Morphogenesis of bovine leukemia virus

80, 42-53 (197’7)

VIROLOGY

Morphogenesis JERO CALAFAT**

of Bovine Leukemia l

AND

Virus

A. A. RESSANGt

*Department of Electron Microscopy, The Netherlands Cancer Institute, Sarphatistraut The Netherlands, and t The Central Veterinary Institute, P.O. Box 6007, Rotterdam, Accepted Februnly

108, Amsterdam, The Netherlands

18,1977

The morphogenesis of bovine leukemia virus (BLV) was studied in short-term cultures of leukocytes from cows with persistent lymphocytosis and in BLV-producing cell lines. Few budding particles were found. They consisted of one shell underneath the cell membrane with granules attached to the inner side. When the shell is completed the budding particle could follow two pathways: It could (a) bud from the cell membrane to give rise to a free immature particle or (b) mature while still in contact with the cell, by condensation of the shell and the granules into a nucleoid, and subsequently bud from the cell membrane as a mature virion. A different pathway of morphogenesis, probably followed by the majority of the virions, is proposed based on the following observations: (1) Low numbers of budding particles on the cell membrane, (2) condensation of electrondense material within the cytoplasm resembling virus particles in the first stage of budding, and (3) immature and mature particles lying free in the cytoplasm. This pathway of morphogenesis supposes the formation of immature and mature particles within the cytoplasm without a budding process. Immunoferritin studies on these BLVproducing cells using bovine and goat anti-BLV sera have shown labeling of the BLV particles. The cell surface, however, was labeled only rarely and then in small areas. This means that on the cell surface of BLV-producing cells very few viral structural polypeptides are present. A comparison of the morphology of BLV and several other oncornaviruses leads to the conclusion that the morphogenesis of BLV is different from that of the type B and C and other similar particles such as Mason-Pfizer monkey virus and bromodeoxyuridine-induced guinea pig leukemia virus. INTRODUCTION

tron-dense central nucleoid (like mature type C) were seen still attached to the cell membrane suggesting that this bovine virus can mature when still attached to the cell membrane. In recent years much work has been done to characterize further this virus which is associated with bovine leukemia and induces leukemia upon inoculation into sheep (Olson et al., 1972). It is now commonly referred to as bovine leukemia virus (BLV). Infected animals develop antibodies against BLV, but this virus has not shown the interspecies reactions shared by other mammalian type C viruses and has shown no antigenic cross reaction with other mammalian oncornaviruses such as mouse mammary tumor virus (MMTV) and Mason-Pfizer monkey virus (M-PMV) (Ferrer, 1972; Ressang et al., 1974; Gilden et al., 1975).

Particles similar to mature type C viruses were originally described in shortterm cultures of blood leukocytes from leukemic cattle (Miller et al., 1969). In longterm cultures from thoracic duct or in huffy coat lymphocytes from cows with leukemia, Ferrer et al. (1971) have found budding and immature particles together with the mature particles (already described demonstrating that these particles are replicated by these cells. In a morphological study on short-term cultures of leukocytes from cows with persistent lymphocytosis we have shown (Calafat et al., 1974a) that maturation of these particles differs from that described for the type C particles. Particles with an elec1 Author dressed.

to whom reprint

requests should be ad42

Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0042-6622

MORPHOGENESIS

OF BOVINE

BLV resembles MMTV and M-PMV virus in having an RNA-dependent DNA polymerase which is preferentially active in the presence of Mg*+ when synthetic templates are used (Gilden et al., 1975). Molecular hybridization between BLVE3HlcDNA and several viral RNAs and vice versa shows that BLV is not related to M-PMV, simian sarcoma virus (SiSV), feline leukemia virus (FeLV), and avian myeloblastosis virus (ALV) (Kettman et al., 1976). The morphological, serological, and biochemical characteristics of BLV suggest that this virus is different from the type C viruses. In the present study we have extended our previous ultrastructural work to define this virus better morphologically. MATERIALS

AND METHODS

Leukocyte cultures. Short-term cultures were prepared from leukocytes from imported Jersey cattle with persistent lymphocytosis as described in detail (Calafat et al., 1974a). Phytohemagglutinin was used at a concentration of 0.02 ml/ml of medium. The cultures were incubated for 48-72 hr at 37”. The cells were harvested by low-speed centrifugation and washed twice in Hanks’ balanced salt solution. The pellet was fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.1, for 1 hr and further processed for electron microscopy as described below. For immunoferritin labeling, the pellet was resuspended in a small quantity of Hanks’ solution. Cell lines. Three BLV-producing cell lines were used: (1) FBL-J5, established by cocultivation of fetal bovine lung fibroblasts and leukocytes from a leukemic cow 55; (2) CDIS-191, produced by the explantate technique from a lymph node from sheep 191 which was infected 2 years earlier with pooled leukemic blood from imported Jersey cows; this sheep was killed in the terminal stage of malignant lymphoma of the lymphoblastic type; (3) FLS (fetal lamb spleen cells), obtained by cocultivation of fetal lamb spleen cells with fragments of neoplastic tissue from a BLVinfected cow; this line is a kind gift from Dr. M. J. van der Maaten. Details on es-

LEUKEMIA

VIRUS

43

tablishment, maintenance, and characteristics of cell lines 1 and 2 have been reported (Ressang et al., 1974). For comparative studies (see Table 1) the following oncornavirus-producing cells were used and maintained under the same conditions as the BLV-producing cells: woolly monkey cells infected with SiSV (SSV-1); cocultivated monkey mammary tumor cell line (CMMT) producing MPMV; both cell lines were a gift from the Pfizer Company through the courtesy of Dr. J. Gruber from the National Cancer Institute, Bethesda, Md.; C57BWKa mouse embryo fibroblasts (BL-5) chronically infected with radiation leukemia virus (RadLV); short-term culture of a C3H/ HeAf mouse mammary tumor producing Gross leukemia virus. The cells were washed twice in Hanks’ solution, fixed in glutaraldehyde, and then scraped off the Falcon flasks with a silicon rubber policeman and centrifuged to make a pellet. This pellet was further processed for electron microscopy. For immunoferritin-labeling studies, the cells were grown in 3.6-cm-diameter Falcon plastic petri dishes until confluency and washed twice with Hanks’ solution before being labeled. Antisera. The following sera were used: serum from an imported Danish cow in the terminal stage of progressive malignant, lymphoma and serum from a goat infected with pooled leukemic blood from imported Jersey cows. Both sera have been shown to react with BLV-producing cells in the indirect immunofluorescence test and in the immunodiffusion test (Ressang et al., 1974). Rabbit antisera to bovine and to goat IgG (Nordic Pharmaceutical, Tilburg, The Netherlands) were coupled to ferritin with glutaraldehyde (Calafat et tzl., 1974b). Labeling procedure. One-tenth milliliter of a leukocyte suspension in Hanks’ solution was incubated with periodic agitation for 1 hr at room temperature in 0.5 ml of undiluted goat anti-BLV serum; the cells were washed twice with Hanks’ solution and then incubated with periodic agitation for 1 hr at room temperature in 0.2 ml of ferritin-labeled rabbit anti-goat IgG diluted 1:20. After three washes the pellets

44

CALAFAT

AND

were fixed in glutaraldehyde. The cell lines grown in 3.5~cm petri dishes were incubated with the antisera and washed in situ at room temperature as described above. Antisera used were: 0.5 ml of bovine anti-BLV serum diluted 1:lO; 0.5 ml of goat anti-BLV diluted 15; 0.5 ml of ferritin-labeled rabbit anti-bovine IgG diluted 1:lOO; 0.5 ml of ferritin-labeled rabbit anti-goat IgG diluted 1:50. After fixation with glutaraldehyde the cells were scraped off the dishes with a rubber policeman and centrifuged. Electron microscopy. All samples were postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.1, for 1 hr, dehydrated, and embedded in a mixture of Epon and Araldite. Thin sections were stained with uranyl acetate and lead hydroxide. Alkaline bismuth subnitrate stain was used in some preparations with ferritin conjugate to enhance the visible size and electron opacity of the ferritin particles. The preparations were examined with a Philips electron microscope, EM300 or EM-301. RESULTS

of BLV BLV particles in short-term cultures of leukocytes (Figs. 1 and 2) were generally located in clusters, often surrounded by cell debris, outside the cell and within cytoplasmic vacuoles. A few budding particles were found in lymphocytes (Fig. 1) and granulocytes (Fig. 2) proving that both kind of cells can produce BLV. In BLV-producing cell lines, BLV particles were mainly located in small groups outside the cell (Fig. 3) and within cytoplasmic vacuoles (Fig. 4). In contrast to those in short-term cultures of leukocytes, the virus particles were not surrounded by cell debris. Only exceptionally were particles found in degenerating portions of the cytoplasm.

Distribution

of BLV In the present cell lines and in shortterm cultures of leukocytes, budding particles localized on the membranes of the cell surface or of the vacuoles were scarce. A

Morphogenesis

RESSANG

single shell with a thickness varying between 40 and 80 A was seen underneath the cell surface or vacuole membrane (Fig. 5). This shell increased in size, and dense granules were seen attached to the concave surface (Figs. 6 and 7); when this stage was completed two pathways of development could be followed: (a) Condensation of the granules into a nucleoid while the particle was still attached to the cell membrane (Fig. 81, followed by budding from the cell membrane in the form of a mature particle; (b) budding from the cell membrane to give rise to a free immature particle (Fig. 9) consisting of an outer envelope with a diameter of about 95 nm and an inner shell with an average thickness of about 90 A. The core was electron translucent and contained granules that very often were attached to the shell. In many of the immature particles the shell was still incomplete (Fig. 10). Probably these particles were still attached to the cell without showing the connection between the cell and the particle in the plane of the section. Later condensation of the granules of these immature particles into a nucleoid resulted in the free mature form. The mature particle (Fig. 111, with a diameter of about 110 nm, consisted of an envelope that sometimes had high electron density (Fig. 2) and quite often had short surface projections (Fig. 1). A central core was often separated from the intermediate layer by a translucent space. This core was electron dense and sometimes granular with a diameter varying between 40 and 90 nm. When the core had a large diameter the translucent space between it and the intermediate layer was small and the core matrix was more dispersed. To be sure that these morphological observations are not due to the methods of culture and preparation for electron microscopy used in our laboratory, we have also studied several oncornavirus-producing cells (see Table 1) following the same conditions as used for BLV-producing cells. The results agree with the data described by other laboratories and summarized in Fig. 22. As an example we show MuLV at different stages of development produced by the short-term culture of a

FIG. 1. Thin section of a lymphocyte. A budding particle (thick arrow) and virus particles outside the cell surrounded by cell debris. The envelope of one of these particles has short projections (thin arrow) ~36,000. FIG. 2. Thin section of a granulocyte. Virus particles and cell debris are present in a cytoplasmic vacuole (V). One of these particles (arrow) has an envelope with high electron density. Glycogen particles (g) can be seen in the cytoplasm. ~36,000. Inset, higher magnification of enclosed area showing a budding particle (arrowhead) with an electron-dense nucleoid. ~80,000. 45

46

CALAFAT

FIG. shown.

FIG.

3. Cell line ~50,000. 4. Cell line

CDIS-191. FBL-JB.

Group Virus

of virus particles

AND

particles (arrows)

C3Hf mammary tumor: a budding particle (Fig. 12) with two shells underneath the cell membrane; an immature C particle (Fig. 13) consisting of two shells wrapped by an envelope; and a mature C particle (Fig. 14) with an envelope and an electrondense nucleoid. In the above description of the development of the BLV particle we have followed

RESSANG

outside

the

in cytoplasmic

cell.

A budding

vacuoles.

particle

(arrow)

is also

~32,000.

the scheme of maturation of the virion by budding from the cytoplasmic membrane, as described for oncornaviruses (Dalton et al., 1975). However, in the BLV-producing cells studied, the number of budding and immature particles per cell, estimated following the formula of Dougherthy and Di Stefano (1968, and the number of these particles per 100 mature particles is low

MORPHOGENESIS

OF BOVINE

LEUKEMIA

VIRUS

FIGS. 5-11. Culture of leukocytes (Fig. 5) and cell line FBL-J5 (Figs. 6-11). Fig. 5. First stadium of the viral budding process. A dense layer (arrow) is formed underneath the surface membrane (arrowhead). ~96,000. Fig. 6. The inner layer increases in size and dense granules (arrowheads) are attached to this shell. x 120,000. Fig. 7. The inner layer is completed, the particle is still attached to the cell (arrow). x 120,000. Fig. 8. Virus particle attached to the cell (arrow) with an electron-dense nucleoid. Fig. 9. Immature virus particle consisting of an outer envelope (1) and an inner shell (2) with attached granules (31. Fig. 10. Immature particle with the inner shell still incomplete (arrow). Fig. 11. Mature virus particle consisting of an outer envelope (11, an intermediate layer (2), and a central granular core (3). Figs. S-11. ~100,000. TABLE

1

PRESENCE OF BUDDING, IMMATURE, PARTICLES IN BLV-PRODUCING CELL SEVERAL ONCORNAVIRUS-PRODUCING

AND MATURE LINES AND IN CELL LINES~

Oneomavirus-proBudding Immature ducing cell line particles per particles per cellb (No. of cell’ (No. of budding parimmature ticles/lOO particles/100

Mature particles per cellb (No.)

mature par- mature partiSiSV-SSV-1 RadLV-B1.5 Gross leukemia virus C3Hf M-PMV-CMMT BLV-BLF.J5 BLV-CDIS-191 BLV-FLS

ticles)

cles)

58 (8.4) 20 (3.2) 38 (7.5)

34 (5.71

53 9 2 4

(85) (1.91) (0.371 (2.15)

110 (17.3)

194 (38) 53 29 16 7

(85) (6.1) (2.95) (3.75)

687 628 507 62 472 542 187

4 At least 200 cell sections examined for each cell line. b Estimated following the formula of Dougherty and Di Stefano (1965).

when compared with the number of these particles in other oncornavirus-producing cells cultured in the same laboratory un-

der the same conditions (Table 1). Two possible explanations for this discrepancy can be given: (1) The maturation process might be very rapid, and (2) the virions might develop via another mechanism. This suggestion of an alternative pathway of BLV development, taking place mainly within the cytoplasm, is supported by several observations: (a) condensation of electron-dense material within the cytoplasm resembling virus particles in the first stage of budding (Fig. 15); (b) formation of complete shells giving rise to immature and mature particles lying free within cytoplasm (Figs. 15 and 16); and (c) transport of mature virion from cytoplasm towards vacuoles or extracellular space (Fig. 17). Filamentous

Forms

Filamentous forms as previously described by Ferrer et al. (1971) were found budding either from the plasma membrane or in clusters from a vacuole membrane (Fig. 18). These filamentous parti-

48

CALAFAT

AND RESSANG

FIGS. 12-14. Culture of C3Hf mouse mammary tumor cells producing Gross leukemia virus. Fig. 12. Budding particle with two concentric shells (2,3) underneath the cell membrane (1). Fig. 13. Free immature C particle consisting of an outer envelope (1) and two concentric shells (2, 3). Fig. 14. Mature C particle showing an envelope (11, and a central electron-dense nucleoid (2). x 120,000.

cles were found only in the cell lines. They generally had a mean outer diameter of about 73 nm and an inner layer along both sides with an average thickness of about 65 A. Electron-dense granules were seen very often between the inner layers. Distribution

of BLV Antigens

When short-term cultures of leukocytes were incubated with goat anti-BLV serum and ferritin conjugate, the envelopes of almost all BLV particles were labeled (Fig. 19), whereas the cell surface was labeled only rarely and then in small areas. The same results were obtained when BLV-producing cell lines were incubated with bovine or goat anti-BLV serum (Figs. 20 and 21). In both cases, virus particles labeled with ferritin were observed in some vacuoles, while other vacuoles contained unlabeled virus particles (Fig. 21). This means that structures that appeared in single sections as cytoplasmic vacuoles containing virus particles in some instances are in open connection with the extracellular space. In other cases they represent real cytoplasmic vacuoles. No labeling of the BLV particles and of the cell surface was observed in the control experiments: (1) short-term cultures of leukocytes of clinically healthy cows with normal hemograms and of fetal bovine lung cells incubated with goat anti-BLV serum followed by ferritin conjugate; and (2) BLV-producing short-term cultures of

leukocytes and cell lines incubated with normal goat serum and ferritin conjugate. DISCUSSION

The morphology of BLV in short-term cultures of leukocytes and in the cell lines studied in this paper is identical. This means that the morphogenetic patterns of this virus are independent of the cell in which it is growing. In this study we used BLV-producing cells from two different species, bovine and sheep; and different kinds of cultivation, short-term cultures of leukocytes growing in suspension and cell lines growing as monolayers. The yield of virus particles seems to be higher in the short-term cultures, because clusters of numerous particles were found outside the leukocytes whereas in the cell line only small groups were present. BLV particles can be produced by lymphocytes and granulocytes; budding particles were found in both kinds of cells. In Fig. 22 the morphogenesis of several oncornaviruses, which can be followed in thin sections, is summarized: At the lefthand side the development of the virus particles, known as C particles, is shown (Laird et al., 1968; De Harven, 1968; Feller et al., 1971; Theilen et al., 1971); and at the right-hand side the morphogenesis of the MMTV, known as B particle, and of MPMV and bromodeoxyuridine-induced guinea pig virus (GPV) is represented (Hageman et al., 1972; Sarkar et al., 1973; Chopra and Mason, 1970; Kramarsky et

FIG. 15. Cell CDIS-191. Condensation of electron-dense material within the cytoplasm resembling vn-us particles in the first stage of budding (C). Mature particles (M) in the cytoplasm. ~76,000. FIG. 16. Cell line CDIS-191. Mature (M) and immature (I) virus particles within the cytoplasm. A virus particle (arrow) seems to leave the cell. ~76,000. FIG. 17. Culture of leukocytes. Three mature virus particles seem to leave the cell. Particle (1) is still embedded in the cytoplasm. Particle (2) is in contact with the surface membrane. Particle (3). wit,h a high electron-dense envelope, seems to be attached to a vesicle. x76,000 49

50

CALAF’AT

AND RESSANG

FIG. 18. Cell line CDIS-191. Clusters of filamentous forms (arrows) budding into a vacuole. An outer envelope (l), inner layer (2), and granules (3) between the inner layers can be seen. ~54,000. Inset, a filamentous particle (arrow) budding from the plasma membrane. ~76,000.

al., 1971; Fong and Hsiung, 1976). In Fig. 23 the two mechanisms of development of BLV as described in Results are summarized. The following differences can be seen if we compare the morphogenesis of the viruses described in both figures. During the

budding process of the oncornaviruses described in Fig. 22, two shells are formed underneath the cell membrane (l-3). When these shells are complete (4) the particle buds off and gives rise to an immature particle (I), consisting of two shells wrapped by an envelope. During the bud-

FIGS. 19-21. Culture of leukocyte (Fig. 19) and cell line FBL-J6 (Figs. 20 and 21) incubated with goat anti-BLV serum and ferritin conjugate. Figs. 19-20. Virus particles are labeled, whereas the cell surface is unlabeled. Fig. 21. Small areas on the cell surface are labeled (marked areas). One virus particle in a cytoplasmic vacuole (arrow) is unlabeled. Fig. 19, ~57,000; Fig. 20, ~80,000; Fig. 21, ~50,000. 51

52

CALAFAT

AND RESSANG

FIG. 22. Schematic representation phogenesis of several oncornaviruses sion).

of the mor(see Discus-

FIG. 23. Schematic representation phogenesis of BLV (see Discussion).

of the mor-

ding process of BLV (Fig. 23, left-hand side), however, only one shell is present underneath the cell membrane (1). To this shell granules are attached (2) which probably are comparable to the second inner shell (of the viruses described in Fig. 22.) that contain the viral RNA. When the shell is completed (31, the particle buds off resulting in an immature particle (I) that also differs from that of the other oncornaviruses in that it consists of only one shell with attached granules inside, surrounded by an envelope. In some cases during the budding process the electron-dense nucleoid can be formed before the particle is freed from the cell (4, 5). In the second pathway of BLV morphogenesis represented in the right-hand part of Fig. 23, some points remain obscure: How does the mature particle leave the cell or enter the cytoplasmic vacuole [Fig. 23(3)]? In the case of short-term cultures of leukocytes, it is possible that the virus particles are freed from the cell as a conse-

quence of cell degeneration. This could also explain why the virus particles outside the leucocytes are very often seen together with cell debris. The immunoferritin studies on BLVproducing cells have demonstrated that both sera used contain antibodies against BLV. The cell surface, however, was only rarely labeled and then in small areas, in contrast to the surface of other oncornavirus-producing cells, where relatively large areas are labeled after incubation with the respective antisera against the viruses (Aoki et al., 1970; Schwarz et al ., 1976; Oshiro et al., 1971; Gelderblom et al., 1972; Calafat et al., 1974b; Gelderblom and Schwarz, 1976). This means that on the cell surface of BLV-producing cells very few viral structural polypeptides are present. This result strengthens the morphological observations on the scarcity of budding particles. This study shows that the morphogenesis of BLV is different from that of the type B and C and of other particles such as MPMV and GPV. ACKNOWLEDGMENTS We thank Mrs. T. Nouwen-Latta, Miss C. Koning, and Miss N. Mastenbroek for excellent technical assistance and Dr. C. A. Feltkamp, Dr. Ph. C. Hageman, and Dr. H. Temmink for critical reading of this manuscript. REFERENCES AOKI, T., BOYSE, E. A., OLD, L. J., DE HARVEN, E., H%MMERL.ING, U., and WOOD, H. A. (1970). G (Gross) and H-2 cell surface antigens: Location on Gross leukemia cells by electron microscopy with visually labeled antibody. Proc. Nat. Acad. Sci. USA 65, 569476. CALAFAT, J., HAGEMAN, P. C., and RESSANG, A. A. (1974a). Structure of C-type particles in lymphocyte cultures of bovine origin. J. Nat. Cancer Inst. 52, 1251-1257. CALAFAT, J., BUIJS, F., HAGEMAN, HILGERS, J., and HEKMAN, A.

P. C., LINKS, J., (1974b). Distribution of virus particles and mammary tumor virus antigens in mouse mammary tumors, transformed BALB/c mouse kidney cells and GR ascites leukemiacells. J. Nat. Cancerlnst. 53,977-992. CHOPRA, H. C., and MASON, M. M. (1970). A new virus in a spontaneous mammary tumor of a rhesus monkey. Cancer Res. 30, 2081-2086. DALTON, A. J., HEINE, U. I., and MELNICK, J. L.

MORPHOGENESIS

OF BOVINE

(1975). Characterization of oncomaviruses and related viruses. (A report.) J. Nat. Cancer Inst. 55, 941-943. DE HARVEN, E. (1968). Morphology of murine leukemia viruses. In “Experimental Leukemia” (M. A. Rich, ed.), pp. 97-129. Appleton-Century-Crofts, New York. DOUGHERTY, R. M., and DI STEFANO, H. S. (1965). Virus particles associated with “nonproducer” Rous sarcoma cells. Virology 27, 351-359. FELLER, U., DOUGHERTY, R. M., and DI STEFANO, H. S. (1971). Comparative morphology of avian and murine leukemia viruses. J. Nat. Cancer Inst. 47, 1289-1298. FEWER, J. F., STOCK, N. D. and PECK-SUN, LIN. (1971). Detection of replicating C-type viruses in continuous cell cultures established from cows with leukemia: Effect of the culture medium. J. Nat. Cancer Inst. 47, 613-621. FERRER, J. F. (1972). Antigenic comparison of bovine type C virus with murine and feline leukemia viruses. Cancer Res. 32, 1871-1877. FONG, C. K. Y., and HSIUNG, G. D. (1976). Oncornavirus of guinea pigs. I. Morphology and distribution in normal and leukemic guinea pig cells. Virology 70, 385-398. GELDERBLOM, H., BAUER, H., and GRAF, T. (1972). Cell-surface antigens induced by avian RNA tumor viruses: Detection by immunoferritin technique. Virology 47, 416-425. GELDERBLOM, H., and S~HWARZ, H. (1976). Relationship between the Mason-Pfizer monkey virus and HeLa virus: Immunoelectron microscopy. J. Nat. Cancer Inst. 56, 635-637. GILDEN, R. V., LONG, C. W., HANSON, M., TONI, T., CHARMAN, H. P., OROSZLAN, S., MILLER, J. M., VAN DER MAATEN, M. J. (1975). Characteristics of the major internal protein and RNA-dependent DNA polymerase of bovine leukemia virus. J. Gen. Viral. 29, 305-314. HAGEMAN, P., CALAFAT, J., DAAMS, J. H. (1972). The mouse mammary tumor viruses. In “RNA Viruses and Host Genome in Oncogenesis” (P. Emmelot and P. Bentvelzen, eds.), pp, 283-300. North-Holland, Amsterdam, London. KETTMAN, R., PORTELELLE, D., MAMMERICKX, M., CLEUTER, Y., DEKEGEL, D., GALOUX, M., CHYS-

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DAEL, J., BURNY, A., and CHANTRENNE, H. (19761. Bovine leukemia virus: An exogenous RNA oncogenie virus. Proc. Nat. Acad. Sci. USA 73, 10141018. KRAMARSKY, B., SARKAR, N. H., and MOORE, D. H. (1971). Ultrastructural comparison of a virus from a rhesus-monkey mammary carcinoma with four oncogenic RNA viruses. Proc. Nat. Acad. Sci. USA 68, 1603-1607. LAIRD, H. M., JARRET, O., CRIGHTON, G. W., and JARRET, W. F. H. (1968). An electron microscopic study of virus particles in spontaneous leukemia in the cat. J. Nat. Cancer Inst. 41, 867-878. MILLER, J. M., MILLER, L. D., OLSON, C., and GILLETE, K. G. (1969). Virus-like particles in phytohemagglutinin stimulated lymphocyte cultures with reference to bovine lymphosarcoma. J. Nat. Cancer Inst. 43, 1297-1305. OLSON, C., MILLER, L. D., MILLER, J. M., and Hoss, H. E. (1972). Transmission of lymphosarcoma from cattle to sheep. J. Nut. Cancer Inst. 49,14631467. OSHIRO, L. S., RIGGS, J. L., TAYLOR, D. 0. N., LENNETTE, E. H., and HUEBNER, R. J. (1971). Ferritin labeled antibody studies of feline C-type particles. Cancer Res. 31, 1100-1110. RESSANG, A. A., MASTENBROEK, N., QUAK, J., VAN GRIENSVEN, L. J. L. D., CALAFAT, J., HILGER~, J., HAGEMAN, P. C., S~UISSI, T., and SWEN, S. (1974). Studies on bovine leukemia. I. Establishment of type C virus producing cell lines. Zentralbl. Vef. Med. B 21, 602-617. SARKAR, N. H., MOORE, D. H., KRAMARSKY, B., and CHOPRA, H. C. (1973). Oncomavimses, 3. The mammary tumor virus. In “Ultrastructure of Animal Viruses and Bacteriophages” (A. J. Dalton and F. Haguenau, eds.) pp. 307-321. Academic Press, New York, London. S~HWARZ, H., HUNSMANN, G., MOENNING, V., and SCHAFER, W. (1976). Properties ofmouse leukemia viruses. XI. Immunoelectron microscopic studies on viral structural antigens on the cell surface. Virology 69, 169-178. THEILEN, G. H., GOULD, D., FOWLER, M., and DUNGWORTH, D. L. (1971). C-type virus in tumor tissue of a woolly monkey (Lagothriz spp) with fibrosarcoma. J. Nat. Cancer Inst. 47, 881-889.