Multiple species-specific and interspecific antigenic determinants of a mammalian type C RNA virus internal protein

lmmunochemistry, 1975. Vol. 12, pp. 6272. Pergamon Press. Printed in Great Britain

MULTIPLE SPECIES-SPECIFIC A N D INTERSPECIFIC ANTIGENIC DETERMINANTS OF A MAMMALIAN TYPE C RNA VIRUS INTERNAL PROTEIN J A M E S DAVIS, R A Y M O N D V. G I L D E N and S T E P H E N O R O S Z L A N Flow Laboratories, Inc., Rockville, Maryland 20852, U.S,A. (Received 20 May 1974) Abstract--The major internal protein, p30, of mammalian Type C viruses contains multiple antigenic determinants in both species-specific and interspecific categories. These were demonstrated by use of p30s modified reversibly by citraconylation, and a p30 fragment isolated after limited trypsin digestion. Six distinct reactivities were demonstrated by direct and absorption analyses in gel diffusion. In certain instances, separation of distinct antibody populations was also achieved by use of immunoadsorbents. Citraconylated p30 (p30c) was deficient in two of three species-specific reactivities, and at least one of three interspecies reactivities. A tryptic fragment from mouse p30 contained one species-specific determinant not shared with p30c and lacked one interspecies determinant contained in p30c. P30c also contained a species-specific determinant not shared with the tryptic fragment. munized with M-MSV tumor homogenates (Gilden et al., 1971).

INTRODUCTION

Previous studies have demonstrated the presence of qualitatively distinct antigenic determinants on the major internal group-specific protein, p30, of mammalian Type C viruses (Gilden et al., 1971 ; Oroszlan et al., 1971). In addition to differential absorption of species-specific and interspecific reactivities in gel diffusion, the use of immunoadsorbents has enabled the separation of antisera into distinct antibody populations corresponding to categories of reaction (Oroszlan et al., 1975). Recent studies with p30 fragments generated by limited tryptic hydrolysis have indicated that individual p30 molecules possess multiple determinants corresponding to the species-specific and interspecific categories (Davis et al., 197.3). In this communication the results of gel diffusion analyses with one major tryptic fragment, and p30 molecules modified reversibly by citraconylation are presented. Utilizing immunoadsorbent procedures to verify the individuality of the reactivities, at least six unique determinants are described: three of the species-specific class, and three of the interspecific class.

C itr acon ylation The method described by Atassi and Habeeb (1972) was used with minor modifications. Purified p30s were dissolved in deionized water to give a final concentration of 400-500 #g protein/ml. To aliquots (0.~l.0ml) of the protein solutions, adjusted with 5 N NaOH to pH 8.0-8.5, citraconic anhydride (Aldrich Chemical Co.. Inc., Milwaukee, Wisconsin) was added in small quantities(5 pl to 0.2 ml) at a time at 5 min intervals. The pH was maintained by the addition of 5 N NaOH using a pH stat (Radiometer, Copenhagen) with constant stirring. A total of 25 pl reagent/0.2 ml protein solution (2.6-3.3 nmole of p30) was used and the reaction mixture was stirred for an additional 15 min after the last addition of citraconic anhydride. Assuming 16 free amino groups per mol of p30.(Summers and Oroszlan, unpublished data), the amount of citraconic anhydride used represented a 5.3-6.7-fold molar excess over the total amino groups. Untreated p30s were diluted to an equal concentration with a buffer prepared as follows: 25#1 citraconic anhydride/0.2 ml H_,O was allowed to completely hydrolyze; the pH was then adjusted to 8"0-8'5 with 5 N NaOH. P30s diluted in this buffer were identical in immunodiffusion to p30s diluted in H20. The citraconylation reaction was reversed by the addition of a 25% (v/v) solution of acetic acid as required to produce a final pH of 5.(r 5"8. This acidified solution was incubated overnight at room temperature before re-establishment of pH 8.0-8.5 with 5 N NaOH.

MATERIALS AND METHODS

Viruses, p30 purification, gel diffusion, and immunoadsorbent procedures are described in the accompanying paper (Oroszlan et al., 1974). Protein determinations Protein concentrations were determined from OD_,~0/as,, measurements by the method of Groves et al. (1968).

RESULTS

Reaction o f citraconylated p30s with species-specific antisera Antisera specific for rat (RaLV), endogenous cat (RD-114), and mouse (MuLV) p30s were tested against

Antisera Antisera used in these studies were from goats andguinea pigs immunized with the purified p30s and from rats im67

68

JAMES DAVIS, RAYMOND V. GILDEN and STEPHEN OROSZLAN

Fig. 1. Immunodiffusion patterns showing reactions of species-specific anti-p30 (gs) sera with purified untreated, and citraconylated p30 proteins. Amisera in center wells. Rp30gps: guinea pig antiserum made against purified rat virus M-MSV(RaLV) gs antigen (p30); RDp30goS: antiserum raised in goat against purified R D 114 gs antigen (p30); Mp30goS: goat antiserum against purified mouse virus gs antigen (p30). Antigens iJl I,eripheral wells as indicated. Abbreviations: R : rat: M : mouse: and R D: R D 114 viruses, c: citraconylated cr: citraconylated and reversed. untreated, citraconylated, and citraconylated-reversed p30s. The species-specificity of the antisera is shown in Fig. 1, and in addition, absorption experiments with heterologous p30s failed to influence the patterns obtained (not shown). In each case, citraconylated p30s (p30c) were antigenically deficient, as shown by partial identity reactions, with respect to untreated p30. Complete serological reactivity was recovered after reversal of the citraconylation reaction by incubation at pH 5.6-5.8 for 18 hr (Figs. 1A-C). The patterns shown in Fig. I predict the separation of species-specific antisera into at least two major populations. This was accomplished in the case of the anti-MuLV serum by adsorption with p30c attached to CNBr activated Sepharose (see legend to Fig. 2). The antibody population not retained on the adsorbent reacted only with p30 (Fig. 2 1), and this reaction was not eliminated by further absorption of this serum

fraction with p30c (Fig. 2- 2). The antibody population which bound to p30c and which was subsequently eluted at acid pH did not distinguish between p30 and p30c determinants, giving identity reactions (Figs. 2 and 3) which were completely removed by prior absorption with intact p30 (Figs. ~4). Reaction q['citraconylated l~30s with interspecie,s reactit,e antisera

Two antisera which detect interspecies determinants as well as species-specific determinants were tested with heterologous p30s and p30c's. Both sera (anti-MuLV, anti-FeLV) show the characteristic reaction of partial identity between homologous and heterologous p30s placed in adjacent wells (Fig. 3). The heterologous p3Oc's are still detected by these antisera but are clearly antigenically deficient with regard to intact heterologous p30s (Fig. 3). In these assays, no differences were

Multiple Species-Specific and lnterspecific Antigenic Determinants

69

Fig. 2. Precipitin reactions of various antibody populations present in goat anti-mouse gs serum (Mp30goS) and separated by immunoadsorption technique (Oroszlan et al., 1974) with citraconylated mouse p30 (p30c) attached to CnBr activated Sepharose. Whenp30c was used to prepare immunoadsorbent, wash with 0.1 M acetic acid was omitted. Wells. (1) Antibody not retained in the immunoadsorbent, (2) Antibody as in Well 1 but adsorbed with mouse p30c by letting the antigen diffuse into the agar before challenging the well with antibody. The line between Well 2 and goat anti-Mgs serum indicates excess of adsorbing antigen. (3) Antibody retained on p30c immunoadsorbent and subsequently eluted with 0.1 M acetic acid, (4) Antibody as in Well 3 but adsorbed with p30c as described above. Other wells are filled with antigens as indicated. See abbreviations in legend to Fig. 1. seen between the determinants detected on the heterologous p30c's. The antigenic deficiency of cross-reactive determinants was reversible in a similar manner as described above for the species-specific reactions.

Relationship of determinants survivinq trypsinization and citraconylation Some species-specific and cross-reacting determinants of MuLV p30 are destroyed by both limited

~. ~ . a ~ : ~

/

Fig. 3. Precipitin reactions of various mammalian Type C virus p30 and p30c antigens with interspeciesspecific antisera. Center Wells. Mu LVgS: antiserum prepared against Tween 80-ether disrupted R-MuLV. Fp30goS: antiserum made by immunization with FeLV electrofocus purified gs antigen (p30). Peripheral Wells. Antigens as indicated. Abbreviations: F. feline, and as in legend to Fig. 1.

JAMES

DAVIS,

RAYMOND

V. GILDEN

and

STEPHEN

OROSZLAN

Fig. 4. Immunodifision reactions showing relationships of various mammalian gs antigens (~30) before and after citraconylation and the mouse p30 fragment obtained by limited trypsin hydrolysis followed by isoelectric focusing (IEFP). All three sera used here react in both intra- and interspecific fashion. In pattern A, MSV I-14 serum (see Materials and Methods) shows a weak but definite reaction with RD I14gs. Abbreviations as noted previously.

gs anugens

in pattern

(p9v~ ano ,ption with IEFP. Ce~trr IV&s. adsorbed with IEFP. Adsorptions were carried 1 pattern A between Well 1 and MSV I-14, and B between Well 2 and MSV I-14 indicate that the adsorbing antigen (IEFP) was present in ... .. reactive wnn .*.... . . 1 **~~~. 1._~. 1 excess to anttnoates it. Anrtgens m peripnerat weus as matcareu

Multiple Species-Specific and lnterspecific Antigenic Determinants trypsinization (Davis et al., 1973) and citraconylation as described above. The relationship of the determinants surviving each procedure was investigated. A rat antiserum designated MSV 1-14 has been previously demonstrated to detect both species-specific and interspecies reactivities on a tryptic polypeptide purified to homogeneity by isoelectric focusing (designated IEFP; Davis et al., 1973). With this serum the line formed with the IEFP was found to spur into the line formed with MuLV p30c (Fig. 4A)I Thus, IEFP lacks determinants found on p30c. A hyperimmune goat antisera prepared against disrupted MuLV shows a slightly different relationship between MuLV p30c and the IEFP. With this serum there is a double spur between the respective immunodiffusion wells (Fig. 4B). Antibodies are present in this serum which recognize determinants on IEFP that are missing on p30c and the reverse is also true. The hyperimmune anti-FeLV p30 serum that recognizes the cross-reacting determinants which survive citraconylation does not form a visible precipitation line with the MuLV IEFP (Fig. 4C). This serum apparently contains a population of cross-reactive antibodies that recognizes MuLV p30c but not IEFP. Comparison o f cross-reactivities of two sera

The cross-reacting determinants detected in immunodiffusion were found to vary with different test sera. Figure 5 shows an example of two sera which have very different populations of cross-reactive antibodies. All of the cross-reactivity of MSV 1-14 is removed by adsorption in the immunodiffusion well with IEFP. The species-specific determinants on un-

71

treated MuLV p30 and p30c survive this adsorption as expected from Fig. 4A. In contrast, a similar adsorption of the goat anti-FeLV p30 with MuLV IEFP does not remove the cross-reactions with either MuLV p30 or MuLV p30c (Fig. 5B). Both the precipitation (Fig. 4C) and adsorption reactions seen with this anti-FeLV p30 sera demonstrate that it lacks antibodies detectable in immunodiffusion to the cross-reacting determinants of MuLV IEFP.

DISCUSSION The present studies have demonstrated at least six antigenic reactivities associated with MuLV p30 which can be visualized in gel diffusion. A summary of these determinants and identifying sera is given in Table 1. The specificity of the direct gel reactions was confirmed where possible by adsorption analyses, thus the negative reactions are not based on valence considerations. The finding of multiple unique determinants on individual protein molecules is to be expected based on an extensive literature. These determinants may be regions of defined sequence or may result from conformational requirements. Thus, the loss of both speciesspecific and interspecies reactivities in citraconylated p30 molecules might be expected from conformational changes while those determinants surviving this procedure and limited trypsinization might be expected to be definable in terms of primary structure. A principal aim of our continuing effort in this area is a more precise definition of the individual p30 determinants by amino acid sequence analysis followed by synthesis

Table 1. Summary of various antigenic determinants present on MuLV gs antigen, p30 Determinant

Antigenic specificity

Detectable in antigens

Not detectable in antigens

Detected with antiserum

Reference to figure

SSa

Species

p30

p30c IEFP

Goat MuLV Goat MuLV gs MSV 1-14

I C, 2 4A, B

SSb

Species

p30 p30c

IEFP

Goat MulV Goat MuLV gs" MSV 1-14

4A, B 5A

SSc

Species

p30 IEFP

p30c

Goat MuLV

4B

ISa

Interspecies

p30

p30c IEFP

Goat FeLV gs

4C

ISb

Interspecies

p30 p30c

IEFP

Goat FelV gs

3B, 4C, 5B

ISc

Interspecies

p30 IEFP p30c (?)b

MSV 1-14

5A'

' Not shown in figures presented. The presence of this determinant on p30c is indicated in Fig. 4A, where the precipitin line with MSV 1-14 shows only a one-way spur between IEFP and p30c (No cross). However, this cannot be accepted as final. c Davis et al. (1973).

72

JAMES DAVIS, RAYMOND V. GILDEN and STEPHEN OROSZLAN

and use in immunoassays. As pointed out in the Introduction of the accompanying paper, p30 has proven an invaluable marker in a variety of studies related to RNA tumor viruses. Precise definition of antigenic regions, especially those shared among a variety of viruses, would be an important first step in the development of new reagents for sensitive tests of human materials for similar reactivity. In the initial work with p30, it was commonly thought of as possessing a single immunologica reacti,city. Subsequently, the existende of both species-specific and interspecific determinants on these molecules was shown (Gilden et al., 1971; Oroszlan c t a l . , 1971) although not readily accepted by many virologists. Now it is clear that these molecules carry multiple determinants corresponding to each category of reaction and type-specific determinants as well (Strand and August, 1974; Gilden et al., in press.). Acknowledgements This work was supported by Contract NOI-CP-3-3247 from the Virus Cancer Program of the National Cancer Institute, National Institutes of Health, Bethesda. Maryland 20014. The authors wish to thank Mr.

David Bova and Mr. Larry Masters for excellent technical assistance.

REFERENCES Atassi M. Z. and Habeeb A. F. S. A. (1972) Methods in En:ymolo~,ty (Edited by Hits. C. H. W. and Timashcff. S. N.k Vot. XXV, Part B, p. 546. Academic Press, New York. Davis J., Gilden R. V. and Oroszlan S. (1973) Virology 56, 411. Gildcn R. V., Oroszlan S. and Hatanaka M. (1974) Viruses, Evolution and Cancer (Edited by Maramorosch K. and Kurstak E.), from Second int. Congress of Comparative Virology, 1973, Montreal: Academic Press, New York. (in press). Gilden R. V., Oroszlan S. and Huebner R. J. (1971) Nature, New Biol. 231, 107. Groves W. E.. Davis F. O. C.. Jr. and Sells B. H. (1968) Analyt. Biochem. 22, 195. Oroszlan S., Huebner R. J. and Gilden R. V. (1971) Proc. hath. Acad. Sci. U.S.A. 68, 901. Oroszlan S., Bova D. and Gilden R. (1975) lmmunochemistry 12, 6l. Strand M. and August T. (1974) J. ! irol. 13, 171.