Chromatographic separation and antigenic analysis of proteins of the oncornaviruses

VIROLOGY 64, 358-366 (1975)

Chromatographic

Separation

and Antigenic

Analysis

of Proteins

of the Oncornaviruses III. Avian Viral Proteins with Group-Specific PAUL FLETCHER,

Sloan-Kettering Institute Wisconsin, Madison,

ROBERT C. NOWINSKI, FLEISSNER

Antigenicity

ELLEN TRESS,’

AND

ERWIN

for Cancer Research, New York, New York 10021, McArdle Laboratory, University Wisconsin 53706, and The Rockefeller University, New York, New York 10021. Accepted November

of

13, 1974

Group-specific viral proteins, ~27, p19, ~15, and ~12, have been purified from different strains of avian leukemia-sarcoma viruses, and their chemical and serological properties have been compared. Functionally homologous proteins from two different viral isolates had similar amino acid compositions. The major constituent of the viral ribonucleoprotein, ~12, possesses the highest content of hydrophilic amino acids (53’S), while the core shell protein, ~27, has the lowest (42%). In isolates from six viral subgroups the equivalent proteins were serologically indistinguishable by immunodiffusion analyses with monospecific antisera. By contrast, for the four different proteins from the same viral isolate, no serological cross reactions were found, nor could structural homologies be demonstrated by amino acid and peptide analyses.

avian gs proteins is occupied by p19, for which no role in virion structure has yet Avian oncornaviruses contain seven been assigned, and for which no obvious major proteins (in addition to reverse transcriptase), four of which bear strong, homolog exists in profiles of mammalian viruses (Nowinski et al., 1972b). Prelimigroup-specific (gs) antigens (Fleissner, nary evidence based on tryptic mapping 1971). In a recently proposed nomenclaindicated a possible relationship in priture, these proteins have been designated mary structure between p19 and p15 (No~27, p19, ~15, and p12 according to their winski et al., 1973). This suggested that apparent molecular weights (August et al., 1974). Some data are now available regard- p19 might be related to ~15 via either a ing the structural roles of ~27, ~15, and p12 gene duplication or a polypeptide cleavage in the virion core: p27 and ~15 appear to mechanism, the latter model being consistplay a role in forming the spherical core ent with the evidence for formation of p27 shell, while ~12 is directly complexed with and ~15 by proteolytic cleavage of a considviral RNA in a structure internal to the erably larger precursor (Vogt and Eisenshell (Bolognesi et al., 1973; Fleissner and mann, 1973). In addition, limitations on Tress, 1973b; Stromberg et al., 1974). Pro- the amount of genetic information present teins analogous to ~27, ~15, and p12 in in avian oncornaviruses (Duesberg et al., their properties and functions are found in 1974) make such a cleavage mechanism mammalian oncornaviruses (Bolognesi et attractive. In this report we describe comparative al., 1973). A peculiar position among the amino acid analyses of gs proteins from two ‘To whom requests for reprints should be ad- avian viral isolates, and more extensive dressed. analysis of tryptic maps, including amino INTRODUCTION

356 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

GROUP-SPECIFIC PROTEINS OF AVIAN ONCORNAVIRUSES

359

Peptide analyses. Protein samples, in acid analysis of individual peptides. The results establish that p19 is a unique poly- buffer containing 6 M GuHCl, 0.02 M peptide, not closely related in primary DTT, and 0.2 M tris(hydroxymethyl)amipH 8.5, structure to the other three gs proteins. In nomethane(Tris) -hydrochloride, addition, serological analyses with antisera were caboxymethylated by addition of prepared against individual viral proteins 0.05 M sodium iodoacetate at room temindicate unique antigenicities for each of perature (excluding light) for 15 min. The the four gs proteins; serological cross reac- reaction was terminated by addition of tions were not observed. Similar data for 0.02 M fl-mercaptoethanol, and the samavian viral proteins have been obtained by ples were dialyzed exhaustively in the cold Herman et al., 1975 and Bolognesi et al., against 0.01 M NH4HC08, pH 8.5. Trypsin-TPCK (Worthington) dissolved in 1975. 0.001 N HCl was added in an amount equal MATERIALS AND METHODS to 2% of the viral protein, and incubation Viruses. The procedures for growth and was carried out overnight at room temperature under nitrogen. The peptide preparapurification of [a6S]-methionine-labeled MC29 avian leukosis virus (AvLV) have tions were then lyophilized. Thin-layer electrophoresis of tryptic been described (Fleissner, 1971). Chicken myeloblasts infected with avian myeloblas- peptides labeled with [*‘S]methionine was tosis viruses (AMV) were obtained from D. performed according to Lazarowitz et al. P. Bolognesi and labeled for 16 hr in (1971). ‘C-Labeled tryptic peptides from AMV suspension culture (Beaudreau et al., 1960) with a “C-labeled amino acid mixture proteins were chromatographed on a 0.9 x (New England Nuclear) prior to virus puri- 49-cm column of Aminex AG-50W-X4 (20-29 pm, Bio-Rad). The lyophilized samfication. Virus of subgroups A (BHTRAV-l), B (BHT-RAV-2), C (B77), D ples were dissolved in 1.1 ml of 0.1 M (CZAV, RSV-SR), E (RAV-0), and F pyridine acetate, pH 2.75, and loaded into (RAV-61) were kindly provided by Dr. H. a 1.0 ml Chromatronix sample injector Temin, University of Wisconsin. Large valve. The sample was then injected onto amounts of AMV as plasma of leukemic the column preequilibrated with the same chicks were supplied by Dr. J. Beard, by buffer. The peptides were eluted with a arrangement with the virus cancer program linear gradient generated by a Buchler of the National Cancer Institute. Varigrad gradient device. Each of two Fractionation of viral proteins. The pro- chambers was filled with 350 ml of 0.1 M cedures for gel filtration chromatography pyridine acetate, pH 2.75, and 2.0 M pyriof viral proteins in 6 M guanidine hydro- dine acetate, pH 6.5. The buffer was chloride (GuHCl) with 0.01 M dithiopumped to the column at 30 ml per hour by threitol (DTT) have been reported (Fleiss- means of a positive displacement Beckman ner, 1971; Nowinski et al., 1972a, 1973). AccuFlow chromatography pump. The colAfter rechromatography of individual pro- umn was maintained at 55” with a circulattein species, they were renatured by dial- ing water bath. Fractions were collected at ysis. When larger amounts of viral proteins 6-min intervals (3.0 ml) for a run time of 20 were fractionated, radioactively labeled hr. The peptide profiles of individual proviral proteins were added to the sample to teins from separate runs were aligned by facilitate analysis of the gel filtration pro- direct measurement of the pH gradient in files. Pooling of peak fractions was done the eluted fractions. Sufficient nonradioacconservatively, i.e., using the fractions tive AMV was mixed with the original with protein concentrations greater than labeled virus to permit amino acid analysis the concentration at the half-height of the of individual peptides in the eluate after peak. For recovery of salt-free proteins, removal of the solvent by evaporation. dialysis was performed against 0.01 M Amino acid analyses. Lyophilized proammonium bicarbonate, pH 8.5, and the tein samples were dissolved in 1.0 ml 6 N samples were lyophilized. HCl and transferred to acid-washed Pyrex

360

FLETCHER

ignition tubes 16 x 125 mm (Corning 9860). The samples were evacuated to < 20 PHg and individually sealed. Hydrolysis was carried out at 110” for 20 hr (Crestfield et al., 1963). The samples were stored at -10” in the sealed tubes until immediately before analysis. The tubes were opened, evaporated to dryness at 50” on a Buchler portable flash evaporator, evacuated with a mechanical vacuum pump, and diluted with 0.2 A4 sodium citrate buffer, pH 2.2. A small volume of dissolved hydrolysate was centrifuged in a Beckman Model 152 Microfuge to remove any non-protein insoluble matter prior to amino acid analysis. Aliquots of the hydrolysates were analyzed in a Gurrum Instrument Corporation Model D-500 Amino Acid Analyzer. Serology. Antisera were prepared in (W/Fu x BN)F, hybrid rats against individual proteins of RSV-SR that were isolated by two cycles of gel filtration in GuHCl. Details of the preparation of these antisera have been described previously (Nowinski et al., 1973). A rabbit antiserum against AMV p12 was kindly provided by Dr. D. P. Bolognesi. Immunodiffusion tests were performed in 2% Noble agar in preformed plates (Immunoplate, Pattern C) that were purchased from Hyland Laboratories. Slides were incubated in a humidified chamber and optimal precipitation occurred within 24 hr of plating.

ET AL. MC 29

40

50

60

70

80

90

100

Frocf~on number

FIG. 1. Gel filtration in 6 M GuHCl and 0.01 A4 dithiothreitol of proteins from strain MC29 avian leukosis virus labeled in uivo with [3”S]methionine. For a description of the labeling conditions and the gel filtration procedure, see Fleissner (1971) and Nowinski et al. (1973). The designations (C) and (N) used in conjunction with p27 and p12 refer to the roles of these proteins in formation of the viral core shell and nucleoprotein, respectively (Fleissner and Tress, 1973b; Bolognesi et al., 1973; Stromberg et al., 1974). Three fractions were pooled from each peak for further analysis (Fig. 2).

where necessary) were subjected to amino acid analysis, together with the corresponding proteins from AMV, with the results shown in Table 1. As previously found by single amino acid labeling, p19 and p12 are lysine- and arginine-rich, respectively; these proteins also have the highest over-all contents of hydrophilic amino acids (Table 2), particularly ~12, which is known to interact directly with viral RNA. Proteins p27 and p15 have a substantial content of branched-chain, hydrophobic amino acids and a lower hydrophilicity, as might be expected for proteins RESULTS which are presumed to interact in the The elution pattern obtained for formation of the viral core shell. While the [35S]methionine-labeled MC29 virus by amino acid compositions of the MC29 progel filtration in 6 M GuHCl is shown in Fig. teins are not identical to the compositions 1. As previously reported (Fleissner, 1971), of AMV gs proteins, they do not differ there are seven prominent proteins re- markedly from the latter, despite the isolasolved by this method. We have shown that tion of these virus strains in widely sepathe first two proteins to elute can be rated geographical areas (Ivanov et al., labeled with radioactive glucosamine, 1964; Eckert et al., 1951). whereas the last five proteins do not incorTo further characterize the viral proteins porate label from this precursor. In this and to search for possible primary-strucreport we are concerned with the four ture relationships, thin-layer electrophorenonglycoproteins designated (by apparent sis of [36S]methionine-labeled tryptic pepmolecular weight) ~27, p19, ~15, and ~12, tides from MC29 virus was carried out. The which appear to be internal virion constitu- patterns obtained are shown in Fig. 2. Peptides with identical mobilities were ents. MC29 proteins purified by gel filtration reproducibly identifiable in the digests of chromatography (and rechromatography, ~27, p19, and ~15. Cross-contamination of

GROUP-SPECIFIC

PROTEINS

OF AVIAN

361

ONCORNAVIRUSES

TABLE 1 PROTEINS AMINO ACID COMPOSITIONS” OFMC29 AND AMV GROUP-SPECIFIC

P27

LYS His Arg ASP Thr Ser Glu Pro Glr Ala ‘12Cys’ Val Met Ile Leu Tyf Phe

Pl9

AMVb

MC29

11.1 2.0 16.8 18.3 18.3 11.6 27.4 25.2 20.2 33.3 2.4 17.1 5.6 14.0 26.9 2.8 4.2

9.9 2.3 17.8 19.2 19.4 12.9 28.9 28.8 20.1 32.0 0.2 16.7 6.2 14.2 23.1 2.8 5.0

AMV

16.0 0.2 7.2 6.2 14.7 16.7 25.5 12.9 20.8 19.2 1.1 9.7 5.3 5.9 17.4 2.7 1.6

P15

P12

MC29

AMVb

MC29

15.0 0.6 7.3 7.0 14.4 16.1 24.2 12.0 19.2 15.1 2.8 10.7 5.4 6.6 16.3 3.4 2.0

3.7 2.9 10.6 13.5 7.0 8.2 11.1 8.4 14.2 9.3 0 9.2 5.5 10.8 16.9 1.1 1.2

5.4 2.4 9.5 12.4 8.6 9.7 11.7 6.7 14.4 8.0 0.8 9.6 5.2 9.6 17.1 1.5 1.4

AMV

6.7 2.0 11.0 10.5 5.3 10.0 16.2 9.6 19.2 9.0 0 5.0 0.9 2.7 6.8 0.2 1.5

MC29

7.9 2.3 11.9 10.0 5.6 10.0 16.6 9.6 18.7 7.7 0.2 4.8 1.5 3.0 7.2 2.0 1.2

a Computed to molecular weights of 27,000, 19,000, 15,000, and 12,000 (August et al., 1974). *The data for AMV p27 is from Niall et al. (1970) after adjustment of molecular weight for their gsa to ca. 27,000. The data for ~15, obtained in the present investigation, compares well to that for gsb of Niall et al. after appropriate molecular-weight adjustment. ’ The half-cysteine and tyrosine values are not corrected for oxidation of these amino acids.

a considerable amount of the radioactive material applied to the origin has not moved during electrophoresis, perhaps because of peptide-peptide interactions and P27 Pl9 P15 P12 limited solubilities in the solvent system AMV 41 43 used. No relationship of p12 to the other 47 53 MC29 42 48 45 53 three gs proteins can be postulated from this data. ’ Calculated according to Capaldi and Vanderkooi In view of the results in Fig. 2, it was (1972) from the data in Table 1. [Hydrophilicity, or considered worth testing in some detail polarity = residues of lys + his + arg + asp + thr + ser + glu as % of total amino acid residues in the whether among the avian viral proteins ~27, p19, and ~15 there might exist some protein.] pair(s) with related primary sequences. proteins in adjacent peaks from the gel This could occur by processes involving filtration eluate is probably not a factor in gene duplication or via the proteolytic these results since the proteins were well cleavage of a common large precursor separated in the isolation procedure and protein-the latter process having been were pooled conservatively (Fig. 1). frhe demonstrated to occur for avian oncorsmall amount of the “middle” p19 peptide naviruses (Vogt and Eisenmann, 1973). On found in the ~15 pattern is an exception, the basis of the amino acid compositions of deriving from a rare proteolytic cleavage of these proteins (Table 1) a simple mechap19 to a size resembling ~15 (cf. nism involving proteolytic cleavage can be Discussion).] The somewhat low yields of ruled out for two possible transitions (~27 the “matching” as well as “nonmatching” + p19 or p19 -+ p15), i.e., the shorter peptides in p27 may be due to the fact that polypeptide chain in each of these pairs has TABLE

2

HY~ROPHILICITIES~ (POLARITIES)OFMC29 ANDAMV GROUP-SPECIFIC PROTEINS

362

p27u

FLETCHER

p19

1315 p12N

FIG. 2. Thin-layer electrophoresis of [%]methionine-containing tryptic peptides from MC29 viral proteins (cf. Fig. 1). Migration was toward the anode (at the top) in pyridine-acetate buffer at pH 3.5 (Lazarowitz et al., 1971).

more residues of particular amino acids than the longer. In the absence of adequate amino acid-sequence data, a closer analysis of peptide maps was necessary to assess other possible mechanisms, including a viral gene duplication. For this purpose we

ET AL.

chose to analyze ~27, p19, and p15 derived from AMV, from which we could obtain larger amounts of material. AMV was grown in cultured myeloblasts in medium labeled with a mixture of “C-labeled amino acids. The labeled purified virus was mixed with a substantial amount of unlabeled AMV prepared from plasma of leukemic chicks, and the viral proteins were purified by gel filtration in 6 M GuHCl. Tryptic peptides were prepared and were chromatographed on a Dowex 50 resin. The resultant profiles of tryptic peptides from the AMV proteins are depicted in Fig. 3. The elution profiles are aligned according to pH determinations on the eluates. In general the number of peptides corresponds quite well to the numbers of arginine and lysine residues in the proteins (Table l), e.g., the large number of peptides derived from p19 reflects the high lysine content of this protein. By inspection p27 seems to differ rather markedly from p19 and ~15; this is in accord with results from cochromatography of differentially labeled tryptic peptides from MC29 viral proteins (not shown). In contrast, a certain number of p15 peptides in Fig. 3 can be tentatively matched with p19 peptides in terms of elution position if not peak height. This finding is consistent with similarities previously noted in cochromatography of [8H]- and [‘Cllysine labeled MC29 p15 and p19 tryptic peptides (Nowinski et al., 1973). Thus, a further analysis was made of peptides from these two proteins. The amounts of peptide materials used for the profiles in Fig. 3 permitted a comparison of individual peptides from the proteins by amino acid analyses. These were carried out for peptides numbered 2-10 in the profile of p15 and for all the peptides of similar elution positions from p19. By this more precise methodology we were unable to identify any common peptides in p19 and ~15. We conclude, therefore, that AvLV ~27, p19, and ~15 are not closely related in their primary sequences. Niall et al. (1971) reached a similar conclusion regarding nonhomology of p27 and p15 (their gs a and gs b) . Conclusions drawn from biochemical

GROUP-SPECIFIC PROTEINS OF AVIAN ONCORNAVIRUSES

363

FIG. 3. Chromatography on Dowex 50 resin of “C-amino acid-labeled tryptic peptides from AMV. Panel A, peptides from ~27; B, peptides from p19, C, peptides from ~15. Elution was carried out with a gradient of pyridine acetate from pH 2.8 to 6.5 (see Methods). Determination of pH in eluted fractions made it possible to align elution profiles of separate column runs. Peptides from ~12 were not compared to those of the other three proteins by this method, since thin-layer electrophoresis of methionine-containing peptides revealed no resemblance of p12 to the rest (Fig. 2).

studies were substantiated by an immunological analysis of individual viral proteins. Antisera prepared against isolated proteins of RSV-SR were examined by immunodiffusion with purified antigens. As shown in Fig. 4, each of the viral proteins bore unique antigenic determinants; serological cross reactions were not observed between any of the viral proteins. The rat anti-p15 sera also gave a weak reaction with p19, but this weak precipitin band formed a line of nonidentity with the major specificity precipitated by anti-p15 and its homologous antigen (~15). The production of antip19 activity in antisera prepared against p15 is probably the result of small amounts of cleaved p19 peptide that contaminated the p15 material used as immunogen (see Discussion). The group specificity of three of these antigens is illustrated in Fig. 5. Antisera prepared against isolated proteins of RSV-SR reacted equivalently with viruses from six subgroups (A, B, C, D, E, and F) of the avian leukemia-sarcoma viruses.

DISCUSSION

Our results indicate that proteins of two AvLV isolates from widely separated geographical regions have very similar, though not identical amino acid compositions (Table 1). Both AMV and MC29 virus preparations have been reported to contain helper virus components which represent the majority of the virion populations (Moscovici and Vogt, 1969; Ishizaki et al., 1972). Thus, the data reported here pertain primarily to the protein components of the helper component. The correspondence between the numbers of sharply resolved tryptic peptides and the numbers of arginine and lysine residues determined for each protein indicate that the analysis was performed on essentially homogeneous proteins. The analysis of tryptic peptides from MC29 and AMV gs proteins shows, in sum, that the four gs proteins do not have a close relationship in primary structure. Similar results have been obtained by Vogt et al. (1974) for AMV proteins by Dowex chro-

364

FLETCHER

ET AL.

Fleissner and Tress, 1973a). ] In immunodiffusion (Fig. 4) each of the four gs proteins from a given viral isolate possesses unique antigenicity, reflecting the structural differences indicated by amino acid and peptide analyses. The anti-RSV-SR ~15 serum contained some antibodies reactive with isolated p19 despite the fact that the immunizing antigen had been subjected to rechromatography in 6 A4 GuHCl. This results from the occasional presence in p15 preparations of a

FIG. 4. Immunodiffusion tests demonstrating serologically distinct antigens in proteins of avian leukosis-sarcoma viruses. The center well of each pattern contains an antiserum prepared against an isolated protein of RSV-SR. The peripheral wells contain proteins from MC29 virus isolated by gel filtration in GuHCl. Antisera against proteins ~27, p19, and p12 were monospecific, and reacted only with the homologous antigens. Antisera against ~15 had predominant activity with ~15, but this serum also reacted to a lesser extent with p19. These two reactions formed lines of complete nonidentity, indicating the serological uniqueness of these specificities.

matography of tryptic peptides. It, therefore, appears that avian oncornaviruses possessan additional polypeptide sequence (p19) compared with mammalian (and reptilian) oncornaviruses. The selective advantage conferred by this extra protein in the avian viruses is not apparent at this time. The avian viral genome has recently been demonstrated to have a nucleotide sequence complexity of approximately 3 x lo8 daltons, allowing for the coding of 3 x 10’ daltons of polypeptides (Duesberg et al., 1974). Thus, AvLV p19 accounts for a significant percentage of the viral genetic information. [Evidence that AvLV gs proteins are, in fact, virus coded derives from the fact that tumors induced by RSV in mammals contain all four gs proteins, as shown by direct immunochemical analysis of the tumor cells and by the detection of antibodies to the gs proteins in the sera of tumor-bearing animals (Fleissner, 1970;

FIG. 5. Immunodiffusion tests demonstrating group-specific antigens in the internal proteins of avian leukosis-sarcoma viruses. The center well of each pattern contains an antiserum prepared against an individual protein of RSV-SR. The peripheral wells contain ether-treated avian leukosis-sarcoma viruses of the envelope subgroups: A (BHT-RAV-1); B (BHT-RAV-2); C (B77); D (CZAV); and E (RAV-0). Included in these tests, but not shown in these figures, were avian leukosis-sarcoma viruses of envelope subgroups: A, B (AMV and MC29); D (RSV-SR), and F (RAV61). Antisera prepared against ~27, p19, ~15, and pl2 (not shown) each identified serologically distinct antigens that were group-specific for all viruses tested.

GROUP-SPECIFIC PROTEINS OF AVIAN ONCORNAVIRUSES

small amount of a p19 fragment, apparently produced by protease activity in the virus preparation (Stromberg et al., 1974). The significance of this phenomenon, which initially lent some support to the hypothesis that p19 and p15 were related by a cleavage mechanism, is not clear. The preservation of group-reactive antigens in several AvLV proteins points to a strong selection for the maintenance of certain recognition sites of these proteins. Virion morphogenesis could provide roles with such selective advantages for these proteins. It is noteworthy that murine leukemia virus ~30, a protein with a role analogous to that of AvLV p27 in the formation of the viral core shell, displays group-specific (and interspecies-specific) properties. Gregoriades has reported that a protein of molecular weight 25,000 (“M” protein) in influenza virus, which resides immediately below the lipid bilayer within the virion, has a relatively low content (42%) of hydrophilic amino acids, and she related this property of the protein to its extractability in chloroform-methanol and its interaction with the viral membrane (Gregoriades, 1973). She also found that AvLV p27 was extractable in the same solvent, which suggested that p27 shares some properties with the influenza M protein. In fact, the amino acid composition of p27 resembles that of M protein, and p27 also has a hydrophilicity of 42% (Table 2). A considerable number of viruses with lipid-containing envelopes possess core proteins with molecular weights of 25,000-40,000, which appear to be closely associated with the inner surface of the viral membrane (Schultze, 1972; M&harry et al., 1971, Harrison et al., 1971). These include, besides oncornaviruses, influenza, parainfluenza, rhabdo-, and togaviruses. Since several instances have been described (McSharry et al., 1971; Huang et al., 1973; Krontiris et al., 1973) in which virions are composed of envelopes specified by one viral genome and cores specified by another, arguing for common recognition mechanisms, it will be of interest to examine further the common properties of “M” or major core (“C”) proteins.

365

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