Hybridoma supernatants were reacted with purified CAEV bound to flexible .... In Hybridoma Technology in Agriculture and Veterinary Research, pp. 121-. 150.
J. gen. Virol. (1987), 68, 2259-2263. Printed in Great Britain
2259
Key words: retroviruses, goat and sheep/antigenic variation/core polypeptide
Antigenic and Structural Variation of the p28 Core Polypeptide of Goat and Sheep Retroviruses By T R A V I S C. M c G U I R E , * A L B E R T A L. B R A S S F I E L D , W I L L I A M C. D A V I S AND W I L L I A M P. C H E E V E R S Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164-7040, U.S.A. (Accepted 12 May 1987)
SUMMARY
The p28 core polypeptides of four isolates of caprine arthritis-encephalitis virus (CAEV) from goats was compared with those of visna virus (VV) and progressive pneumonia virus (PPV) from sheep. Monoclonal antibodies recognized p28 epitopes common to all six retrovirus isolates, a p28 epitope on four CAEV isolates, but not VV and PPV isolates, a p28 epitope on four CAEV isolates and VV, but not PPV and a p28 epitope unique to the CAEV isolate used for immunizing the mouse spleen donor. Comparison of two-dimensional maps of tyrosine containing tryptic peptides of p28 demonstrated that three CAEV isolates had similar maps while a fourth CAEV isolate, VV and PPV had several peptides different from the three closely related CAEV p28s and from each other.
Distinct, but related, retroviruses of the lentivirus subfamily occur in goats and sheep. In goats, caprine arthritis-encephalitis virus (CAEV) causes encephalitis in young kids (Cork et al., 1974), progressive arthritis in older goats (Crawford et al., 1980) and, in some instances, pneumonia and mastitis (Cork & Narayan, 1980; Crawford et al., 1980). Visna virus (VV) is the prototype of sheep lentiviruses and causes encephalitis (Gudnadottir, 1974). Retroviruses isolated from sheep with non-suppurative pneumonia are closely related to VV, but were designated maedi and progressive pneumonia virus (PPV) (Gudnadottir, 1974). In addition to pneumonia, PPV isolates cause lesions in the brain, mammary gland and joints (Oliver et al., 1981 ; Cutlip et al., 1985). These lentiviruses are not host-specific as PPV causes lesions in goats and CAEV causes lesions in sheep (Banks et al., 1983). Competitive genomic RNA/cDNA hybridizations show 15 to 30 ~ genome sequence homology among VV, PPV and CAEV isolates (Roberson et al., 1982; Gazit et al., 1983). Heteroduplex and molecular hybridization analyses of cloned CAEV and VV demonstrate that the greatest conservation of nucleotide sequences is in the gag and pol gene regions and in two other smaller regions (Pyper et al., 1986). Antigenically, some common determinants are shared among all the proteins and glycoproteins of CAEV, VV and PPV (Gogolewski et al., 1985). Most animals infected with CAEV make antibodies to the p28 major core polypeptide, and these antibodies are used to diagnose CAEV infection (Crawford et al., 1980; Roberson et al., 1982). Immunodiffusion and immunoprecipitation assays with sera from animals infected with CAEV, VV and PPV do not differentiate the p28s of these viruses (Weiss et al., 1977; Dahlberg et al., 1981 ; Roberson et al., 1982; Gogolewski et al., 1985). In this study, monoclonal antibodies (MAbs) and peptide maps were used to compare p28s from VV, PPV and four CAEV isolates. The CAEV-63 and -89 isolates were obtained by explantation of synovial membranes from goats with arthritis (Crawford et al., 1980) and CAEV-52 was from a brain explant culture from a naturally infected goat. The CAEV-Co isolate was provided by Dr L. C. Cork, Johns Hopkins University, Baltimore, Md., VV was provided by Dr A. Haase, University of Minnesota, 0000-7629 © 1987 SGM
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Short communication
(a) 1 2 3 4
5 6
(b) 1 2 3 4 5 6
5
(e) 1 2
(c)
l
2
3 4 5
200•
92-5• 69 • 46• 30~
14.3•
(d) 1 2
3
4
6
34
56
(f) 1 2 3 4 5 6
4669 30,-
....
Fig. 1. Immunoprecipitation of 35S-labelled goat and sheep lentiviruses with five MAbs made to CAEV-63. Lanes I to 6 of each panel represent MAbs 5A1, 10A1, 8BI, 13B1 and 12A1 and a control MAb respectively. Lane 7 of (b) is non-immunoprecipitated [35S]methionine-labelled CAEV-Co. (a to f ) Immunoprecipitation results with [3sS]methionine-labelled CAEV-63, CAEV-Co, CAEV-52, CAEV-89, VV and PPV, respectively. The lanes from each panel are cut from the same autoradiograph of the same gel. Mol. wt. values given are x 10-3.
Minneapolis, Mn., and PPV was provided by Dr R. C. Cutlip, U.S. Department of Agriculture, Ames, Ia., U.S.A. The C A E V isolates were grown in primary foetal goat synovial membrane cultures (Crawford et al., 1980) while VV and PPV were grown in sheep choroid plexus cells. Unlabelled and [35S]methionine-labelled viruses (Johnson et al., 1983) were purified by ultracentrifugation on 5 to 45~o sucrose gradients (Cheevers et al., 1981). BALB/c mice were immunized with p28 separated by gel filtration (P-100, Bio-Rad from 10 mg of purified, NP40-disrupted CAEV-63. Spleen cells from these mice were fused with myeloma cells (Davis et aL, 1983). Hybridoma supernatants were reacted with purified C A E V bound to flexible microtitre plates (Dynatech Laboratories, Arlington, Va., U.S.A.), washed three times with T E N buffer (20 mM-Tris-HC1, 5 mM-EDTA, 100 mM-NaC1, 5 mg/ml bovine serum albumin pH 7.4), incubated with rabbit antiserum to mouse immunoglobulin (Ig), washed three times, incubated with ~2SI-labelled protein A and washed three times. Individual wells were cut out and counted. Five hybridomas were cloned twice and the Ig isotype was determined to be IgG1 in double immunodiffusion with commercial antisera (Bionetics Laboratory Products Division, Charleston, S.C., U.S.A.); the Ig concentration was measured by single radial immunodiffusion (Johnson et al., 1983).
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Short communication Table 1. Monoclonal antibody binding to virus-infected cells assayed by indirect immunofluorescence MAb (0.1 mg/ml) 5A1 10AI 8B1 13B1
12A1
, CAEV-63 + +
Virus used to infect cells ~' CAEV-Co VV PPV + + + + + -
+
+
+
-
+ +
+ -
-
-
None
-
The panel of five MAbs was evaluated for binding to [35S]methionine-labelled viral proteins in an immunoprecipitation assay (Johnson et al., 1983; Gogolewski et al., 1985). All five antibodies (5A1, 8BI, 10A1, 12A1 and 13B1) precipitated only p28 from 35S-labelled CAEV-63 proteins as determined by autoradiography of precipitates separated by SDS-PAGE (Fig. 1a). When 35S_labelled CAEV-Co was evaluated, antibody 12A 1 failed to bind while the remaining four antibodies bound p28 (Fig. 1 b). Results with two additional isolates of CAEV, 52 and 89, were similar to those with CAEV-Co (Fig. 1 c, d). The p28 bands immunoprecipitated by antibody 13B1 in lanes 4 of Fig. l(c) and (d) are faint, but were clearly visible on the autoradiographs. Antibodies 5A1, 8B1 and 10AI bound 35S-labelled VV p28, while 12A1 and 13B1 did not (Fig. l e). Antibodies 5A1 and 10A1 bound 35S-labelled PPV p28, while 12A1, 13B1 and 8BI did not (Fig. l f ) . The reactivity of the five MAbs with virus-infected and uninfected cells was evaluated by indirect immunofluorescence (Cheevers et al., 1981) using fluorescein isothiocyanateconjugated rabbit antibodies to mouse Ig. The reactivity of the antibody panel in immunofluorescence tests (Table 1) was the same as in immunoprecipitation tests (Fig. 1) except that antibody 10A1 failed to bind PPV in immunofluorescence tests. The results from the MAb studies indicated that antibody 5A1 recognized a p28 epitope common to all six isolates in both immunoprecipitation and immunofluorescence assays. This antibody may be useful to detect sheep and goat retroviruses in culture and in infected tissues. Another antibody (12A 1) recognized an epitope unique to CAEV-63 which was the virus isolate used for immunization of the spleen donor mice. Monoclonal antibody 13B 1 bound to all CAEV isolates, but not to VV and PPV; this antibody may differentiate CAEVs. Monoclonal antibody 8B1 bound all CAEV isolates and VV, but not PPV. Demonstration of antigenic differences among the p28s from goat and sheep retrovirus isolates is similar to findings with murine retroviruses (Chuat et al., 1985). For peptide mapping, the p28s were cut from Coomassie Brilliant Blue-stained 7.5 to 17.5~o SDS-PAGE gels of purified viruses. The p28 was the predominantly stained band for each virus and was easily separated from the other proteins. Slices were washed with 25 ~ isopropyl alcohol and 10 ~ methanol, dried and labelled with 125i (Elder et al., 1977). Labelled slices were washed, dried and digested with TPCK-treated trypsin (Elder et aL, 1977). After digestion, the supernatant was removed from the gel slices, lyophilized, resuspended in electrophoresis buffer and the peptides were separated by thin-layer electrophoresis and chromatography (Whittaker & Moss, 1981) on 20 x 20 cm cellulose plates (EM Reagents, Darmstadt, F.R.G.). The plates were dried and exposed to X-Omat film. Comparison of CAEV-63 (Fig. 2a), CAEV-Co (Fig. 2b) and CAEV-52 (Fig. 2c) revealed similar p28 peptide maps, while CAEV-89 p28 (Fig. 2d) had differences. Also, differences in p28 peptides occurred when the related p28s of CAEV (63, Co and 52; Fig. 2a, b, c) were compared with VV (Fig. 2e) or PPV (Fig. 2f), when CAEV-89 (Fig. 2d) was compared with VV (Fig. 2e) or PPV (Fig. 2f) and when VV (Fig. 2e) was compared with PPV (Fig. 2f). Structural differences provide a plausible explanation for the differential reactivity by some of the MAbs with p28s. Similar structural differences occur in the major core polypeptide of primate lymphotropic retrovirus isolates (Jurkiewicz et al., 1986) and mouse retrovirus isolates (Gautsch et al., 1978). In contrast, equine infectious anaemia virus isolates have similar p26s (the major
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Short communication
Fig. 2. Two-dimensional maps of 125I-labelled tryptic peptides of isolated p28 of goat and sheep lentiviruses. (a to f ) p28s from CAEV-63, CAEV-Co, CAEV-52, CAEV-89, VV and PPV, respectively. (*) represents the origin and (1")is at the end of the first chromatography step. Electrophoresis was to the right and chromatography was upward.
core polypeptide) with the exception of a stable change in the p26 of one isolate after passage in horses (Montelaro et al., 1984). In conclusion, the p28 peptide maps show divergence among CAEV isolates and between VV and PPV, preventing differentiation of goat from sheep lentiviruses. In contrast to the results with p28 peptide maps, one MAb differentiates goat from sheep lentiviruses. The expert technical assistance of Linda Norton, Deta Stem, Mary Jo Hamilton and Jan Carlson is gratefully acknowledged. This work was supported by funds from the United States Public Health Service grant AM 27680 and United States Department of Agriculture, Animal Health Special Research grant 84-CRSR-2-2442. REFERENCES BANKS, K. L., ADAMS,D. S., McGUIRE,T. C. & CARLSON,L L. (1983). Experimental infection of sheep by caprine arthritis-encephalitis virus and goats by progressive pneumonia virus. American Journal of Veterinary Research 44, 2307-2311. CHEEVERS, W. P., ROBERSON,S., KLEVJER-ANDERSON,P. & CRAWFORD, T. B. (1981). Characterization of caprine arthritis-encephalitis virus: a retrovirus of goats. Archives of Virology 67, 111-117. CHUAT, J.-C., GRCE, M., PILON, C. & GUILLEMIN,M.-C. (1985). Monoclonal antibodies to murine retrovirus protein p30. Journal of General Virology 66, 1855-1862. CORK,L. C. & NARAYAN,O. (1980). The pathogenesis of viral leukoencephalitis-arthritisof goats. I. Persistent viral infection with progressive pathological changes. Laboratory Investigation 42, 596-602. CRAWFORD, T. B., ADAMS,D. S., CHEEVERS,W. P. & CORK, L. C. (i980). Chronic arthritis in goats caused by a retrovirus. Science 207, 997-999. CUTLIP, R. C., LEHMKUHL,H. D., BRODGEN,K. A. & BROLIN,S. R. (1985). Mastitis associated with ovine progressive pneumonia virus infection in sheep. American Journal of Veterinary Research 46, 326-328. DAHLBERG,J. E., GASKIN,J. M. &PERK,K. (1981). Morphological and immunological comparison of caprine arthritis encephalitis and ovine progressive pneumonia viruses. Journal of Virology 39, 914-919. DAVIS, W. C., PERRYMAN, L. E. & MeGUIRE, T. C. (1983). The identification and analysis of major functional populations of differentiated cells. In Hybridoma Technology in Agriculture and Veterinary Research, pp. 121150. Edited by N. J. Stern & H. R. Gamble. Totowa: Rowman & AUanheld. ELDER, J. H., PICKETT, R. A., HAMPTON, J. & LERrqER, R. A. (1977). Radioiodination of proteins in single polyacrylamide gel slices. Tryptic peptide analysis of all major members of complex multicomponent systems using microgram quantities of total protein. Journal of Biological Chemistry 252, 6510~6515.
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(1978). Structural markers on core protein p30 of murine leukemia virus: functional correlation with Fv- 1 tropism. Proceedingsof the National Academy of Sciences, U.S.A. 75, 4170-4174. GAZIT, A., YANIV, A., DVIR, M., PERK, K., IRVING, S. G. & DAHLBERG, J. E. (1983). The caprine arthritis-encephalitis virus is a distinct virus within the lentivirus group. Virology 124, 192-195. GOGOLEWSKI, R. P., ADAMS, D. S., Mc'GUIRE, T. C., BANKS, K. L. & CHEEVERS, W. P. (1985). Antigenic cross-reactivity between caprine arthritis-encephalitis, visna and progressive pneumonia viruses involves all virionassociated proteins and glycoproteins. Journal of General Virology 66, 1233-1240. GUDNADOTTIR,M. (1974). Visna-Maedi in sheep. Progress in Medical Virology 18, 336-349. JOHNSON, G. C., BARBET, A. F., KLEVJER-ANDERSON, P. & Mc-'GUIRE, T. C. (1983). Preferential immune response to virion surface glycoproteins by caprine arthritis-encephalitis virus-infected goats. Infection and Immunity 41, 657-665. GAUTSCH, J. W., ELDER, J. H., SCHINDLER, J., JENSEN, F. C. & LERNER, R. A.
JURKIEWlCZ, E., NAKAMURA, H., SCHNEIDER, J., YAMAMOTO, N., HAYAMI, M. & HUNSMANN, G. (1986). S t r u c t u r a l
analysis of p19 and p24 core polypeptides of primate lymphotropic retroviruses (PLRV). Virology 1.50, 291-298. MONTELARO,R. C., PAREK8,R., ORREGO,A. & ISSEL,C. J. (1984). Antigenic variation during persistent infection by equine infectious anemia virus, a retrovirus. Journal of Biological Chemistry 259, 10539-10544. OLIVER, R. E., GORHAM, J. R., PARISH, S. F., HADLOW, W. J. & NARAYAN, O. (1981). O v i n e p r o g r e s s i v e p n e u m o n i a :
pathologic and virologic studies on the naturally occurring disease. American Journal of Veterinary Research 42, 1554-1559. PYPER, J. M., CLEMENTS, J. E., GONDA, M. A. & NARAYAN, O. (1986). S e q u e n c e h o m o l o g y b e t w e e n c l o n e d c a p r i n e
arthritis encephalitis virus and visna virus, two neurotropic lentiviruses. Journal of Virology 58, 665-670. (1982). Caprine arthritisencephalitis virus is distinct from visna and progressive pneumonia viruses as measured by genome sequence homology. Journal of Virology 44, 754-757. WEISS, M. J., ZEELON, E. P., SWEET, R. W., HARTER, D. H. & SPIEGELMAN, S. (1977). Immunological cross-reactions of the major internal protein component from 'slow' viruses of sheep. Virology 76, 851-854. WaITrAKER, R. G. & MOSS, B. A. (I981). Comparative peptide mapping at the nanomole level. Analytical Biochemistry 11, 56-60. ROBERSON, S. M., McGUIRE, T. C., KLEVJER-ANDERSON, P., GORHAM, J. R. & CItEEVERS, W. P.
(Received 30 December 1986)
