptides.

Acid Cleavage-In an attempt to determine where the additional amino acids in p270 were located, acid-catalyzed cleavage of the two polypeptides at aspartylprolyl bonds was carried out. Separation of the products of p27 and p270 after hydrolysis a t 37°C in 50% formic acid is illustrated in Fig. 1. It is clear that both polypeptides p27 and p270 generate two fragments with approximate molecular weights of 12,000 (A- I), 15,000 (A-II), 12,000 (AR-I), and 17,000 (AR-II), respec- tively.

TABLE I Amino acid comuositwn of structural uolyve&des p27 and p270

Amino acid P27 p27a

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

residues/mokcuk 9 10 3 3

17 19 19 21 16 19-20 11 12 28 28 22 27 22 19-20 30 34

N.D." N.D. 16 17 5-6 7 13 15 25 27 3 3 5 5

Total no. of residues 245 268 N.D., not determined.

A B C D E F

p 27-

A-I I - - AR -E A - I - - A R - I

- P 270

FIG. 1. Separation of the products of p27 and p270 after hydrolysis at 37°C in 50% formic acid. A and F = standards (see text); B = RSV-PR-C; C = acid digest of p27; D = acid digest of ~270; E RSV-PR-E-95c.

TABLE I1 Quantitative yields of PTH-derivatives

Peptide Cycle

A-I A-I1 AR-I AR-I1

0 (-10 m o l ) (-10 m o l ) (-8 nm01) (-8 nm01) 1 Pro (1.8) Pro (2.0) Pro (1.7) Pro (1.9) 2 Val (2.6) Arg (1.1) Val (2.0) Arg (1.1) 3 Val (2.8) Ser ( 4 ' ' Val (2.1) Ser (-)" 4 ne (2.4) Pro (1.9) Ile (1.9) Pro (1.3) 5 Lys (1.9) Ala (2.2) Lys (1.7) Ala (1.9)

7 Glu (2.0) Gly (1.2) Glu (1.9) ClY (0.9)

9 Pro (1.0) Gly (1.6) Pro (1.0) Gly (0.9) 10 Ala (1.9) Ala (1.9) Ala (1.8) Ala (1.4)

6 Thr (-)a Asn (1.4) Thr (-1" Asn (1.0)

8 Gly (1.7) Gln (1.6) Gly (1.9) Gln (1.0)

Repetitive 95% 96% 96% 97% yield " (-), values not quantitated.

TABLE III Partial amino acid sequence of structural polpeptides p27 and

P270 Poly- Amino acid sequence

rfi NHrtenniMl C O O H - t e d d

10 p27 h.o-Val-Val-ne-Lys-Thr-Glu-Gly- X -Ala- -Ala-Ala-Met

~ 2 7 ~ Pro-Val-Val-ne-Lys-Thr-Glu-Cly-Pro-Ala- -Ala-Ala-Met

TABLE IV NH2-tenninal amino acid sequence of acid-cleaved fragments

Peptide NH2-termid amino acid sequence"

1 10 A- 1 Pro-Val-Val-Ile-Lys-Thr-Glu-Cly-Pro-Ala- AR-1 Pro-Val-Val-Ile-Lys-Thr-Glu-Gly-Pro-Ala-

A-I1 Pro-Arg-(Ser)-Pro-Ala-Asn-Cly-Gln-Gly-Ala- AR-I1 Pro-Ara-(Ser)-Pro-Ala-Asn-Clv-Gln-Gly-Ala-

" Unconfirmed residues are enclosed in parantheses.

Amino Acid Sequence-To determine which fragment rep- resented the NH2- and COOH-terminal portion of the intact molecule the cleaved fragments were eluted from a prepara- tive gel and their NH2-terminal sequence was determined on microgram quantities (150 to 300 pg) with an average repeti- tive yield of 96% (Table 11). This sequence for p27 and p270 and their acid-cleaved fragments is shown in Tables I11 and 1V. Table I11 also shows the amino acid sequences of p27 and p270 at their COOH-terminal end as determined by carboxy-

6964 Acid-catalyzed Hydrolysis of Structural Polypeptide of Avian Retroviruses

n LU

MET

5 10 15 20 25 30 35 40 45 50 55 GO

0

C

5 10 15 20 25 30 35 40 45 50 55 60 TIME IN MINUTES

FIG. 2. Quantitative results of the digestion of 1 to 2 nmol of p27 (A), p27,, (B), and radioactivity release (a with carboxy- peptidase A.

peptidase A digestion. The kinetics of amino acid release which established these assignments is shown in Fig. 2.

DISCUSSION

The studies reported here clearly demonstrate that the p270 polypeptide derived from RAV-0 possesses additional amino acids (-23) in the COOH-terminal peptide of the molecule. Evidence for additional amino acids is presented in Table I. The values obtained for individual residues per molecule for p27 correlate well with the results reported by Herman et ai. (13) and Fletcher et al. (14) for the p27s of avian myeloblas- tosis virus, and with those of Niall et al. (15) for avian

myeloblastosis virus gs-a if the amino acid values are cdcu- lated for a molecular weight of 27,000. In addition, the NH2- terminal amino acid sequence of p27 (ASV) obtained in these and earlier studies by us (5) is identical with that reported by Niall et al. (15).

The common NH2-terminal amino acid sequence of p27 and p270 supports the idea that these 2 molecules are closely related. Furthermore, an identical amino acid sequence at the NH2 terminus of p27 and p270 (Table 111) rules out the possibility that the additional amino acids are located at these ends of the polypeptide. The conservation of an aspartylprolyl bond that can be cleaved at low pH in both molecules has allowed us to locate the additional residues in the COOH- terminal peptide of the RAV-0 ~270. A-I and AR-I are of the same size (M, = 12,000) and possess the NHZ-terminal se- quence of the uncleaved polypeptides, indicating that they originate from the NHz terminus of p27 and p27,,, respectively. A-I1 and AR-11, the COOH-terminal fiagments, on the other hand, exhibit a difference in molecular weight equivalent to that of their parent molecules. Since A-I1 and AR-I1 share a common NHz-terminal sequence, the additional amino acids in p270 must therefore reside COOH-terminal to this cleavage site. Alternatively, since RAV-0 may represent the ancestral progenitor to the exogenous sarcoma and leukemia viruses, the p27 polypeptide of these exogenous viruses may have resulted from the deletion of information at this end of the RAV-0 molecule.

The results of COOH-terminal sequence analysis rule out a terminal addition of amino acids to p270 or an alteration in the Pr76 proteolytic cleavage site since both exogenous and endogenous polypeptides have identical COOH termini. Al- though the COOH-terminal amino acid sequence -Ala-Ala- Met obtained in these studies is different from that reported earlier by Allen et al. (19), it is consistent with the p27 amino acid sequence deduced from DNA sequence studies.2 Further- more, the presence of methionine at the COOH terminus has been confirmed unequivocally by the rapid and maximal release of radiolabeled methionine within 15 min of digestion of p27 with carboxypeptidase A (Fig. 2c). It is of interest that Vogt et al. (20) identified a methionine-labeled tryptic peptide in avian myeloblastosis virus p27 that appeared to be located at the COOH-terminal end of the molecule in pactamycin- mapping experiments. Since this peptide was not present in Pr76 but was replaced by a new peptide “X,” the authors concluded that the methionine-containing peptide might span the COOH-terminal cleavage site for p27; this is consistent with the data presented in this communication.

Acknowledgments-We thank Thomas Cornelius, Jane Ellen Smith, Karen Buttler, and Edgar Hill for expert technical assistance and Abhoyjit Bhown for computer analysis assistance.

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Acid-catalyzed Hydrolysis of Structural Polypeptide of Avian Retroviruses 6965

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