Antigenic Analysis of Isolated Polypeptides from Visna Virus

INFECTION AND IMMUNITY, June 1976, p. 1728-1732 Copyright 1976 American Society for Microbiology Vol. 13, No. 6 Printed in USA. Antigenic Analysis of Isolated Polypeptides from Visna Virus P. D. MEHTA,* F. H. LIN, AND H. THORMAR New York State Institute for Basic Research in Mental Retardation, Staten Island, New York 1314 Received for publication 22 January 1976 The antigenic activity of 1 Visna polypeptides separated by gel filtration in the presence of 6 M guanidine hydrochloride (GuHCl) was examined with rabbit antisera made specific for Visna virus. The results showed that the first (GuHCl 1) and the ninth (GuHCl 9) polypeptide peak reacted with the antisera when examined in immunodiffusion, passive hemagglutination, and complement fixation tests. Whole virus, GuHCl 1, and GuHCl 9, when tested with the antisera, appeared to be immunologically identical in the immunodiffusion test. However, GuHCl 1 reacted weakly with the antisera by all three techniques as compared with GuHCl 9 and whole virus. GuHCl 9, when subjected to polyacrylamide gel electrophoresis containing.1% sodium lauryl sulfate, revealed the presence of one polypeptide with a molecular weight of 25,. By the same method, GuHCl 1 was found to contain an aggregate of four different polypeptides, the major one having a molecular weight of 25,. The results indicate that the antigenic activity of both GuHCl 1 and GuHCl 9 was associated with a single polypeptide having a molecular weight of 25,. Visna virus is the cause of a slow infection affecting the central nervous system of sheep (12). Recent studies from our laboratories (4, 9) have demonstrated that rabbits immunized with concentrated purified Visna and Maedi viruses form precipitating and low titers of neutralizing antibodies in addition to high titers of passive hemagglutination (PHA) and complement fixation (CF) antibodies. Our recent studies (7) have also shown that the purified Visna protein, after being disrupted by guanidine hydrochloride (GuHCl) and heating, was resolved into 1 polypeptide peaks by agarose column chromatography in 6 M GuHCl. Since it has been reported (2) that viral polypeptides separated by gel filtration in 6 M GuHCl recover their antigenic activity after the removal of the denaturing agent, we initiated a study of the antigenic properties of the isolated polypeptides of Visna virus using Visna-specific antisera made in rabbits. In addition, the homogeneity of some of the isolated polypeptides was studied by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium lauryl sulfate (SLS). The results showed that the antigenic activity resided in a single polypeptide with molecular weight of 25,. MATERIALS AND METHODS Cell cultures and virus. Cultures of sheep choroid plexus cells were used for production of virus. They were grown in 25-ml Falcon tissue culture flasks and maintained in Eagle basal medium with.2% bovine serum albumin as described previously (6). The cells were inoculated with Visna virus strain K796. Infectious tissue culture fluid was harvested, and the virus was concentrated by zinc acetate precipitation and purified on a potassium tartrate gradient as described previously (5, 6). Agarose gel filtration of Visna proteins in GuHCl. The gel filtration of Visna proteins was done as described previously (7), except that.7-ml fractions instead of 1-ml fractions were collected. Briefly, the purified virus containing about 5 mg of protein was dissolved in 8 M GuHCl (Ultrapure, Schwarz/Mann, Orangeburg, N.Y.) and heated in boiling water for 3 min. The polypeptides were chromatographed on a 6% agarose (Bio-Gel A-5M, 2 to 4 mesh, Bio-Rad Laboratories, Rockville Center, N,Y.) gel column (.9 by 85 cm) equilibrated in 6 M GuHCl. Each fraction was dialyzed exhaustively against distilled water for 72 h. Absorbance of the dialyzed fractions was measured at 28 nm, using distilled water as reference. Preparation of antisera. Antisera to purified Visna virus was prepared (9) in rabbits by injecting.5 mg of antigen first with complete Freund adjuvant into the footpads and then intramuscularly. The antisera were made specific for virus antigen by suitable absorption with lyophilized preparations of sheep serum, sheep liver, and bovine serum albumin (9). The absorbed sera failed to show any precipitin line in the Ouchterlony analysis when tested against sheep serum, sheep liver, bovine serum albumin, and a concentrated preparation of tissue culture fluid from uninfected sheep cells. Serological tests. The PHA test was carried out by the method of Stavitsky (13). Briefly, sheep erythrocytes were treated with tannic acid and coated with 5- to 1-,ug/ml solutions of Visna virus 1728

VOL. 13, 1976 and with pooled viral polypeptide fractions, respectively. All antisera were inactivated at 56 C for 3 min. Readings were recorded after 2 to 3 h at room temperature and after incubation overnight at 4 C. The CIF test was performed by the microtiter technique as described by Sever (11). Purified virus or viral polypeptides were used as antigens. In the procedure,.25 ml of antigen (protein content, 5 to 1 Ag/ml),.25 ml of specific antiserum to Visna and.5 ml of complement (2 U) were mixed and incubated overnight at 4 C. After incubation,.5 ml of sensitized sheep erythrocytes was added, and the mixture was incubated at 37 C for 3 min. CF titers were determined under conditions where no anti-complementary activity could be demonstrated. The immunodiffusion test was carried out according to the method of Ouchterlony (1). Determination of protein. Protein determinations of pooled polypeptides were performed by the method of Lowry et al. (8), and that of the whole virus was carried out by the modified Lowry method as described by Wang and Smith (14). In both methods bovine serum albumin was used as standard. Polyacrylamide slab gel electrophoresis. A 5 to 2% gradient of polyacrylamide slab gel containing.1% SLS was prepared according to the method of Baum et al. (1). About 1 ug of viral protein was precipitated with 5% trichloroacetic acid. The precipitate was washed once with 2 ml of cold acetone. The protein was dissolved in 4,ul of a buffer containing 62 mm tris(hydroxymethyl)aminomethanehydrochloride, ph 6.8, 2% SLS, 2% sucrose, 5% 2- mercaptoethanol, and.1% bromophenol blue. The solution was heated in boiling water for 2 min. Electrophoresis was carried out at 15 V/gel (1 cm high and 15 cm wide) for 14 h at room temperature. The gel was stained with Coomassie brilliant blue ANTIGENIC ANALYSIS OF VISNA POLYPEPTIDES 1729 for 3 h and destained with 7.5% glacial acetic acid containing 25% methanol. RESULTS Agarose gel filtration and immunological properties of Visna polypeptide peaks. The viral protein was resolved into 1 polypeptide peaks by agarose gel filtration in 6 M GuHCl. The profile of absorbance at 28 nm is shown in Fig. 1. For the sake of convenient identification, the peaks will be referred to as GuHCl 1 through 1. The 1 peaks observed in the present experiment are similar to those detected earlier using radioactively labeled virus (7). Before the appropriate fractions of each peak were pooled, each fraction, after being dialyzed, was examined in the Ouchterlony test against rabbit anti-visna serum. Initially, only fractions 13 to 14, comprising the peak of GuHCl 9, showed a precipitin line against specific rabbit anti-visna sera. However, after the appropriate fractions were pooled and concentrated, GuHCl 1, in addition to GuHCl 9, showed a precipitin line in Ouchterlony analysis with the same antisera. The results of immunological cross-reactions between whole Visna virus and isolated GuHCl fractions are shown in Fig. 2a and b. Whole Visna virus (well 1) showed two precipitin lines against rabbit anti-visna serum. The diffuse top precipitin line of whole virus showed a reaction of complete identity with GuHCl 9 (well 2) and GuHCl 1 (well 3) against rabbit anti-visna 1'4 7.2. E C.82.6 A_ I8 4 5 FRACTION I I I I I I 1 11 12 13 14 15 NUMBER FIG. 1. Gel filtration of Visna viral protein on an agarosegel column in 6 M GuHCI. The polypeptide peaks (GuHC1 1 and GuHCl 9), represented by the broken lines, indicate a positive reaction against rabbit anti- Visna serum.

173 MEHTA, LIN, AND THORMAR b INFECT. IMMUN. ^ FIG. 2. (a) Immunodiffusion- plate of rabbit anti-visna serum (center well) when tested against isolated GuHCl polypeptides. Outer wells contain: (1) whole Visna virus, 5 mg/ml; (2) GuHCl 9, 5 pg/ml; (3) GuHCl 1, 2 jg/ml; (4) GuHCl 6 through 8, 2 pg/ml; (5) GuHCl 3, 1 pg/ml; (6) GuHCl 1, 1 pg/ml. (b) Represents a schematic diagram of (a). serum. The other precipitin line near the antiserum well (center well) in whole Visna virus indicated the presence of another antigenic component. However, none of the GuHCl fractions showed this additional component. It was noteworthy that GuHCl 1, which had higher protein concentration than GuHCl 9 and the Visna virus preparation, reacted rather poorly in the Ouchterlony test. No polypeptide peaks other than GuHCl 1 and GuHCl 9 showed any precipitin line with rabbit anti-visna sera. The pooled fractions were further tested in PHA and CF tests using the same antisera. Again, only GuHCl 1 and GuHCl 9 showed detectable activities in these tests. The PHA and CF titers of one of the antisera tested against GuHCl 1, GuHCl 9, and whole purified Visna virus are shown in Table 1. The anti- Visna serum showed almost equal titers against GuHCl 9 and the Visna virus preparation in the PHA and CF tests. The antiserum also showed significant titers against GuHCl 1. However, the titers were somewhat lower than those seen against Visna virus and GuHCl 9. Except for GuHCl 1 and GuHCl 9, the other fractions did not show any detectable titer in the above tests. GuHCl 1 and GuHCl 9 did not show any positive reaction when tested in Ouchterlony and CF tests against sera from Visna-infected sheep. This was not surprising, since whole purified Visna virus preparation reacted very TABLE 1. PHA and CF antibody titers ofrabbit anti- Visna serum as tested against whole Visna virus, GuHCl 1, and GuHCl 9 Sample PHA Titer CF Whole Visna virus 6,4 512 GuHCI 1 1,6 64 GuHCl 9 12,8 512 poorly in these tests against one of the sheep sera that had very high neutralizing antibodies (titer of 1:8,). Thus, in the Ouchterlony test, whole Visna virus, at a protein concentration of 1 mg/ml, formed a very faint precipitin line against the sheep serum. In the CF test, the sheep serum had a titer of only 1:8 with purified Visna virus. The PHA test was not performed with sheep serum since it caused a nonspecific agglutination of sheep erythrocytes. Electrophoresis of GuHCI polypeptides. The viral polypeptides GuHCl 1, GuHCl 6 through 8 (pooled), and GuHCl 9 were analyzed on 5 to 2% PAGE-SLS, as described in Materials and Methods. GuHCl 1 had a major polypeptide component with a molecular weight of 25,, as measured by the relative rate of migration in the gel (Fig. 3). In addition, three other major polypeptide components were visible in the gel, with molecular weights of 1,, 8,, and 13,, respectively. The electrophoretic pattern of GuHCl 9 revealed

VOL. 13, 1976 ANTIGENIC ANALYSIS OF VISNA POLYPEPTIDES 1731 BSA --* W~~4 Ova. - O- Chy. - o RNase --o A * I B C D FIG. 3. Electrophoresis of GuHCl 1, GuHCl 6 through 8 and GuHCI 9. The polypeptides were dialyzed, precipitated by trichloroacetic acid, and electrophoresed as described in the text. (A) Standard proteins: 1 pg each ofbovine serum albumin (BSA; molecular weight, 68,); ovalbumin (Ova; molecular weight, 45,); chymotrypsinogen A (Chy; molecular weight, 25,); and pancreatic ribonuclease A (RNase; molecular weight, 13,). (B) 3 pg ofguhci 1. (C) 2 pg ofa mixture ofguhcl 6 through 8. (D) 1 pg ofguhcl 9..: + only one polypeptide with a molecular weight of 25,. Thus, GuHCl 9 appeared to be homogeneous. The mixture of GuHCl 6 through 8 was separated by PAGE into three different polypeptides with molecular weights of 1,, 8,, and 71,, respectively. DISCUSSION Analysis of the Visna GuHCl 9 by gel filtration in 6 M GuHCl and by PAGE-SLS indicates that this polypeptide is homogeneous. Its molecular weight was previously estimated to be about 12, by agarose gel filtration in 6 M GuHCl (7) but was found to be 25, when measured by its relative migration in PAGE- SLS. The same polypeptide having a molecular weight of 25, appeared to be a major component of GuHCl 1. It should be pointed out that a polypeptide with a molecular weight of 25, was also reported as a major Visna protein by Haase and Baringer (3). Since a mixture of GuHCl 6 through 8 was run on PAGE, no conclusion can be drawn as to the homogeneity of each individual peak. However, the molecular weights of the three polypeptide bands observed in PAGE are high (Fig. 3) compared with those estimated previously by gel filtration (7). The native antigens of Visna virus were recovered from the GuHCl eluates by dialysis against water. This result is very similar to that observed in oncornaviruses (2). In addition, the antigenic activity of the recovered polypeptides with the antisera measured by the PHA and CF tests was high compared with whole virus (Table 1). Only two polypeptide peaks, GuHCl 1 and 9, reacted with rabbit anti- Visna sera in immunodiffusion, PHA, and CF tests. The protein content of peak GuHCl 1 was at least four times higher than that of GuHCl 9. However, it reacted as a weaker antigen than GuHCl 9 when tested against rabbit anti-visna sera, indicating that the active antigen was partly inaccessible to the antibody due to aggregation with the other high-molecular-weight polypeptides. Since GuHCl 1 and GuHCl 9 showed a single precipitin line with complete identity in the Ouchterlony test, it was concluded that the 25,-dalton polypeptide was the active antigen in both peaks. The other main groups ofpolypeptides, namely, the glycopolypeptide GuHCl 3 (7) and GuHCl 6 through 8, did not react with the rabbit anti-visna virus sera, indicating that GuHCl 3 and GuHCl 6 through 8 are immunologically different from GuHCl 9. Whether or not the individual polypeptides (GuHCl 6-8) are antigenic when injected separately into rabbits needs further study. It is of interest to note that GuHCl 9, which is antigenically active with the rabbit antisera, failed to show positive reaction with sera obtained from Visna-infected sheep, although these sera showed high neutralizing activities against Visna virus. These results indicate that the antigenic components that formed antibodies in rabbits and those that cause the formation of antibodies in sheep after inoculation with Visna virus or during the natural course of the disease are different. ACKNOWLEDGMENTS The technical assistance of Arlene Kane and Mark Edelstein is gratefully acknowledged. We wish to thank H. M. Moon for performing polyacrylamide gel electrophoresis. LITERATURE CITED 1. Baum, S. G., M. S. Horwitz, and J. V. Maizel, Jr. 1972. Studies of the mechanism of enhancement of human

1732 MEHTA, LIN, AND THORMAR adenovirus infection in monkey cells by simian virus 4. J. Virol. 1:211-219. 2. Fleissner, E. 1971. Chromatographic separation and antigenic analysis of proteins of the oncornaviruses. I. Avian leukemia-sarcoma viruses. J. Virol. 8:778-785. 3. Haase, A. T., and J. R. Baringer. 1974. The structural polypeptides of RNA slow viruses. Virology 57:238-25. 4. Karl, S. C., and H. Thormar. 1971. Antibodies produced by rabbits immunized with visna virus. Infect. Immun. 4:715-719. 5. Lin, F. H., and H. Thormar. 197. Ribonucleic aciddependent deoxyribonucleic acid polymerase in visna virus. J. Virol. 6:72-74. 6. Lin, F. H., and H. Thormar. 1971. Characterization of ribonucleic acid from visna virus. J. Virol. 7:582-587. 7. Lin, F. H., and H. Thormar. 1974. Substructures and polypeptides of visna virus. J. Virol. 14:782-79. 8. Lowry,. H., N. J. Rosebrough, A. L. Farr, and R. J. INFECT. IMMUN. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 9. Mehta, P. D., and H. Thormar. 1975. Comparative studies of visna and maedi viruses as antigens. Infect. Immun. 11:829-834. 1. Ouchterlony,. 1958. Diffusion-in-gel methods for immunological analysis, p. 1-78. In P. Kallos and B. H. Waksman (ed.), Progress in allergy. S. Karger, Basel. 11. Sever, J. L. 1962. Application of a microtechnique to viral serological investigations. J. Immunol. 88:32-329. 12. Sigurdsson, B., P. A. Palsson, and H. Grimsson. 1957. Visna, a demyelinating transmissible disease of sheep. J. Neuropathol. Exp. Neurol. 16:389-43. 13. Stavitsky, A. B. 1954. Micromethods for the study of proteins and antibodies. J. Immunol. 72:36-367. 14. Wang, C. S., and R. L. Smith. 1975. Lowry determination of protein in the presence of Triton X-1. Anal. Biochem. 63:414-417.