VIROLOGY

99,

192-196

(1979)

Characterization

HYUNE Laboratory

of

of Reverse Transcriptase from Virus by Radioimmunoassay

M. RHO AND ROBERT

Tumor Cell Biology,

National Accepted

Cancer

Institute,

Feline

Leukemia

C. GALLO’ NIH,

Bethesda,

Maryland

20205

August 27, 1979

Reverse transcriptase (RT) of feline leukemia virus (FeLV) has been purified and characterized by radioimmunologic techniques. The radioimmunoassay detects about 2 ng of the purified enzyme. Competition immunoassays revealed that the enzyme possesses antigenic determinants not shared by other known feline viral proteins or any proteins present in fetal calf serum. Group-specific antigenic determinants of FeLV RT were demonstrated in homologous competition immunoassays. In this assay, antigenicity of RT among subgroups of FeLV were indistinguishable. Heterologous immunoassays showed that the enzyme was broadly cross-reactive with other mammalian type-C viral polymerases, indicating that these DNA polymerases share interspecific antigenic determinants. An analysis of natural cat sera confirmed earlier observations that infection of cats by FeLV can result in serologically detectable antibodies to specific reverse transcriptase.

Reverse transcriptases (RT) have been purified and characterized from several mammalian type-C viruses, and in general, have shown to consist of a single polypeptide of 70,000 to 84,000 daltons (1, 2). The relatedness of RT from various retroviruses has been extensively studied by immunological enzyme activity neutralization assays, and by these tests RT of mammalian type-C viruses can be distinguished but show evidence of group-specific antigenic relatedness (3). More recently in the case of RT from baboon endogenous virus, enzyme binding assays have shown that RT may exhibit type-specific as well as group-specific antigenie determinants (4). Recently, we have become interested in the detailed biochemical and immunological properties of RT from various strains of feline leukemia virus (FeLV): first, because antibodies to this protein can be a sensitive indicator of natural infection of cats by FeLV (5); and second, because IgG molecules which react with RT from mammalian type-C viruses have been discovered in the surface of some human leukemia blood cells (6). In some types of leukemia, these interactions are apparently stronger with RT from FeLV. ’ To whom reprint requests should be addressed. 0042-6822/79/150192-05$02.00/O Copyright All rights

Q 1979 by Academic Press, Inc. of reproduction in any form reserved.

The characteristics of RT from FeLV do not appear to substantially differ from RT of other mammalian type-C viruses, consisting of a single polypeptide of 70,000 to 72,000 daltons (7, 8), and exhibiting antigenie relatedness to RT from some other mammalian type-C viruses as determined by RT activity neutralization assays (3). Although these assays have been very useful, more precise determinations of relatedness of a given protein can be determined by radioimmunological assays. Such assays are also of use for following epidemiological patterns of infections, and these assays can be used to detect catalytically inactive RT, such as RT biosynthetic precursors (9) which can sometimes be the only detectable form of RT in a cell (IO), as well as enzymatitally active molecules. In contrast, RT neutralization assays detect only catalytically active forms of enzyme. While these assays have been abundantly described for several structural proteins, there are very few reports of radioimmunoassays for RT. Panet et al. (11) first described a sensitive radioimmunological technique for RT of avian type-C virus and similar assays for RT of murine type-C virus were described very recently (12, 13). Here we describe the results of the purification of RT from FeLV, 192

SHORT COMMUNICATIONS

its characterization by radioimmunoassays, and the use of these assays for detection of antibodies to FeLV RT in the sera of naturally infected cats. The FeLV RT was purified by ion exchange and nucleic acid affinity column chromatography procedures (14, 15). The purified enzyme contains DNA polymerase and RNase H activities. The purity of the RT was analyzed by SDS-PAGE before and after radioiodination. The SDS-PAGE analysis shows a single polypeptide with a molecular weight of 72,000 (Fig. 1 inset). The purified FeLV RT was iodinated by the chloramine-T method (17). The lz51-labeled probe was next examined by SDS-PAGE. As shown in Fig. 1, the labeled probe migrated as a single, sharp peak of 72,000 molecular weight and was greater than 95% pure by gel analysis. To establish the antigenicity of the labeled probe, the ability of various antisera to precipitate the labeled RT was examined. As shown in Fig. 2A, more than 90% of the labeled FeLV RT was precipitated by an

FRACTION

193

.

antiserum prepared against FeLV RT, suggesting that the immunological properties of the enzyme were preserved during the purification and lz51-labeling procedures. Antisera prepared against partially purified RT of R-MuLV, SSAV, BaEV, and RD-114 also precipitate the radiolabeled RT, but with substantial differences in titers. Antiserum prepared against AMV RT did not precipitate any of the labeled probe. To further test the purity of the labeled probe, antisera prepared against other FeLV proteins (gp’70, ~30, and ~15) and fetal calf serum were also used to test for precipitation of the labeled probe (Fig. 2B). None detectably precipitated the labeled ‘RT, although each antiserum was able toZprecipitate its respective lz51-labeled protein (data not shown). These results suggest that the FeLV RT’was significantly pure, and possesses antigenic determinants not shared by other known viral proteins nor by proteins from fetal calf serum. Mammalian type-C viral proteins contain a wide range of antigenic determinants that

NUMBER

FIG. 1. SDS-PAGE analysis of lz51-labeled FeLV RT. Purified FeLV RT was radiolabeled with lz51 by the chloramine-T method (17) with a minor modification. The reaction mixture (100 ~1) contained 0.5 pg of RT, 2.0 mCi of lz51 (New England Nuclear), and 30 pg of chloramine-T in a buffer consisting of 50 n&f Tris-HCl (pH 7.8) and 300 mM KCl. The free iodine and 1251-labeled RT were separated by Bio-Gel P-10. Iodinated enzyme (30-50 &i/pg) was diluted in 50 n&f Tris-HCl (pH ‘7.8), 300 mikf KCl, 0.5% Triton X-100, 0.5% bovine serum albumin, 0.05.% sodium aside, and 10% glycerol. All radioimmunoprecipitation assays were’ carried out in this buffer system. Around 100,000 cpm of ‘251-labeled FeLV RT was applied to a 10% cylindrical polyacrylamide gel at 2.5 mA/gel. After electrophoresis the gel was sliced and the radioactivity was measured. The inserted figure shows SDS-PAGE analysis of purified RT from FeLV. RT was subjected to electrophoresis on a 10% slab gel, and the gel was stained with Coomassie blue as described (16); The following proteins were used as markers: bovine serum albumin, molecular weight 68,000; ovalbumin, molecular weight 45,000; and chymotrypsinogen, molecular weight 25,000.

SHORTCOMMUNICATIONS

a-1 RECIPROCAL

OF SERUM

too

101 COMPETINO

OlLUllON

102 ANTIOEN

103

,ng,

. A .

10' ANTIORN

In01

A r

.

i

\

D COYPITINQ

A

io2

103

COMPETINO

.

104 ANTIOEN

. v

P .

A

i

. .

'7 . .i

:

105 ,ng,

FIG. 2. Radioimmunoprecipitation and radioimmunoassay for FeLV RT. (A) Immunoprecipitation of L251-labeled FeLV RT. The ability of various antisera to precipitate Y-labeled FeLV RT was determined by incubating around 10,000 cpm of the labeled probe with serial twofold dilutions of each goat antiserum. After incubation for 18 hr at 4”, antigen-antibody complexes were precipitated by adding the proper amount of anti-goat IgG rabbit serum as described (1.2). The radioactivity in the pellets was measured in a gamma counter, and the results were expressed as the percentage of total lz51 counts per minute precipitated at each dilution. Antisera tested included: anti-FeLV RT (O), anti-R-MuLV RT (O), anti-SSAV RT (A), anti-BaEV RT (0), anti-RD-114 RT (A), anti-AMV RT (*), anti-FeLV gp’70 (m), anti-FeLV p30 (V), and anti-fetal calf serum (0). (B) Homologous competition radioimmunoassay for FeLV RT. Purified FeLV proteins (RT, gp70, ~30, ~15) and fetal calf serum protein were evaluated for their ability to compete with L251-labeled FeLV RT for limiting amounts

SHORT

COMMUNICATIONS

FIG. 3. Specificity of natural cat serum as determined by competition radioimmunoassays. Serial twofold dilutions of detergent-disrupted viruses were tested for their ability to replace in a competition immunoassay utilizing limited amounts of natural cat serum to precipitate ‘251-labeled FeLV RT. The titer of natural cat serum for 10% precipitation of labeled probe was 1:4000. Type-C viruses included, FeLV (FL-74) (0); R-MuLV (A), RD-114 (A); SSAV (0); and BaEV (0).

cross-react with respective proteins of different viruses. A double-antibody competition radioimmunoassay was developed with lz51-labeled FeLV RT and limiting amounts of antisera prepared against FeLV RT. In this homologous immunoassay, only FeLV RT fully competed; the sensitivity was around l-3 ng (Fig. 2B). The RT purified from FeLV, subgroup C, also competed fully with a similar degree of sensitivity. However, FeLV gp’70, ~30, ~15, and fetal calf serum proteins were unable to compete. These results demonstrate again that FeLV RT possesses antigenic determinants not

195

shared by other FeLV-coded proteins and fetal calf serum. The ability of detergent-disrupted virions to compete with the precipitation of antibody to FeLV lz51-labeled FeLV RT purified from Theilen strain FeLV (FL-74) was next examined with a limiting amount of antisera. As shown in Fig. 2C, only FeLV (FL-‘74) and individual FeLV subgroups (A, B, and C) competed fully with the labeled probe for antiserum binding sites. The Theilen strain of FeLV (FL-74) consists mainly of subgroups A and B, and negligible amounts of subgroup C (the ratio of subgroup component is 104:103:100 for subgroups A, B, and C, respectively) (18); Sarma and Reddy, personal communication). Thus, RT from subgroup C was also purified, labeled, and tested in competitive radioimmunoassays. Similar results were obtained (data not shown). Other (especially rodent) mammalian type-C viruses partially competed. In contrast, the mammalian type-B, type-D, and avian type-C viruses tested did not compete. These results indicate that: (i) FeLV RT possesses group-specific antigenie determinants; (ii) FeLV RT also possesses determinants shared with RT from other mammalian type-C viruses; and (iii) the antigenicity of RT among FeLV subgroups, A, B, and C is indistinguishable by these assays. However, small differences in type-specificity or structural relatedness of RT among subgroups cannot be rulled out by this study alone. Further studies, e.g., peptide fingerprint or radioimmunoassay with highly specific antibody, would be necessary before a firm conclusion can be drawn. Several interspecific antigenic determinants on RT have been classified in the mammalian type-C viruses by enzyme in-

of goat anti-FeLV RT. The results are expressed as the percentage of total lZsI counts per minute in the antigen-antibody precipitate normalized to 100% binding in the absence of competing antigen. Proteins tested included FeLV RT (O), FeLV gp’i’0 (O), FeLV p30 (A), FeLV p15 (A), and fetal calf serum proteins (Cl). (C) Group-specific determinants of FeLV RT. Serial twofold dilutions of detergentdisrupted viruses were tested for their ability to compete with Y-labeled FeLV (FL-74) RT in the homologous competition radioimmunoassay described in (B). Viruses tested included: FeLV (FL-74) (0); FeLV, subgroup A (FeLV-A) (A); FeLV-B (Cl); FeLV-C (0); R-MuLV (0); AKR (A); RALV (*); RD-114 (V); BaEV, SSAV, and GaLV (SF) (v); and MPMV, AMV, and MMTV (a). (D) Interspecies antigenic determinants of FeLV RT. Serial Twofold dilutions of detergent-disrupted vii-ions were tested for their ability to compete with lZ51-labeled FeLV RT in the heterologous immunoassay using limiting amounts of goat anti-R-MuLV RT. Viruses tested included: FeLV (FL-74) (0); R-MuLV (0); SSAV (A); BaEV (A); RD-114 (V); AMV (0); MPMV (W); and MMTV (W.

196

SHORT

COMMUNICATIONS

hibition studies (3, 4). The reactivities of various mammalian type-C viruses were tested in a heterologous competition radioimmunoassay using 1251-FeLV RT and limiting amounts of antisera prepared against R-MuLV RT. As shown in Fig. 2D, all mammalian type-C viruses tested competed fully in this assay. In contrast, mammalian type-B and type-D viruses, and avian type-C viruses, were unable to compete in the heterologous assay (Fig. 2D). These results demonstrate that the heterologous competition radioimmunoassay detects the broadly shared interspecies antigenic determinants of mammalian type-C viral RT. We had previously reported that IgG from sera of some cats naturally infected by FeLV neutralize FeLV RT (5). The ability of detergent-disrupted virions to compete in the competition radioimmunoassays was next examined using ‘251-labeled FeLV (FL-74) RT and limiting amounts of natural cat serum, which was obtained from a nonviremic cat that was antibody positive to feline oncornavirus membrane antigen (FOCMA). As shown in Fig. 3, only FeLV could fully compete, suggesting that natural antibodies to FeLV RT are found in cat serum and they possess the appropriate specificity toward FeLV RT. Therefore, the radioimmunoassays should be useful for seroepidemiological analysis of these animals and especially in discriminating between an immune response to RT of FeLV and one to RT from the feline endogenous virus, RD-114. ACKNOWLEDGMENTS The authors wish to thank Drs. R. Gallagher, J. Krakower, and M. Sarngadharan for helpful discussions, and Dr. M. Essex for generously providing cat

sera. The excellent technical is gratefully acknowledged.

assistance

of M. Sharata

REFERENCES 1. VERMA, I. M., Biochem. Biophys. Acta 473, 1-38 (1977). 2. SARNGADHARAN, M. G., ROBERT-GUROFF, M., and GALLO, R. C., Biochem. Biophys. Acta 516, 419-487 (1978). 3. SHERR, C. J., FEDELE, L. A., BENVENISTE, R. E., and TODARO, G. J., J. Viral. 15, 1440-1448 (1975). 4. ROBERT-GUROFF, M., and GALLO, R. C., J. Gen. Viral. 43, 241-246 (1979). 5. JACQUEMIN, P. C., SAXINGER, C., GALLO, R. C., HARDY, W. D., JR., and ESSEX, M., Virology 91, 472-476 (1978). 6. JACQUEMIN, P. C., SAXINGER, C., and GALLO, R. C., Nature (London) 276, 230-236 (1978). 7. TRONICK, S. R., SCOLNICK, E. M., and PARKS, W. P., J. Viral. 10, 885-888 (1972). 8. KHAN, A. S., DEOBOGKAR, D. N., and STEPHENSON, J. R., J. Biol. Chem. 253, 8894-8901 (1978). 9. JAMJOOM, G. A., NASO, R. B., and ARLINGHAUS, R. B., Virology 78, 11-34 (1977). 10. WITTE, 0. N., and BALTIMORE, D., J. Viral. 26, 750-761 (1978). II. PANET, A. D., BALTIMORE, D., and HANAFUSA, T., J. Viral. 16, 146-152 (1975). 12. KRAKOWER, J. M., BARBACID, M., and AARONSON, S. A., J. Viral. 22, 331-339 (1977). 13. KRAKOWER, J. M., and AARONSON, S. A., Virology 86, 127-137 (1978). 14. ABRELL, J. W., and GALLO, R. C., J. Viral. 12, 431-439 (1973). 15. GRANDGENETT, D. P., and RHO, H. M., J. Viral. 15, 526-533 (1975). 16. LAEMMLI, V. K., Nature (London) 277, 680-685 (1970). 17. GREENWOOD, F. C., HUNTER, W. M., and GLOVER, J. S., Biochem. J. 39, 114-123 (1963). 18. SARMA, P. S., and LOG, T., Virology 54, 160-169 (1973).