Vol. 25, No. 3

JOURNAL OF VIROLOGY, Mar. 1978, p. 738-749

0022-538X/78/0025-0738$02.00/0

Printed in U.S.A.

Copyright © 1978 American Society for Microbiology

Endogenous Mink (Mustela vison) Type C Virus Isolated from Sarcoma Virus-Transformed Mink Cells CHARLES J. SHERR,* RAOUL E. BENVENISTE, AND GEORGE J. TODARO Laboratory of Viral Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda,

Maryland 20014

Received for publication 19 September 1977

A previously described type C virus stock (designated PP-1R), isolated by cocultivating baboon cells with mink cells transformed by Kirsten sarcoma virus (64J1), has been further cloned and characterized. End point-diluted stocks of PP-1R have been obtained that are free of focus-forming activity and lack both Kirsten sarcoma and primate type C viral sequences. Nucleic acid hybridization experiments show that the cloned virus (MiLV) is an endogenous, genetically transmitted virus of the mink (Mustela vison). MiLV replicates in canine, feline, and 64J1 mink cells but not in an untransformed mink cell line. Multiple viral gene copies can be detected in the DNA of normal mink cells in culture and in normal mink tissues; related endogenous viral genes are also detected in several related Mustela species. The virus codes for a p30 protein very closely related antigenically to that of feline leukemia virus but contains p15 and p12 proteins that are antigenically distinct. The mink cell line, MvlLu, and its Kirsten sarcoma-transformed derivative, 64J1, express relatively low levels of type C viral RNA related to MiLV and normally do not produce detectable levels of MiLV p30 protein or complete, infectious viral particles. Infection of sarcoma virus-bansformed mink cells with baboon type C virus, however, can augment the level of expression of endogenous mink viral RNA and can result in the synthesis and packaging of mink viral RNA and p30 antigen in extracellular virions. Since the MvlLu cell line and its transformed derivatives have become widely used in studies of retroviruses, the possibility of activating endogenous mink viral genes should be considered by investigators working with these cells.

The mink epithelioid cell line MvlLu, derived from fetal lung cells of the Aleutian mink (Mustela vison), supports the replication of many different groups of type C viruses, including the feline leukemia viruses (FeLV), the endogenous feline viruses (RD-114 group), the endogenous baboon viruses (M7/M28 group), the infectious primate viruses isolated from gibbons and a woolly monkey, and several different classes of murine xenotropic viruses (20). The relative permissiveness of these cells for type C viral replication has made them particularly useful as an indicator cell line in cocultivation experiments designed to obtain new type C isolates (22, 24, 50). The epithelioid morphology and high degree of contact inhibition exhibited by these cells has facilitated their use in assays for transformation by type C sarcoma viruses (20, 33) and in the isolation of a new class of focus-forming murine leukemia viruses (19). Nonproducer clones, derived from MvlLu celLs transformed by murine and feline sarcoma viruses, have been used to construct viral pseudotypes containing sarcoma viral genomes (20, 32, 33) and to develop sar-

coma virus-specific DNA transcripts (38-40). Morphologically flat revertants selected from clones of nonproductively transformed cells

have proven useful in assays for the enumeration of infectious, nontransforming leukemia viruses (32). More recently, MvlLu cells and certain of their transforned derivatives have been used to study relationships between hormone binding and type C virus-mediated transformation (49) and the expression of cell surface antigens encoded by leukemia (28) and sarcoma (48) viruses. MvlLu cells have been continuously propagated in our laboratory since 1973 and have thus far failed to spontaneously produce endogenous mink type C virus. These cells have been used in numerous cocultivation experiments and have remained virus negative throughout hundreds of passages in culture (50). Recently, however, we described two novel type C isolates (designated PP-1R and EP-1R) obtained from cocultivation experiments performed using primate cells and mink cells transformed by Kirsten sarcoma virus (45). We have now cloned and more completely characterized a component derived

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from the PP-1R virus stock that contains neither Kirsten sarcoma nor detectable primate type C viral sequences. The cloned isolate (MiLV) is an endogenous, genetically transmitted type C virus of mink that can, at least in certain circumstances, be derived from mink cell cultures. MATERIALS AND METHODS

rabbits by previously published methods (47). Goat antiserum to Tween-ether-disrupted FeLV was obtained from the Office of Resources and Logistics, Virus Cancer Program, Bethesda, Md. Purification and radiolabeling of viral proteins. Type C viruses were radiolabeled with [3P]phosphoric acid as described (44). Disrupted MiLV or FeLV virions were externally labeled with u261 (43), using the chloramine T method (17), to approximate specific activities of 1 ,tCi/.ug. Disrupted 3P2 and 125Ilabeled virions were cochromatographed on columns (90 by 5.0 cm) in the presence of 6 M guanidine hydrochloride (27), and individual viral proteins were identified by the distribution of "I label in the column effluent; viral p30, p15, and p12 proteins were dialyzed, lyophilized, and resuspended for radiolabeling as previously described (43). The purification of p30 proteins from the M7 baboon virus, the simian sarcoma-associated virus, the endogenous rat virus, RT21c, and the Rauscher murine leukemia virus has been previously described (46, 47). Purified,viral proteins relabeled with "I by the chloramine T method had specific activities of approximately 5 jiCi/,ug, were greater than 95% precipitable with trichloroacetic acid, and gave single sharp bands of appropriate molecular weight in polyacrylamide gels containing sodium dodecyl sulfate (21). Labeled proteins used in radioimmunoassays were stored in 0.05 M phosphate buffer, pH 7.5, containing 0.15 M NaCl and 1% bovine serum albumin and were used within 4 days of labeling. All proteins were greater than 90% precipitable with appropriate antisera, and titrations showed no loss of antigenicity of the labeled preparations during this period. Radioimmunoassays. Antiserum titrations and competitive radioimmunoassays were performed by a double-antibody method for precipitating immune complexes (30, 41). Competition assays were initiated with 50% of the iodinated test antigen bound in immune complexes; 10,000 cpm of tracer were used per 0.5-ml reaction tube (47). Purified viral proteins or detergent-disrupted vinuses were used as competing antigens, and immune complexes were precipitated with a titered excess of antiserum to the appropriate 7S globulin (46). Radioactivity in precipitates was quantitated in a Beckman 300 gamma counter (±3% error) at a 65% counting efficiency. The data are plotted as percent displacement of the labeled test antigen from immune complexes (linear ordinate) versus micrograms of competing protein (log scale abscissa). Standard curves were developed by using purified competing proteins as shown in Fig. 4 and 5. Points on the competition curves represent the averages of triplicate determinations for each competing antigen in each assay system. Proteins were quantitated by the method of Lowry et al. (25), with bovine serum albumin as a standard. Molecular weight of viral RNA subunits. Canine FCf2Th cells infected with MiLV were labeled with [3P]phosphoric acid (44), and virus was concentrated from culture supernatants and banded isopycnically in sucrose. The labeled viral RNA was extracted and centrifuged in linear sucrose velocity gradients as descibed (14). The pooled genomic RNA was precipitated with ethanol, denatured (14), and

Cells and culture conditions. The epithelioid mink cell line MvlLu, derived in 1964 by A. J. Kniazeff, W. A. Nelson-Rees, and N. B. Darby from fetal lung cells of the Aleutian mink (M. vison), was obtained in 1972 at the 41st passage from reference seed stocks frozen at the American Type Culture Collection (CCL 64). The 64J1 and 64F2 cell lines are clonal derivatives of MvlLu nonproductively transformed by Kirsten and feline sarcoma viruses, respectively (20). The rabbit SIRC cell line (CCL 60) was also obtained from the American Type Culture Collection. The canine (FCf2Th) and feline (FEC) cell lines were obtained from the Naval Biomedical Research Laboratories (Oakland, Calif.). Other cells used in these studies were rhesus monkey lung celLs, DBS-FRhL-1 (52); rat kidney cells, NRK (15); the murine SC-1 cell line (18); and human rhabdomyosarcoma cells, A204 (16). All cells were grown in plastic tissue culture flasks or in roller bottles in Dulbecco modified Eagle medium supplemented with 10% calf serum and were transferred with 1% trypsin in phosphate-buffered saline. Viruses. Previously characterized type C viruses used in these studies included the endogenous baboon virus (M7) isolated from Papio cnocephahu (5), the endogenous cat virus, RD-114 (26), and the Rickard strain of FeLV (36). The M7 virus was grown in human A204 cells or in mink 64F2 and 64J1 cells as indicated in Results. The RD-114 virus was grown in 64J1 or A204 cells, and FeLV was grown in cat FEC cells. The PP-1R virus, obtained from a cocultivation experiment performed with baboon cells and the mink 64J1 cell line (45), was grown in canine FCfT cella Further cloning of virus and the derivation of MiLV from the PP-1R stock was performed as described in Results. For synthesis of DNA transcripts of the virl RNA genomes, vinuses were concentrated from fresh, 4-h harvests of culture supernatants as described previously (10). For purification of viral structural proteins, 24-h harvsts from cells chronically producing virus were collected and frozen before concentration of viral particles from pooled culture supernatants. All vinus were concentrated by centrifugation and banded isopycnically in sucrose at 1.16 g/mL Infection of host cells with virus was performed as previously described (23).

Supernatant reverse transriptase assay.

These assays were performed as descrbed (23), using a polyriboadenylate template, oligodeoxythymidylatei2 18 primer, and 0.6 mM manganese as the divalent cation. The results are expressed as counts per minute of [3HJITMP incorporated into radioactive polydeoxythymidylate product in a 60-min linear reaction. Antisera. Antisera to detergent-disrupted MiLV and to the purified viral p30 proteins of MiLV and other type C viruses were raised in New Zealand white

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SHERR, BENVENISTE, AND TODARO

applied to cylindrical 0.5% agarose-1.5% polyacrylamide gels (31). [3H]RNA standards (28S, 18S, and 4S) were purchased from Sigma Chemical Co. (St. Louis, Mo.) and were admixed with 39P-labeled viral RNA before denaturation and electrophoresis. Electrophoresis was performed at 4 mA/tube for 60 min at 4°C. Gels were fractionated into 1-mm slices, and radioactive material was eluted and counted by liquid scintillation (2). Preparation ofDNA transcripts. [3H]thymidinelabeled DNA transcripts of viral RNA were prepared in the presence of actinomycin D as described (4, 11), using limiting concentrations of magnesium as the divalent cation (37). Labeled transcripts were resuspended in 0.2 N KOH and 5 mM EDTA, heated at 37°C for 30 min, and centrifuged in 5 to 20% linear alkaline sucrose gradients; [3H]DNA transcripts larger than 19S (approximately 7,000 bases) were used for molecular hybridization studies (11). The specific activity of the [3H]DNA was approximately 1.8 x 107 cpm/,ug. Hybridization of 70S 32P-labeled MiLV RNA with an equimolar concentration of [3H]MiLV DNA resulted in protection of 84% of the viral RNA from pancreatic RNase digestion (4, 11); with a 2.5 molar excess of [3H]DNA, 95% of the MiLV RNA was hybridized. Less than 2% of the MiLV [3H]DNA was resistant to digestion with the single-strand-specific nuclease, Si (1). Labeled mink cellular DNA was prepared by labeling MvlLu cells for 72 h with 120 ,uCi of [3H]thymidine per ml of culture medium. The DNA was extracted (4), and the nonrepetitive cellular DNA was isolated by removing the highly reiterated DNA sequences that anneal by a Cot of 200 (approximately 40% of the total DNA) by fractionation on hydroxyapatite (8). The specific activity of this DNA was 2.4 x 104 cpm/ug). Nucleic acid hybridization. Hybridization reactions were performed as described (4, 8), and the extent of hybridization was determined with the single-strand-specific nuclease, Si (1). Cot values were calculated as suggested by Britten and Kohne (12) and corrected to a monovalent cation concentration of 0.18 M (13). DNA and RNA were extracted from cultured cells and tissues as described (4); DNA was sonically treated to a mean size of 5 to 7S as determined by sedimentation in alkaline sucrose (10). Thermal stability measurements were performed as described (8-10).

RESULTS Isolation of MiLV. The PP-1R virus was initially isolated by cocultivation of baboon cells from the species Papio papio with the Kirsten sarcoma virus-transformed mink cell line, 64J1. Stocks of PP-1R, diluted beyond the end point for focus formation, still contained RNA sequences that hybridized to cycled DNA probes prepared to the Kirsten sarcoma virus as well as to a portion (about 12%) of the baboon type C viral genome. Nucleic acid hybridization studies also showed that the end point-diluted stocks of PP-1R contained additional RNA sequences of mink host cell origin (45).

J. VIROL.

To derive viral stocks free of Kirsten sarcoma viral sequences, serial fivefold dilutions of PP1R virus were used to infect cultures of FCf2Th canine thymus cells, and supernatants from each culture were assayed for viral polymerase activity. Eight replicate cultures of canine cells were infected at each viral dilution. Virus was taken 6 weeks after infection from one of two flasks that were positive for supernatant DNA polymerase activity at limiting dilution. This stock was used to infect new canine thymus cells, and the procedure was repeated. Recloned virus obtained at limiting dilution 8 weeks after the second round of infection (one of eight flasks positive for supematant polymerase activity) was used in subsequent studies. Canine cells infected with high-titer cloned virus lacked RNA sequences that hybridized (at Crt 6 x 104) to [3H]DNA transcripts prepared to the Kirsten sarcoma virus or to baboon type C virus (see below). To determine the host range of the recloned virus, culture supernatants from chronically infected canine cells were used to infect several indicator cell lines, and viral replication was monitored by a supernatant reverse transcriptase assay. The host range of the virus (designated MiLV) was compared with that of the original PP-1R virus stock. The host ranges of MiLV and PP-1R were identical, and each virus preparation replicated in dog, cat, and transformed mink cells (64J1), but not in normal MvlLu mink cells (Table 1). As opposed to the PP-1R virus, which took 2 months to yield significant levels of supernatant polymerase activity, MiLV yielded significant levels of supernatant viral polymerase activity 2 weeks after infection. These results suggest, then, that serial passage in canine thymus cells selected for a virus that replicated to high titer in each of the permissive indicator cell lines. Size of the MiLV viral genome. To determine the size of the MiLV RNA genome, canine cells producing the virus were radiolabeled with [3P]phosphoric acid, and virus concentrated from the culture supernatant was banded isopycnically in sucrose at a density of 1.16 g/ml. Virions obtained from the gradient were lysed, and the radioactive material was subjected to velocity sedimentation in a linear sucrose gradient. A portion of the 32P radioactivity sedimented in the region characteristic for 70S viral RNA (Fig. 1, inset). When the 70S RNA was pooled, denatured, and electrophoresed in an

agarose-acrylamide gel (Fig. 1), a single sharp radioactive band was observed. By comparison to the mobilities of 3H-labeled ribosomal and transfer RNA standards applied to the same gel, the molecular weight of the denatured viral

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VOL. 25, 1978 TABLE 1. Host ranges of PP-IR and MiLV

Host cell line

Species

3

Supematant reverse transcriptase activity (cpm x 10-3 of [3H]TMP incorporated)a

PP-lRb MiLVc 225 Mink (trans133 formed) Mink (untrans