Serological analysis of reverse transcriptase of the Mason-Pfizer monkey virus

VIROLOGY

69,

335-338 (1974)

Serological

Analysis

of Reverse

Mason-Pfizer A. YANIV,

Transcriptase

Monkey

Virus

T. OHNO, D. KACIAN, D. COLCHER,’ J. SCHLOM,2 AND S. SPIEGELMAN

Znstitute of Cancer Research, Columbia

S. WITKIN,

University College of Physicians New York, New York 100% Accepted January

of the

and Surgeons,

24, 197.4

Antiserum prepared against partially purified DNA polymerwe from MasonPfizer monkey virus neutralized the endogenous DNA polymerase of that virus and of X381, an agent morphologically indistinguishable from MPMV, isolated from cultures prepared from the lactating mammary gland of a rhesus monkey. The antiserum did not inhibit the DNA polymerase activities of avian myeloblastosis virus, feline leukemia virus, Rauscher murine leukemia virus, Friend murine leukemia virus, murine mammary tumor virus, and simian sarcoma virus-l.

Mason-Pfizer monkey virus (MPMV), isolated from a spontaneous mammary carcinoma of rhesus monkey, possesses many of the biochemical, biophysical, and morphological properties of the known oncogenic RNA viruses of animals (I-11). Previous studies have demonstrated that the immunologic analysis of polymerases can be used to classify oncogenic RNA viruses into several distinct groups: viper (12, IS), avian leukosis-sarcoma (12,14,15), lower mammalian leukemia-sarcoma (la18)) nonhuman primate leukemia-sarcoma (15-l 7), and the mouse mammary tumor virus (12-14). We report here that antisera against partially purified DNA polymerase of MPMV does not inhibit the DNA polymerases of chicken, mouse, cat, and monkey C-type viruses, nor the type-B mouse mammary tumor virus, but does affect the DNA polymerase of X381 virus, an agent morphologically indistinguishable from MPMV, isolated from the lactating mammary gland of a rhesus monkey (19). 1 Also Meloy Laboratories, Inc., Springfield, VA. * Also National Cancer Institute, National Institutes of Health, Bethesda, MD 20014.

Mason-Pfizer monkey virus was propagated in suspension cultures of the normal human lymphocyte cell line NC-37 and partially purified at the J. L. Smith Memorial for Cancer Research, Pfizer Inc., as described (7). The virus was further purified by centrifugation through an S-ml column of 20% glycerol in TNE buffer (0.01 M TrisHCl [pH 8.31, 0.15 M NaCl, 0.002 EDTA) onto a pad of 100% glycerol at 98,OOOgfor 60 min at 4 C. The virus was taken up in TNE and spun to equilibrium in a continuous 20-50% sucrose gradient in TNE at 98,000g for 16 hr. The virus band at the density of 1.14-1.18 g/ml was then diluted, pelleted at 98,OOOgfor 45 min at 4 C, and used immediately for DNA polymerase purification. Avian myeloblastosis virus (AMV), BAl strain A, was obtained from Dr. J. W. Beard; SSV-1 from Pfizer and from Dr. F. Deinhardt; Friend leukemia virus (FLV) and feline leukemia virus (FeLV) from Dr. D. Bolognesi; Rauscher leukemia virus (RLV) from Dr. E. H. Bernstein at University Labs.; and X381 from Pfizer. All were purified as described above. MPMV DNA polymerase was prepared essentially as described (20) for the AMV

335 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

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enzyme except that it did not adhere to the DEAE-cellulose column. The flowthrough was taken and carried through further purification steps. Owing to the limited quantity of material available, the expected yield of enzyme protein per preparation was less than 25 pg. Purity of the preparation was, therefore, not monitored by gel electrophoresis, nor were rigorous determinations of specific activity made. Three preparations of MPMV DNA polymerase were used for inoculation. Solubilized, partially purified virions were the source of other DNA polymerases. Viruses were disrupted in 0.2% Nonidet P-40, 50 mM DTT, 8 mM Tris-HCl (pH 8.3), for 15 min at 0 C immediately before use, and the standard DNA polymerase assay was performed as described in the legend to Fig. 2. Antiserum against MPMV DNA polymerase was prepared in New Zealand white rabbits. The antigen was emulsified with an equal volume of Freund’s complete adjuvant and injected into the two hind footpads.

Two additional inoculations were given at 2-wk intervals in the same sites. The animal was bled 2 wk after the third inject,ion. Two additional cycles of immunization were performed on the same schedule in order to achieve the desired titer. Sera were fractionated by chromatography on Sephadex G-200 in 0.1 M Tris-HCl (pH 8.0). Serum (1.5-2.0 ml) was applied to a 2.5 X 90-cm column, and 2-ml fractions were collected at a rate of 6 ml per hr. Rabbit gamma globulins were identified serologically by immunodiffusion with goat anti-rabbit IgG antiserum. The relevant fractions were concentrated by ammonium sulfate precipitation (50 % saturation) and dialyzed against 0.1 M Tris-HCl (pH 8.0). The protein concentration of IgG fractions was measured by the method of Lowry (21). Reaction mixtures for Ohe neutralization of DNA polymerase activity (total volume 55 ~1) contained purified IgG fraction (25200 pg), 0.01 M Tris-HCl (pH 8.3), 0.15 M pot’assium chloride, 25 pg of bovine serum albumin (BSA), and 1 unit (see legend to

Minutes

1. A. Inhibition of endogenous DNA polymerase activity of MPMV by RNase. Reactions (50 pl) were prepared containing detergent-disrupted MPMV with RNase A (0-O) at 80 rg/ml or without nuclease (O-O). After 30-min preincubation, endogenous reaction conditions were established. The endogenous reaction (final volume 170 pl) contained in rmoles: Tris-HCl (pH 8.3), 10.9; MgClz , 1.4, KCl, 8.2; dithiothreitol, 3.0; dATP, dGTP, dCTP, each 0.27; [3H]dTTP (2.4 X lo4 cpm/pmole), 0.05. Aliquots of 25 ~1 were removed at the indicated times, and the acid-precipitable radioactivity wae determined. B. Effect on anti-MPMV DNA polymerase IgG on endogenous DNA polymerase activity of MPMV. Reaction mixtures (50 ~1) were prepared each containing 70 pg of anti-MPMV DNA polymerase IgG (0-O) or 75 rg normal rabbit IgG (O-O) and detergent-disrupted MPMV. After 30-min preincubation at 37 C, endogenous react,ion conditions were established. Aliquots (25 ~1) were removed at the designated times, and the acid-precipitable radioactivity was determined. FIG.

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Fig. 2) of DKA polymerase. After 15 min at 37 C, standard polymerase assay conditions were established. The acid-precipitable radioactivity was taken as a measure of the DNA polymerase activity not neutralized by the added IgG. IgG with anti-MPMV DNA polymerase activity was obtained after the first immunization cycle and substantial anamnestic response was observed after the second and the third inoculation schedules. Since 50 rg IgG (obtained after the third immunization cycle) per reaction neutralized approximately 50 % of DNA polymerase activity, this quantity was used for all subsequent neutralization assays. No neutralization was observed with as much as 200 pg IgG obtained from sera taken prior to immunization. Incubation of the purified viral polymerase at 37 C in the absence of the components required for DNA synthesis resulted in rapid loss of activity. The addition of BSA preserved the activity of AMV polymerase (22). With the MPMV enzyme, approximately 80 % of the enzyme activity was recovered when 25 pg of BSA were used per standard lOO+l reaction mixture. The addition of BSA also eliminated stimulation of DNA polymerase activity by low levels of normal IgG. Preincubation for 15 min at 37 C with test IgG was required to neutralize more than 50 % of the enzyme activity. This 15-min preincubation was used in all subsequent studies. No neutralization was observed over a 90-min period with normal IgG obtained from the same rabbit prior to immunization. Neutralization of the endogenous DNA polymerase of Mason-Pfizer virus is shown in Fig. 1B. The reaction was completely RNase sensitive (Fig. lA), supporting the conclusion that synthesis from the viral RNA is being inhibited. The ability of the anti-MPMV DNA polymerase IgG to neutralize the DNA polymerase activity of other oncornaviruses is shown in Fig. 2. (Poly dT)* (poly rA) was used as template, and purified virion preparations were used as the enzyme source. Anti-MPMV DNA polymerase did not inhibit the DNA polymerase activity of avian

myeloblastosis

virus (AMV),

Rauscher and

150

0Microgram

IgG

FIG. 2. Serological comparison of the DNA polymerase of MPMV with those of other oncornaviruses as determined by neutralization assay using increasing amounts of anti-MPMV enzyme. The antigen content in reaction mixtures was standardized according to the DNA polymerase activity. One unit of activity was defined as the amount of enzyme necessary to render acidprecipitable 1 pmole of [aH]TTP per min with (poly dT). (poly rA) template. Standard polymerase reactions were performed using (poly dT) * (poly rA) template. The assay mixture (total volume 100 pl) contained the following in micromoles: Tris-HCI (pH 8.2), 5.0; magnesium chloride, 0.6; and potassium chloride, 4.0; dithiothreitol, 0.04; 0.02 each of deoxyadenosine 5’-triphosphate [3H]deoxythymidine 5’-triphosphate (200-300 cpm/pmole); (poly dT). (poly rA) template, 1300. Reactions were incubated at 37 C for 30 min and assayed for acid-precipitable counts. Control normal rabbit serum IgG was tested at comparable concentrations for determining percent neutralization. Friend murine leukemia viruses (RLV and FLV), feline leukemia virus (FeLV), murine

mammary tumor virus (MMTV), or simian sarcoma virus (SSV-1). The latter two viruses were tested at higher levels than shown. The DNA polymerases of these were not inhibited with as much as 200 pg of antiserum. Anti-MPMV DNA polymerase

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did inhibit to an appreciable extent, however, the DNA polymerase activity of X381 ACKNOWLEDGMENTS This study was supported by Grant CA-02332 and Contract Nos. NOl-CP3-3258 and l-CP-43223 within the Virus Cancer Program of the National Cancer Institute, National Institutes of Health. REFERENCES AHMED, M., MAYYASI, S. A., CHOPRA, H. C., ZELLJADT, I., and JENSEN, E. M., J. Nat. Cancer Inst. 46, 1325-1334 (1971). I., JENSEN, E. M., 2. CHOPRA, H. C., ZELLJADT, M.~soN, M. M., and WOODSIDE, N. J., J.

1.

Nat. Cancer Inst. 46,127-137 (1971). 3. JENSEN, E. M., ZELLJ~~DT, I., CHOPRA, H. C., and MASON, M. M., Cancer Res. 30, 23882393 (1970). B., SBRKAR, N. H., and MOORE, 4. KRAMARSKY, D. H., Proc. Nat. Acad. Sci. USA 68, 16031607 (1971). 5. MANNING, J. S., and HACKETT, A. J., J. Nat. Cancer Inst. 48,417-422 (1972). R. C., EDYNAK, E., and SARKAR, 6. NOWINSKI, N. H., Proc. Nat. Acad. Sci. USA 68, 160% 1612 (1971). S., Proc. Nat. 7. SCHLOM, J., and SPIEGELMAN, Acad. Sci. USA 68, 1613-1617 (1971). 8. CHOPRA, H. C., Bibl. Haematol. 39, 228-235 (1972). D., MARSHALL, S., and GALLO, R., 9. GILLESPIE, Nature (London) New Biol. 236, 227-231 (1972).

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