Jun 11, 1973 - Radioimmune precipitation (RIP) tech- niques allowed precise ... California Medical School, Davis, California 95616. ..... arthropod-bome groupof animal viruses. Trans. N. Y. ... 583-605. In F. L. Horsfall and I. Tamm,(ed.),.
JOURNAL OF VIROLOGY, Nov. 1973, p. 1034-1042 Copyright 0 1973 American Society for Microbiology
Vol. 12, No. 5 Printed in U.S.A.
Antigenic Components of Group A Arbovirus Virions J. M. DALRYMPLE, S. N. VOGEL, A. Y. TERAMOTO,1 AND P. K. RUSSELL Department of Virus Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20012 Received for publication 11 June 1973
Three group A arboviruses, Sindbis (SIN), western (WEE) and eastern equine encephalitis (EEE), were selectively degraded with a nonionic detergent to yield a core particle and a soluble envelope component. Antigenic analysis by using radioimmune precipitation techniques revealed marked antigenic similarity among the core particles of the three viruses. The soluble envelope component exhibited antigenic specificity similar to that of intact virions. A close relationship between SIN and WEE envelopes was shown, whereas EEE envelope antigen appeared antigenically specific. These data indicate that nucleocapsids of group A arboviruses contain an antigenic determinant common to the group; the envelope contains virus-specific antigens as well as antigens which relate members of a subgroup.
Antigenic relationships among the group A arboviruses have been established primarily on the basis of complement fixation, hemagglutination inhibition, and neutralization test results by using unpurified antigen preparations derived from infected suckling mouse brain (7, 8, 10, 11, 28). These tests have exhibited varying degrees of both group and type specificity. The individual antigenic determinants contributing to the observed reactions have not been clearly defined, although both group- and type-specific determinants must reside on the virion surface because type-specific as well as cross-reactions have been observed by using purified virion preparations (12, 13, 21, 22, 26). Group-specific nucleoprotein antigens have been described for other RNA viruses (3, 14, 18, 19, 23), but only limited information (12) has been obtained for arboviruses. Group A arboviruses have been disrupted by a variety of methods to yield envelope and nucleoprotein antigens (1, 2, 4, 15, 16). Radioimmune precipitation (RIP) techniques allowed precise determination of antigenic relationships between subunits of degraded virions of three group A arboviruses. This investigation describes the antigenic relationships of the RNA-rich "core" particle and the detergent-solubilized "envelope" components.
equine encephalitis (WEE), MacMillan strain (17), and the Cambridge strain of eastern equine encephalitis (EEE) virus (6). Viruses were propagated in primary chicken embryo cell cultures infected with suckling mouse brain seed virus at a multiplicity of infection of approximately 10. Procedures for protein radiolabeling with 3H-amino acid mixtures and virion purification by ammonium sulfate precipitation and sucrose gradient centrifugation have been previously described (13). RNA radiolabeling included the addition of "4C-uridine (New England Nuclear Corp., Boston, Mass.) to infected cell cultures in conjunction with the 3H-amino acid mixture. Preparation of virus subunit antigens. Purified virion preparations were treated with the nonionic detergent Nonidet P-40 (Shell Chemical Co., N.Y., N.Y.) at a final concentration of 0.1% and incubated at 4 C for 10 min with frequent mixing. Separation of virion components was accomplished by rate-zonal centrifugation on 10 to 40% sucrose density gradients. Gradients were prepared by using a programmed gradient pump (Instrumentation Specialties Co., Lincoln, Neb.) with RNase-free sucrose (Schwarz/Mann, Div. of Becton, Dickinson & Co., Orangeburg, N.Y.) in a buffer consisting of 0.05 M Tris-hydrochloride, pH 7.4, 0.1 M NaCl, and 0.3% heat-inactivated fetal bovine serum. Centrifugation at 200,000 x g in an SW 50 L rotor (Spinco) for 50 min resulted in two separable radioactive components. Polyacrylamide gel electrophoresis (PAGE). Separation and identification of virion polypeptides was performed on 7.5% acrylamide gels according to the method of Maizel (20). Samples for PAGE analysis MATERIALS AND METHODS were heated at 100 C for 10 min in 1% sodium lauryl Virus propagation. Viruses selected for these stud- sulfate and 1% B-2-mercaptoethanol prior to gel ies were Sindbis (SIN) strain AR339 (29), westem analysis. Gel slices were incubated in a scintillation cocktail for 12 h at 37 C prior to counting. The liquid ' Present address: Department of Pathology, University of scintillation counting cocktail consisted of 3% ProtoCalifornia Medical School, Davis, California 95616. sol and 4.6% Liquifluor (New England Nuclear Corp., 1034
GROUP A ARBOVIRUS VIRIONS
VOL. 12, 1973
Boston, Mass.) in spectroquality toluene (Matheson, Coleman and Bell, Norwood, Ohio). Radioimmune precipitation (RIP) tests. The application of radioimmune precipitation procedures to the study of group A arboviruses has been previously described (13). Antisera to these viruses consisted of mouse hyperimmune ascitic fluids (MHAF) prepared according to the method of Brandt et al. (5) and modified by the use of Sarcoma 180 cells to induce ascites (24). Antisera to degraded virus components were prepared as above with the exception that all immunogen preparations were treated with 0.1% Formalin for 12 h at 37 C, followed by 96 h at 4 C to insure complete inactivation of infectivity. Residual infectious virus could not be detected by either plaque assay in primary chicken embryo cells or intracerebral inoculation of suckling mice. RESULTS
Antigenic specificity of intact virions. Purified virion preparations of each of the three viruses were examined by RIP tests by using antisera (MHAF) from animals immunized with infected suckling mouse brain suspensions (Fig. 1). Virions were precipitated maximally by their homologous antiserum over a wide range of antibody dilutions; however, reciprocal crossreactions were demonstrable between SIN and WEE viruses at somewhat lower antiserum dilutions. Precipitation of EEE virus appeared specific by comparison in that very little EEE virus was precipitated by SIN or WEE antisera. Conversely, EEE antisera did not react appreciably with SIN or WEE viruses. These results agree with the previously reported close relationship between WEE and SIN viruses and the serologically separate EEE virus (9). To investigate the hypothesis that antibodies of differing specificities resulted from separate antigen moieties in the immunizing antigen preparation, virion components were prepared for antigenic analysis. Characterization of subunit antigens. Treatment of 3H-amino acid-labeled SIN virion preparations with the nonionic detergent NP-40 and subsequent rate-zonal centrifugation in a sucrose gradient resulted in two separable protein peaks (Fig. 2A). No remaining intact virions were evident in treated preparations, indicating complete disruption by the detergent to a particle of approximately 140S and a soluble fraction. Untreated virions centrifuged on an identical gradient resulted in a single peak of radioactivity near the bottom of the gradient. Similar NP-40 treatment of preparations labeled with 8H-uridine yielded a single peak of radioactivity (Fig. 2B), indicating that all of the virus RNA was contained in the particle sedimenting at 140S. Detergent treatment of virions labeled with 3H-glucosamine resulted in a ra-
1035
dioactive soluble fraction (Fig. 2C). The absence of 3H-uridine-labeled RNA in the nonsedimenting component, and the absence of 3Hglucosamine label in the sedimenting particle indicated a high degree of purity of each preparation. Intact SIN virions and the products of NP-40 disruption (Fig. 2A) were analyzed by PAGE. SIN virion preparations exhibited the characteristic polypeptide composition previously described (27): the 53,000 mol wt membrane glycopeptide and the smaller 30,000 mol wt core associated polypeptide (Fig. 3). The RNA-rich, non-glycosylated, 140S particle contained only the 30,000 mol wt polypeptide, whereas the nonsedimenting glycosylated fraction consisted entirely of the larger 53,000 mol wt protein. Purified virion preparations of WEE and EEE virus were similarly treated with essentially identical results. PAGE analysis of core and envelope preparations indicated that each was essentially free of contamination by the other. Serological reactivity of envelope and core antigens. The RIP reaction of core preparations from each of the three viruses with homologous and heterologous antisera is shown in Fig. 4. Nucleocapsid antigens were cross-reactive to the extent that differentiation of the homologous reaction was impossible. Although end point titers were not as great as those observed with these antisera and virion antigens, crossreactivity was increased, especially with EEE nucleocapsid antigen. The SIN and EEE antisera had high titers of anti-core antibody and effectively demonstrated the extent of the cross-reactivity between core antigens. The WEE antiserum, on the other hand, exhibited some specificity, but extensive cross-reactions with SIN and EEE nucleocapsids were evident. Soluble envelope antigens were examined in the same manner, and the resulting RIP profiles are presented in Fig. 5. Envelope antigens were more specific than core antigens and strongly resembled intact virions in RIP reactivity. Homologous reactions were most prominent; the expected cross-reaction was observed with SIN and WEE viruses, and EEE virus envelope antigen was relatively specific. Again, as observed with nucleocapsid antigens, the maximum precipitation and antibody titers were lower than observed when using intact virions. Reactivity of antisera to virion, envelope, and nucleocapsid antigens. SIN virions, as well as nucleocapsid and envelope antigens, were used as separate immunogens for the preparation of specific antibody. Reactions of MHAF prepared against each of the SIN anti-
100
% RIP 40
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60
100 80 60 % RIP
40 20
100 80 60 % RIP
40 20
1.0
5.0 4.0 3.0 2.0 LOG I/ANTISERUM DILUTION
6.0
FIG. 1. Radioimmune precipitation profiles of purified radioactive (0) SIN, (0) WEE, and (A) EEE virions with mouse hyperimmune ascitic fluids. Virus antisera were anti-SIN (top panel), anti-WEE (middle), and anti-EEE (lower panel). Rabbit antimouse serum was used as the secondary precipitation antibody. Percent precipitation is plotted as percentage of RIP versus the log of the reciprocal of the virus antibody dilution. 10:36
A
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0 0tx 0 L
I0
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. 100
90
6
I
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toi it
30
1
40 50 60 70 FRACTION NUMBER
I
I
80
I
I
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100
FIG. 2. Sedimentation of detergent-disrupted SIN virus. Purified SIN virions intrinsically labeled with (A) 3H-amino acids, (B) 3H-uridine, and (C) 3H-glucosamine were treated with 0.1% NP-40 at 4 C for 20 min prior to centrifugation. Centrifugation was performed in a 4.8-ml 10 to 40%o (wt/vol) sucrose gradient on a 0.1-ml 70%o sucrose cushion at 200,000 x g for 50 min. Fractions (0.05 ml) were collected volumetrically from the bottom of the tube. 10:37
12 II 10
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4 3 2
5
10
15 20 25 30 35 40 45 50 55 60 65 70
5
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15 20 25 30 35 40 45 50 55 60 65 70
5
10
IS
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20 25 30 35 40 45 50 55 60 65 70 FRACTION NUMBER FIG. 3. Polyacrylamide gel electrophoresis of 3H-amino acid-labeled SIN virion (top), soluble envelope (middle), and nucleocapsid components (bottom). Prior to electrophoresis, samples were heated at 100 C for 10 min in 1% SLS and 1% 2-ME. Electrophoresis was performed in 8% gels in a buffer containing 0.1% SLS at 60 V until a bromophenol blue dye marker had migrated through the entire gel length. Migration was from left to right. Gels were frozen and sliced into 1-mm fractions. 1038
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8060% RIP
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20 0 1.0
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100 80 60% RIP
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%RIP
LOG I/ANTISERUM DILUTION FIG. 4. Radioimmune precipitation profiles of purified radioactive nucleocapsid preparations from (0) SIN, (0) WEE, and (A) EEE virus, with (A) SIN, (B) WEE, and (C) EEE virus mouse hyperimmune ascitic fluids. Antigen concentrations were adjusted to approximately 250 counts per min per 0.05 ml.
1(39
100
% RIP 40 20-
_
0
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B
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00_
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5.0 c
80 60 % RIP 40
20
0
T
20 3.0 4.0 6.0 5.0 LOG l/ANTISERUM DILUTION FIG. 5. Radioimmune precipitation profiles ofpurified radioactive soluble envelope components of (0) SIN, (0) WEE, and (A) EEE detergent disrupted virus with (A) SIN, (B) WEE, and (C) EEE mouse hyperimmune ascitic fluids. Equivalent antigen concentration was estimated by radioactivity at approximately 250 counts per min per 0.05 ml.
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GROUP A ARBOVIRUS VIRIONS
VOL. 12, 1973
TABLE 1. Specificity of antisera prepared to Sindbis virion component antigens Antisera Antigen
Sindbis virion Sindbis nucleocapsid Sindbis envelope WEE virion
Antiinfective virus (%)
AntiAntiAntivirion envelope nucleocapsid (%) (%)
98a 94
70 60
80 0
50 90
96 92
25
40 40
0 0
aMaximum precipitation occurring at optimal antibody concentration in the standard RIP test.
gens are shown in Table 1. A SIN virus MHAF prepared in the conventional manner, by using infected suckling mouse brain as the immunogen, has been included for comparison. This antiserum reacted with all antigens tested, including the cross-reactive WEE virion, and precipitated all antigens nearly 100%. Antisera to Formalin-inactivated SIN virions (antivirion) likewise reacted with all antigens, but maximum RIP was lower, especially with the envelope antigen. Antisera to envelope antigen reacted with both SIN virion and envelope antigens and even exhibited a cross-reaction with WEE virion, but did not react with nucleocapsid antigen. Antisera to SIN virus nucleocapsid gave maximum RIP with homologous nucleocapsid antigen and did not react with either envelope antigen or WEE virion; however, it did unexpectedly react with SIN virion.
DISCUSSION Detergent disruption of purified group A arbovirus virions allowed the resolution of two virus components which differed markedly in their antigenic specificity. Core particles, which contained all of the virus RNA as well as the 30,000 mol wt polypeptide, were broadly crossreactive when using antisera prepared with infected suckling mouse brain immunogens. The cross-reactions of antisera (MHAF) to SIN and EEE viruses with heterologous core antigens were of nearly the same magnitude as the homologous reactions, although no cross-reactions between these two agents occurred with intact virions or envelope antigens. The WEE antisera also cross-reacted extensively with core antigen; however, the homologous titers were significantly higher than heterologous titers. This group-reactive nucleocapsid antigen appears analogous to the common ribonucleo-
1041
protein antigen of influenza viruses (19) and certain RNA tumor viruses (3, 14, 23). Soluble envelope components, which contained only the larger 53,000 mol wt glycopeptide, exhibited antigenic relationships similar to those of the intact virion. SIN and WEE viruses were closely related and did not cross-react appreciably with EEE virus antisera. Although all radioactive envelope protein migrated as a single peak when using this method of polyacrylamide gel electrophoresis, convincing evidence has been presented which indicates that two separate virus glycopeptides are present in the 53,000 mol wt component (25). It is reasonable to suggest that once separated, one or both of these glycoproteins may be virus-specific antigen(s). The RIP reactions of antisera to envelope and nucleocapsid showed no antigenic relationship between envelope and core antigens. Antienvelope reacted with both homologous SIN and closely related WEE virions as expected; however, anti-SIN nucleocapsid also reacted with intact SIN virion. It seems unlikely that SIN nucleocapsid antigen is exposed on the surface of the intact virion because WEE virion did not react, even though this antiserum reacted strongly with cores prepared from all three viruses.
1. 2.
3. 4.
5.
6.
7. 8.
9.
10.
LITERATURE CITED Acheson, N. H., and I. Tamm. 1970. Purification and properties of Semliki Forest virus nucleocapsids. Virology 41:306-320. Appleyard, G., J. D. Oram, and J. L. Stanley. 1970. Dissociation of Semliki Forest virus into biologically active components. J. Gen. Virol. 9:179-189. Bauer, H., and W. Schafer. 1966. Origin of group-specific antigen of chicken leukosis virus. Virology 29:494-497. Bose, H. R., and B. P. Sakik. 1970. Immunological activity associated with the nucleocapsid and envelope components of an arbovirus. J. Virol. 5:410-412. Brandt, W. E., E. L. Buescher, and F. M. Hetrick. 1967. Production and characterization of arbovirus antibody in mouse ascitic fluid. Amer. J. Trop. Med. Hyg. 16:339-347. Byrne, R. J., G. R. French, F. S. Yancey, W. S. Gochenour, P. K. Russell, H. H. Ramsburg, 0. A. Brand, F. G. Scheider, and E. L. Buescher. 1964. Clinical and immunologic interrelationship among Venezuelan, eastem and westem equine encephalomyelitis viruses. Amer. J. Vet. Res. 25:24-31. Casals, J. 1944. Immunological relationships among central nervous system viruses. J. Exp. Med. 79:341-359. Casals, J. 1957. Viruses: the versatile parasites. I. The arthropod-bome group of animal viruses. Trans. N. Y. Acad. Sci. 19:219-235. Casals, J., and D. H. Clarke. 1965. Arboviruses; Group A., p. 583-605. In F. L. Horsfall and I. Tamm, (ed.), Viral and rickettsial infections of man, 4th ed. J. B. Lippincott Co., Philadelphia. Clarke, D. H., and J. Casals. 1955. Improved methods for hemagglutination studies with arthropod-bome viruses. Proc. Soc. Exp. Biol. Med. 88:96-99.
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