Reptilian C-type virus: Biophysical, biological, and immunological properties

Reptilian

C-Type

Virus: Biophysical,

Biological,

The oncogenic potential of RKA-containing viruses of the C-type in their natural hosts has been reported for several mammalian and avian species (1, a. Evolutionary considerations lead to the

and Immunological

Properties

inability tfJ isolate them at w-ill from certain species (including man) is not at present a serious obstacle to the above hypothesis. However, continued isolation of C-type viruses from new species, especially, but

FIG. 1. C-t,ype particles in VSW cult,ures. Thin sections stained with Reynold’s lead citrat,e and many1 acetate were examined with an Hitachi IIU 11E elect ran microscope. (A) Nltmerons pari icles with densenucleoids; (B) Budding particle (arrow) and apparent intravscuolar localix:ltion of particles; (C) particles at high magnificatioll.

obvious suggestion t,hat members of this family of viruses are involved in induction of malignancies in other animal species as well. The C-type viruses are known under certain conditions to have no measurable expression (3) even Lvithirl species where their presence is dell established so that

not necessarily, in association Lvith maJignancy is predicted. Recently, Zeigel and Clark (,G) have reported particles which closely resemble C-type viruses in a tissue culture line established from the spleen of a Russell’s viper (I’ipera ~.~selli) kvhich had :L large myxofibroma in the connective 187

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COMMUNICATIONS TABLE ETHER SENSITIVITY

1

OF THE VIPER C-TYPE

Acid-insoluble 3H-uridine (count/min)

Treatment

Intact-banded virus Virus + RNaseb Banded virus + ether Banded virus + ether

VIRUS*

+ RNase

14,330 12,119 13,250 136

a *H-uridine-labeled and purified virus was precipitat.ed on Millipore filters by 10% trichoroacetic acid after various treatments. The data show that the viral RNA is only susceptible to RNase digestion after ether treatment. * 1 pg/ml, 15 minutes, 37”.

f%ACTlON NUMBER

FIG. 2. Incorporation

of 3H-uridine into viper C-type virus RNA and inhibition of viral synthesis by actinomycin D. VSW cultures were incubated with 3H-uridine, 10 &i/ml, for 24 hours. The supernatant fluid was removed, clarified by low speed centrifugat,ion, and then centrifuged to equilibrium in 1560% (w:v) sucrose gradients. Fractions were collected by bottom puncture and assayed for radioactivity and refractive index. The peak activity in each run occurred at 1.16 g/cc. Incubation with actinomycin D, 0.5 pg/ml A--A, and 0.1 pg/ml b-,m was carried out for 24 hours concurrently with radioactive labeling. Control (no actinomycin), @---a. In addition to the radioactive incorporation data, visual inspection of gradient tubes showed a distinct opalescent band in the 1.16 region for control cultures (electron microscopic examination showed numerous C-type particles in this band) and no band in the drug-treated cultures. The radioactivity at the top of gradient tubes represents low molecular weight, nonviral RNA from the tissue culture medium.

tissue anterior to the heart. This represents the first report of a reptilian C-type virus. With the cooperation of Dr. Clark, we have studied certain biological, biophysical, and immunological properties of this virus for comparative purposes.

The viper cell line (VSW) was obtained from Dr. H. F. Clark, Wistar Institute of Anatomy and Biology, PhiladeIphia, Pennsylvania, in passage 77. It has been maintained at 30” using Eagle’s basal medium with 10 % fetal bovine serum and has undergone multiple passages in our laboratory. The presence of C-type particles in this cell line was readily confirmed by electron microscopic examination. Numerous C-type particIes were seen, both free in the extracellular spaces and also apparently within vacuoles (Fig. 1). Definitive budding particles could also be found without difficulty. The particle dimensions, 100-110 rnp diameter, and special features of this C-type virus, especially the condensed nucleoid, are consistent with the observations of Zeigel and Clark (4). Utilizing radioactive labeling and virus purification procedures in general use for avian and mammalian C-type viruses (5, G), several biophysical and biological properties of the viper virus were determined. The virus contains RNA and has a buoyant density in sucrose of 1.16 g/cc (Fig. 2). The viral RNA in the intact particle is resistant to RNase digestion, but after ether treatment the nucleic acid becomes RNase sensitive (Table 1). Extraction of high molecular weight RNA (~705) which characteristic of avian and murine E-type viruses (7) was readily accomplished from virus pellets (Fig. 3). As is true of its mammalian and avian counterparts (8, 9) synthesis of the viper virus is in-

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189 TABLE

2

~ONREACTIVITY OF LT~~~;:n C-TE-PIN \~r~ws PREPARATION WITH MURINK AND Av-IAN C-TYPE ~~‘IRION ANTISKIM

i\ntigen

VSW-20% cell suspension Viper virus-O.3 mg/ml Viper virus-ether disrupted AKR virus (mouse)-0.2 mg/ml (intact and ether disrupted) RAV-1 (chicken)-0.2 mg/ml (ether disrupted) 6

2

4

6

I3

10

FRACTION

12

14

16

18

NO.

FIG. 3. Sedimentation profile of RNA extracted from 3H-uridine-labeled viper (0-m) and 14C-uridine-labeled Rauscher leukemia virus (X---X). Supernatant fluids of 24-hour labeled cultures were clarified by low speed centrifugation, and then virions were pelleted through 20% sucrose (IS). RNA was extracted according to the method of Solymosy et al. (14) using tris(hy(.05 droxymethyl)aminomethane-hydrochloride M, pH 7.6) buffered sodium dodecylsulfate (1%) containing diethylpyrocarbonate (3.39&) to inhibit ribonuclease activity. Viper and Rauscher virus RNA’s were mixed and sedimented at 40,000 rpm (Spinco SW 50 rotor, 5”, 80 minutes) through a 5-2OGj, w:v sucrose gradient. After centrifugation, equal fractions were collected by bottom puncture of gradient tubes, and radioactivity was determined in a Beckman LS-250 liquid scintillation counter. Corrections for 14C act!ivity in the 3H channel (set for minimal 14C spillover) were based on dat,a obtained from gradient centrifugation of 14C-RLS’ alone. The direction of sedimentation is from right to left. The peaks of activity at the top of tube (right) represent the low molecular weight RNA generally associated with purified virus after extended labeling periods (13).

hibited by actinomycin D (Fig. 2). Whether or not the dependence on DNA transcription (inferred from the actinomycin results) is based on a “nonspecific” cellular activity or indicates a DNA provirus state (10) for the C-type viruses remains to be decided. The avian and murine C-type viruses are

Serum ~-~ -.--. MSV- KSVKat,,, tumored hamste+

a Broad-reacting serum for the murine C-type viruses (II, 15). * This serum reacts with the group-specific antigen of the avian C-type viruses (16). c Reciprocal of complement-fixation titer with 4 units of antiserum. Viruses were purified by sucrose gradient centrifugation. Protein content was estimated from absorbance at 280 and 260 rnp in a Beckman spectrophotometer.

characterized by a species-specific, groupreactive antigen detectable by complement-fixation (21) and gel-diffusion (12) assays. The possibility of antigen sharing between the viper virus and these viruses was tested in both types of assay. Purified virus (both intact and ether disrupted) was tested at concentrations which would have given CF titers of 1:64-l: 128 and gel diffusions titers of 1: 8 with homologous antisera. The viper virus preparations did not show reactivity (Table 2) in either of these assays and thus may be assumed to possess a “group-specific” (gs) antigen distinct from that of the well-characterized avian and murine viruses. By means of isoelectric focusing a protein fraction has been isolated from disrupted purified viper virus which appears similar in some physical characteristics to the murine gs antigen (S. Oroszlan et al., unpublished data). Immunization of guinea pigs with this fraction resulted in the production of antiserum apparently specific for the viper virus. This antiserum reacted with ether-disrupted viper virus and not with similar preparations of murine and

190

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COMMUNICATIONS

avian C-type viruses. This provides additionaI evidence for a distinctive viper internal antigen. The reptilian C-type virus described by Zeigel and Clark (4) thus conforms in a number of biological and biophysical properties to the well known avian and murine C-type viruses. Attempts to demonstrate oncogenic activity in newborn animals of several mammalian species are negative to date while similar attempts in avian and reptilian species have not yet been attempted. While demonstration of oncogenic potential has not yet been achieved, it does appear significant that the cell line in which the viper virus was observed was derived from a tumorous snake. The widespread occurrence of members of the C-type virus family along with increasing demonstration of oncogenic potential of indigenous viruses in new hosts such as the cat (17) provides the basis for an intensive effort to isolate similar viruses from man. The recent demonstration of C particles in a human liposarcoma-derived culture (18) further strengthens this view. ACKNOWLEDGMENTS This work was partially supported by Contract NIHt%97 from the Etiology Area, SVCP, National Cancer Institute, National Institutes of Health. We gratefully acknowledge the technical assistance of Mr. T. Stanley and Miss Carol Owen. REFERENCES 1. BEARD, J. W., ed. Int. Conf. on Avian Tumor viruses, National Cancer Institute Monograph 1964 17 (1964). 2. RICH, M. A., and MOLONXY, J. B., ed. Conf. on Murine Leukemia, National Cancer Institute Monograph 1966 22 (1966). S. IICJEBNER, R. J., and TODARO, G. J., Proc. Nat. Acad. Sci. C.S. 64, 1087-1094 (1970); HUEBNER, R. J., TODARO, G. J., SARMA, P., HARTLEY, J. W., FREEMAN, A. E., PETERS, R. L., WHITMIRE, C. E., MEIER, H., and GILDEN, R. V., Proc. Second Znt. on Tumor Viruses, Paris, 1970. Swp. Submit,ted for publication. 4. ZEIGEL, R. F., and CLARK, H. F., In “Proc. 27th Annual Electron Microscopy Sot. of

America Meeting” (C. J. Arceneaux, ed.) p. 220. Claitors, Baton Rouge, La., 1969; Zeigel, 1~. F., and Clark, II. F., J. Xat. Cancer h-t. 43, 1097-1099 (1969). 5. DUESBERG, P. H., and ROBINSON, W. S., Proc. Nat. ;Icad. Sci. CT. S. 55, 219-227 (1966). 6. O’CONNOR, T. E., RAUSCHER, F. J., and ZEIC;I’:L, R. F., Science 144, 1144-1147 (1964). 7. ROIXNS~N, W. S., ROBINSON, H. L., and DunsBERG, P. H., hoc. Nat. AcUd. ,%i. c:.s. 58, 825-834 (1967). 8. D~I,:sI~>:I~G, P. H., and ROBINSON, W. S. Virology 31, 742-746 (1967). 9. TEMIN, H. M., Virology 20, 577-582 (1963); BADI~;I~, J. P., Virology 22, 462-468 (1964). 10. TEMIN, H. M., Proc. Abut. Acad. Sci. U.S. 52, 323-329 (1964). il. I~UEBNER, R. J., Proc. *brat. Acad. Sci. U.S. 58, 835-842 (1967). f2. GEERING, G. L., OLD, L. J., and BOYSE, E. A., J. Exp. Med. 124, 753-772 (1966). IS. BADGER, J. P., and STECK, T. L., J. Viral. 4, 454-459 (1969). 14. SOLYMOSY, F., GULYAS, A., and FARKAS, G. L., Eur. J. Biochem. 5, 520 (1968). fb. HARTLEY, J. W., ROLE, W. P., CAPPS, W. I., and H~EDNER, R. J., J. Viral. 3, 126 (1969). f6. HUEBNER, R. J., ARMSTRONG, D., OKUYAN, M., SARMA, P. S., and TURNER, H. C. Proc. Nat. Acad. Sci. C.S. 51, 742 (1964). f7. JAKI~ETT, W. F. H., CRAIVFORD, E. M., MARTIN, W. B., and DAVIE, F., A’ature London 202, 567 (1964) ; KAXVAICAMI, T. G., THXILEN, G. H., DUNGX-ORTH, D. L., MUNN, R. J., and BEALL, S. G., Science 158, 1049 (1967); RICKARD, C. G., POST, J. E., NORMKA, F., and BARI~, L. M., J. Nat. Cancer Inst. 42,987 (1969). 18. MORTON, D. L., HALL, W. T., and MALMGREN, It. A. Science 165, 813 (1969). RAYMOND Ti. GILDEN YONG KI LEE STEPHEN OROSZLAN JOHN L. WALKXR Flow Laboratories, Inc. 12601 Twinbrook Parkway Rockville, Maryland 10852 ROBERT J. HUEBNER National Cancer Institute Viral Carcinogenesis Branch Arational Institutes of Health Bethesda, Maryland %‘OOl4 Accepted March S, 1970