Control of RFM Strain Endogenous Retrovirus in RFM Mouse Cells

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1 JOURNAL OF VIROLOGY, Jan. 1983, p X/83/ $02.00/0 Copyright 1983, American Society for Microbiology Vol. 45, No. 1 Control of RFM Strain Endogenous Retrovirus in RFM Mouse Cells RAYMOND W. TENNANT,t* JAMES A. OTTEN, TSEI-WEI WANG,f RUEY-SHYAN LIOU, ARTHUR BROWN,t AND WEN K. YANG Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee Received 15 June 1982/Accepted 21 September 1982 RFM/Un mice express an endogenous type C retrovirus throughout their life span in many tissues; primary or established embryo fibroblast cell cultures do not express a virus but can be induced by exposure to 5-iodo-2'-deoxyuridine. All of our sources yielded a single ecotropic virus (RFV) which appeared to be related more closely to the endogenous N-tropic virus (WN1802N) of BALB/c mice than to Gross leukemia virus on the basis of two-dimensional gel electropherograms of virion proteins. No xenotropic or recombinant viruses were isolated by cocultivation techniques. RFV is N-tropic, and RFM/Un cells possess the Fv-l" allele, as indicated by restriction of B-tropic virus and susceptibility to Gross strain N- tropic virus. However, RFM cells are highly resistant to RFV and other endogenous N-tropic viruses. This resistance is expressed by two-hit titration kinetics and by inhibition of viral linear duplex DNA formation. This is similar to the effects of the Fv-1 locus, but preliminary work has shown no apparent genetic linkage between the two restrictions. The relative strength of the restriction, the presence of a single class of ecotropic virus, and the absence of recombinant viruses suggest that in RFM mice virus is expressed only in cells in which it is induced and not by cell-to-cell transmission. After irradiation, RFMIUn mice (6, 22, 24) develop a high incidence of myeloid leukemia or thymic lymphomas (3, 28-30), and the pathological features of the myeloid leukemia closely resemble chronic granulocytic leukemia in humans (2, 28, 29). A number of factors that influence the development of RFM/Un myeloid leukemia have been described (30); for example, the incidence of the disease is significantly altered in animals subjected to different types of microbial environments, possibly as a result of associated variations in granulocytopoiesis (31). Evidence implicating a retrovirus in the etiology of the disease has been reported (13, 25), but no detailed biological characterization of a putative myeloid leukemia virus has been presented. The studies reported here indicate that RFM/Un mice, which have an Fv-Jn genotype, have only one inducible endogenous N-tropic virus locus and possesses an apparently unique gene, which restricts infection of RFM/Un cells by endogenous N-tropic viruses. t Present address: Cellular and Genetic Toxicology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC t Present address: University of Tennessee, Knoxville, TN Present address: Biotech Research Laboratories, Inc., Rockville, MD MATERIALS AND METHODS Cells. Cells were routinely grown in 100-mm tissue culture dishes (Corning Glass Works, Corning, N.Y.) and were fed twice weekly with McCoy medium (GIBCO Laboratories, Grand Island, N.Y.) containing 10% fetal calf serum (Flow Laboratories, Inc., McLean, Va.), 2 mm glutamine, 100,g of streptomycin per ml, and 100 U of penicillin per ml. A trypsin- EDTA solution (0.2%) was used for subculturing cells that were kept subconfluent by subculturing at 4- to 7- day intervals. Cells were incubated at 37 C in an atmosphere containing 5% CO2. Routine testing of the cultures for mycoplasma showed no evidence of contamination. The AKR cells were obtained originally from Wallace P. Rowe, National Institutes of Health (NIH), as a cloned non-virus-producing cell line and were routinely monitored for spontaneous virus activation. The origins of NIH/3T3 cells, BALB/c 3T3 (clone A31) cells, SC-1 cells, and CCL64 (mink) cells have been described previously (20, 27). The RFM/3T3 and C3H cell lines were developed from primary embryo cultures in our laboratory and were maintained as 3T3 lines (1). NIH Swiss mouse embryo cells were prepared from embryos of Swiss mice (Microbiological Associates, Walkersville, Md.) of 15 to 17 days' gestation. Viruses. The preparation of RFV virus is described below. The other viruses used in our experiments were WN1802N, WN1802B, and Gross leukemia virus (GLV), which were gifts from Janet Hartley, and AKR virus, which we prepared in our laboratory by inducing AKR cells with 5-iodo-2'-deoxyuridine (IUdR). All

2 48 TENNANT ET AL. viruses were assayed on various Fv-1n and Fv-1b cells to determine their host ranges and were also tested for the presence of xenotropic virus by infecting CCL64 cells. All virus stocks were stored at -190 C in the vapor phase of a liquid nitrogen freezer. Assays for virus-infected cells were performed by the XC plaque assay (23) or by a fluorescent antibody assay (27) for viral antigens. Modified Hirt extract, agarose gel electrophoresis, and diazobenzyloxymethyl paper transfer methods. Cells were plated at a density of 1.5 x 106 cells per 100-mm dish and were infected 18 to 24 h later with virus at a multiplicity of infection of 0.5. Infected cells were harvested at different times postinfection by lysing the cells in a Hirt solution at 60 C. The lysates were adjusted to contain 1 M NaCl and then incubated for 8 to 16 h at 4 C. After centrifugation for 60 min at 25,000 rpm to remove the precipitate, the supernatant was digested with pronase and concentrated in a dialysis bag by using polyethylene glycol The nucleic acid was isolated by phenol-chloroform extraction, separated by electrophoresis in an agarose gel, and transferred to diazobenzyloxymethyl paper. Virus-specific DNA was detected by hybridization with specific 32P-labeled copy DNA to WN1802B virion RNA, followed by autoradiography. Further details of these procedures have been described elsewhere (33). Isoelectric focusing and gel electrophoresis. WN1802B, WN1802N, and GLV were grown in the presence of [3H]leucine and were harvested at 3-h intervals. The viruses were first purified by centrifugation at 30,000 x g in an SW27 rotor through a 20% sucrose cushion. The resulting pellets were suspended in TNE buffer and placed on 12-ml 15 to 20%o sucrose J. VIROL. gradients, which were centrifuged overnight at 30,000 x g in an SW41 rotor; fractions were collected, and the virus-containing fractions were identified by scintillation counting. Two-dimensional gel electrophoresis was performed by the method of O'Farrell (19). Briefly, purified viral samples were dissolved in lysis buffer A (9.5 M urea, 2% Nonidet P 40, 2% ampholines, 5% P-mercaptoethanol). The first-dimension gel, containing a sample of 60,000 to 100,000 cpm, was run for 17 h at 400 V, followed by electrophoresis for 1 h at 800 V in thinwall Corning glass tubes (1.3-mm inside diameter by 12 cm). A control tube was included in each run to be used for ph gradient measurements. Immediately after the completion of the first-dimension run, the gel was extruded from the tube and layered onto a uniform 12% polyacrylamide gel (with stacking), followed by fluorography. The gel was run at 25 ma/gel until the dye reached the bottom. After drying, the gel was exposed to Kodak X-O-matic-R film with intensifying screens. The parallel first-dimension control gel was cut into 1-cm sections; each section was suspended in 1 ml of water to extract ampholines, and the ph was then determined for each section. RESULTS Expression of endogenous retrovirus in RFM/Un tissues and cells. The spleens, thymuses, and bone marrows of both weanling and young adult mice from control and irradiated (300 R) RFM/Un mice were tested for virus expression. Table 1 shows that the virus proteins detected by the immunofluorescence assay TABLE 1. Analysis of RFM/Un tissues for virus (control, irradiated, and chemically induced) Cocultivation with:' Tissue source Thymus SC-1 cellsc Mink cells sectiona Bone Bone Thymus Spleen marrow Other Thymus Spleen marrow Other Weanling mice (3-4 weeks old) Young adults (6 months old) Irradiated (250 R) young adults Cultured primary embryos RFM/3T3 RFM/3T3 + IUdRd + Thymic epithelium Thymic epithelium + IUdRd + Myeloid leukemia + Thymoma + + a Frozen sections (4 to 5,um) of thymus tissue were acetone fixed to a slide and stained with fluoresceinconjugated anti-moloney leukemia virus antisera. b Tissues were removed from the animals, dispersed, and cocultivated with indicator cells (SC-1 or CCL64) in the presence of 2 ILg of polybrene per ml. After three subcultures, supernatant was removed, filtered, and inoculated onto SC-1 and CCL64 cells. Cover slips were examined after 48 h for virus production by immunofluorescence. c When virus was detected in the SC-1 cells or the RFM/Un tissues, tropism was determined by inoculating SME (Fv-1") or BALB/c (Fv-1b) cells, followed by an XC plaque assay. d Cells were treated with 50,ug of IUdR per ml for 24 h. The cultures were washed once, and indicator cells were added in the presence of polybrene. Cover slips were harvested and assayed for virus protein by immunofluorescence.

3 VOL. 45, 1983 CONTROL OF RFM ENDOGENOUS RETROVIRUS 49 and the infectious virus detected by the XC plaque assay were present in the hematopoietic tissues from all of the different age groups examined. In addition, a radioimmunoassay (16) showed the presence of gp7o and p30 proteins in the spleens, thymuses, and sera of weanling mice (data not shown). In contrast, cultured primary RFM/Un embryo cells did not express virus protein or infectious virus. When cells from secondary or tertiary cultures or established RFM/3T3 cells were treated with 50,ug of IUdR per ml for 24 h, they were induced to express virus protein and infectious virus (21). Virus was also detected in cultured RFM/Un thymoma cells and in cultured and transplantable myeloid leukemic spleen cells (13). These latter tumor cells produced neoplasms of the respective histological types when they were inoculated into young RFM/Un mice. Virus protein was not detected in adherent epithelial (nonneoplastic) cells cultured from a spontaneous thymoma unless the cells were treated with 50,ug of IUdR per ml. Although virus protein could be detected by immunofluorescence assay of treated cells, it was difficult to detect virus by direct XC plaque assay. To enhance the detection of virus, we cocultivated RFM/Un cells with SC-1 mouse cells or CCL64 mink cells (9). Virus was detected (by XC plaque induction and immunofluorescence) only in SC-1 cells, and all isolates demonstrated preferential growth on NIH Swiss (Fv-1n) cells as opposed to BALB/c (Fv-Jb) cells, confirming the N-tropism of the various tissue isolates (21). No virus of B-tropism, xenotropism, or mixed tropism was detected in any tissues or isolates when we used techniques which have been successful in demonstrating such viruses in cells from other mouse strains (9). If IUdR-induced RFM/Un cells were not cocultivated with SC-1 cells, the percentage of immunofluorescent cells rapidly declined, and it was possible to establish a culture that continuously produced virus only if the cells were subcultured after IUdR treatment. These results suggested that the spread of infectious virus was very inefficient and that stabilization of the induced state may require cell proliferation (unpublished data). Virus isolated directly from normal and neoplastic cells (tissues and cultured cells) or induced from virus-negative cells by IUdR treatment was inoculated by subcutaneous and intrathymic routes into newborn RFM/Un, C3H, and NIH Swiss mice, and these animals were observed for up to 18 months. In no case was there any consistent pattern of neoplastic or other disease which could be associated with virus inoculation. Characterization and host range of the RFM/Un endogenous virus. We used an IUdRinduced line of RFM/Un cells (RFM/3T3) developed in our laboratory as the principal source of endogenous virus for characterization. RFM/3T3 cells were treated with medium containing 50,ug of IUdR per ml for 24 h; the IUdR was then removed, and the cells were cocultivated with SC-1 cells. The addition of hydrocortisone (10-6 M), insulin (0.02 U/ml), and polybrene (2.0 p.g/ml) to the medium significantly improved detection of induction, presumably by promoting the spread of the induced virus to the SC-1 cells (11). The cocultivated cells were subcultured four times in the presence of the additives described above, and at each subculture the expression of virus was monitored by an immunofluorescence assay (20, 27). At the fourth subculture, 90 to 100% of the cells expressed viral antigen, and at this time membrane-filtered (0.45 pum) supernatant was added to SC-1 cells for detection of infectious virus. At 48 h, 90% of the SC-1 cells were positive for viral antigen, indicating the presence of infectious virus. Supernatant from the infected SC-1 cells was passed to another group of SC-1 cells, and from these latter cells a seed pool of virus (designated RFV) was prepared for further study. RF/J and some other mouse strains carrying the n allele of the Fv-J locus have been found to be more resistant to infection by some N-tropic viruses than the prototype strains (W. P. Rowe, personal communication). Table 2 shows the comparative titers of four N-tropic viruses in cells having known Fv-1 genotypes. We compared the titration patterns in six cell lines of the endogenous RFM/Un virus (RFV) and three other viruses: WN1802N, an endogenous N- tropic virus from BALB/c mice; AKR, an endogenous N-tropic virus of mouse strain AKR; and GLV, a leukemogenic, laboratory-passaged virus. All four viruses infected SC-1 (Fv- -) cells to comparably high degrees and were highly restricted in BALB/c 3T3 clone A31 (Fv-1b) cells. All three of the endogenous viruses were restricted by 3 to 4 logs in RFM/3T3 cells, whereas GLV infectivity was reduced only about 10-fold. These results demonstrated a significant restriction of RFM/3T3 cells for endogenous N-tropic viruses, particularly RFV derived from RFM/Un cells, relative to infection by the exogenous N-tropic virus GLV. A plot of the percentage of cells infected versus the multiplicity of infection (based upon the titer in SC-1 cells) illustrated the kinetics of RFV infection of RFM/Un cells (Fig. 1). The patterns of infection of SC-1, NIH Swiss, C3H, and AKR cells, which are all Fv-1n, were comparable to the pattern observed for other N- tropic viruses. Infection of BALB/c clone A31 cells (Fv-Jb) showed two-hit kinetics as expect-

4 50 TENNANT ET AL. J. VIROL. TABLE 2. Relative sensitivities of four N-tropic virus stocks on cells of different Fv-1 genotypes Cells Fv-l Titer (PFU/ml)a Cellsgenotype WN1802N RFV AKR GLV C3H nn 4.4 x x x 105 NDb RFM/3T3 nn 1.3 x x x x 105 AKR nn 9.4 x x x x 106 NIH/3T3 nn 5.4 x x x x 106 BALB/c 3T3 bb 7.2 x x x x 103 clone A31 SC x x x x 106 a The cells were plated in 35-mm wells, and 18 h later the medium was removed and the cells were treated with DEAE-dextran (25,ug/ml) for 30 min. The DEAE was removed, and the cells were washed and then infected with virus for 1 h. The virus was removed, and 5 days later XC plaque titrations were performed. b ND. Not determined. ed, but infection of RFM/Un (Fv-J') cells was restricted to even a greater degree and demonstrated a two-hit slope. Therefore, the restriction of RFM/Un cells for infection by the endogenous virus is a significant barrier to the extracellular spread of the virus. Although the characteristics of this restriction are similar to the characteristics of the Fv-Jn locus for B- tropic virus, and the Fv-1 nr allele for certain virus strains, our initial experiments involving BALB/c x RFM/Un crosses did not indicate genetic linkage (data not shown). Analysis of RFV viral proteins by gel electrophoresis. The two-dimensional gel electrophoresis patterns of the major gag gene proteins, p30, p15, p12 and p1o, showed characteristic multiple spots distributed within a specific ph range. Figure 2 shows the two-dimensional pattern for the RFV virus along with the patterns for WN1802B, WN1802N, and GLV for comparison. Differences are apparent in the patterns, but RFV appears to resemble most closely the endogenous N-tropic virus of BALB/c cells (WN1802N). Viral DNA formation. Kinetic analysis of proviral DNA formation using measurements of the linear (form III) and circular (forms I and II) viral DNAs in Fv-1 restrictive and permissive cells has been used in our laboratory to study the mechanism of restriction of murine retroviruses by the mouse Fv-J locus (33). This technique was also used to analyze the infection of B- tropic (WN1802B) virus, as well as GLV in RFM/3T3 cells and in NIH Swiss 3T3 cells (Fig. 3). Although NIH Swiss cells showed some inhibition of the formation of linear duplex DNA by B-tropic (WN1802B) virus compared with other Fv-I cells, as we previously reported (33), RFM/3T3 cells showed nearly complete inhibition. However, when these cells were infected with the laboratory-adapted virus GLV, forms I and III were both present by 12 h postinfection. Specifically, the appearance of the two species of covalently closed circular DNAs was quantitatively depressed in Fv-J restrictive cells according to the two-hit patterns of virus dose response. To characterize more fully the restriction of the RFV virus in RFM/Un cells, SC-1 (Fv-1-), RFM/3T3 (Fv-J ), and Swiss mouse embryo (SME) (Fv-10) cells were infected with RFV, and the kinetics of DNA formation were followed by Hirt extraction at 6, 12, and 30 h postinfection. Figure 4 shows that linear duplex (form I) DNA formation by RFV was preferentially inhibited in RFM/3T3 cells compared with formation in SC-1 or SME cells. Infection of SC- 1 and SME cells with RFV generated all forms of DNA by 12 to 30 h postinfection. These results indicate that RFMIUn cells possess a restrictive allele of the Fv-ln gene for B-tropic virus, as well as a significant restriction for its c:0 z J) Ji 0.1 ao0- QOO1 - QOOO1 0 au A A 0 aaa~ ol oa01 Xoo MULTIPLICITY OF INFECTK)N FIG. 1. Titration patterns of RFV in cells of various mouse strains. Cultures were plated in 35-mm wells, and 18 h later the medium was removed and the cells were treated with DEAE-dextran (25,ug/ml) for 30 min. The DEAE was removed, and the cells were washed and infected with dilutions of RFV for 1 h. The virus was removed, and after 5 days XC plaques were counted. Symbols: A, SC-1 cells; *, NIH Swiss cells; 0, BALB/c cells; O, C3H cells; A, AKR cells; 0, RFM cells. 0

5 VOL. 45, 1983 CONTROL OF RFM ENDOGENOUS RETROVIRUS 51 I C IMW.t.A. _ :rl Nw*.w S. C A: p.,, -4 -i 21 I. I.' I-. '- 1. * C C..1 i. L. I 1. 'I -D 4_. I 4 Downloaded from FIG. 2. Two-dimensional gel electrophoresis patterns of RFV, WN1802N, WN1802B, and GLV. The viruses were produced in medium containing [3H]leucine (10 mci/ml) in leucine-free medium and were processed as described in the text. Two-dimensional gel electrophoresis procedures were performed by the method of O'Farrell. (A) RFV. (B) WN1802N virus. (C) WN1802B virus. (D) GLV. own endogenous N-tropic virus (RFV) reflected in the depressed formation of linear duplex viral DNA. DISCUSSION The etiological role of retroviruses in radiation-induced leukemogenesis has been extensively analyzed in mouse strain C57BL/6 (4, 8, 14, 15). At least three distinct host range types of virus have been isolated from both normal cells and radiation-induced C57BL/6 tumors, and a recombinant virus, which preferentially infects thymocytes (thymotropic virus), has been described (4). The characteristics of this thymotropic agent suggest an etiological role in radiation-induced thymic lymphomas in C57BL/6 mice (15). In the RFM/Un strain we have been able to demonstrate only one (Ntropic) host range class of virus in both normal and tumor cells. Low levels of this virus are expressed in hematopoietic tissues throughout the life span of RFM/Un mice, and no measurable increase in expression is associated with irradiation. The virus exhibits no apparent cellspecific tropism (i.e., thymotropism). Although it may be preferentially expressed in hematopoietic cells, virus from normal or neoplastic nonhematopoietic cells infects embryo fibroblasts. We have inoculated various aged RFM/Un mice with virus isolates, but no myeloid leukemia or thymic lymphoma has been induced in mice held for at least 18 months. We tested only the potential tumorigenicity of direct isolates without recourse to in vivo serial passage, which frequently results in the emergence of highly oncogenic retroviruses. Therefore, the relationship between the expressed or induced endogenous virus and myeloid leukemia is unclear. However, the results obtained thus far indicate that in the RFM/Un strain, radiation-induced leukemogenesis may involve some mechanisms on May 8, 2018 by guest

6 52 TENNANT ET AL. J. VIROL. FIG. 3. Formation of viral DNA intermediates in NIH/3T3 and RFM/3T3 cells. The autoradiograms show the viral DNA intermediates in Fv-P0 mouse cells inoculated with Gross N-tropic virus (N-virus) or WN1802B B- tropic virus (B-virus) at a multiplicity of infection of 0.5. At 6, 12, 24, and 48 h after virus inoculation, the cells were extracted for unintegrated viral DNAs by using a modified Hirt extraction procedure. DNA preparations were separated by electrophoresis in a 0.7% agarose gel, transferred to diazobenzyloxymethyl paper, and hybridized with 32P-labeled viral copy DNA. The positions of the linear duplex form (III), closed circular supercoiled duplex forms (I), and open circular duplex forms (II) of viral DNA are indicated. HindIlI-generated phage XH DNA fragments were used as molecular weight markers for linear DNA duplexes. of leukemia induction or pathogenesis different from the mechanisms in the C57BL/6 strain. One key difference in the RFM/Un strain is the significant restriction of infection by endogenous N-tropic viruses, particularly the virus derived from RFM/Un cells, compared with infection by exogenous N-tropic Gross virus. The RF/J strain has also shown evidence of other potential genetic resistance elements (17, 18, 21) which inhibit tumor induction by chemicals. The two alleles of the Fv-1 locus (n and b) have been well defined, and Rowe (personal communication) has also found evidence of another type of allele (called nr) in cells from strains NZB, NZW, 129, and RF/J, which showed lowered sensitivity to N-tropic virus than the prototype Fv-J' NIH Swiss strain (21). The characteristics of RFM endogenous virus restriction appear to be similar to the characteristics of those alleles of the Fv-1 locus which appear to involve the virion p30 protein (10) as a target (in particular, in two-hit infection kinetics and in effect on proviral DNA synthesis). However, two major differences are that RFM endogenous virus restriction is a restriction of N- tropic virus in an Fv-ln genotype cell and that the synthesis of linear duplex DNA is inhibited, rather than the synthesis of the circular form, which appears to be a target of Fv-1-mediated resistance (33). The insensitivity of GLV to restriction by RFM cells while being restricted in Fv-lb cells suggests that there are discrete virion targets. The long passage history of GLV compared with the passage histories of endogenous viruses suggests that the difference resulted from adaptation to growth in cultured cells. Preliminary results do not indicate that the RFM endogenous virus resistance effect is linked to the Fv-1 locus, but further studies are in progress to confirm this observation. This effect may represent another nonlinked form of Fv-J. RFM restriction may be important in the pathogenesis of viral leukemia since it prevents or at least significantly reduces the frequency of intercellular spread of the virus. This means that the virus which is expressed in RFM/Un tissues probably arises by induction of endogenous virus, analogous to the activation of viral genes by IUdR or radiation (20, 21). RFM restriction may also be important in preventing the emergence of recombinant viruses, such as MCF (9), which may arise from recombination between spreading virus and other endogenous viral loci. We do not know if the restriction functions with equal efficiency in all cells and tissues of RFM/Un mice, but the absence of any recombinant viruses suggests that it is widespread. Therefore, if RFV is etiologically involved in spontaneous or radiation-induced hematopoietic neoplasia in this strain, it is unlikely that intercellular spread is a key element in the process. It is possible that the endogenous virus plays no role in radiationinduced leukemogenesis, particularly since simple intercellular spread of either spontaneously

7 VOL. 45, 1983 CONTROL OF RFM ENDOGENOUS RETROVIRUS 53 SC-I RFM/Un hr _I - 111~~~~~~~~~~~~~~~~~~~~4 _ SME ACKNOWLEDGMENTS This research was sponsored jointly by the Program of the National Cancer Institute und Agreement Y01 CP and the Office ( Environmental Research, U.S. Department of contract W-7405-eng-26 with the Union Carbid W LITERATURE CITED H 1. Aaronson, S. A., and G. J. Todaro Development of 3T3-like lines from BALB/c mouse embryo cultures: Mo I. Wt. transformation susceptibility to SV4o. J. Cell. Comp. X t06 Physiol. 72: BrIll, A. B., M. Tomanap, and R. M. Heyssel _ 15 0 Leukemia in man following exposure to ionizing radiation: 5,0 V a summary of the findings in Hiroshima and Nagasaki, and a comparison with other human experience. Ann. Intern. Med. 56: Clapp, N. K., E. B. Darden, and M. C. Jernigan Relative effects of whole-body sublethal doses of 60-MeV * - 4q 3 protons and 300-kVp x-rays on disease incidences in RF T mice. Radiat. Res. 57: Decdeve, A., C. Sato, M. Lieberman, and H. S. Kaplan Selective thymic localization of murine leukemia * 29 virus-related antigens in C57BL/Ka mice after inoculation with radiation leukemia virus. Proc. Natl. Acad. Sci. U.S.A. 71: Dojuku, R., J. L. Biedler, B. A. Spengler, and L. J. Old Trisomy of chromosome 15 in spontaneous leukemia of AKR mice. Proc. Natl. Acad. Sci. U.S.A. 72: Furth, J., H. R. Seibold, and R. R. Rathbone Experimental studies on lymphomatosis of mice. Am. J. Cancer 19: Hanna, M. G., R. W. Tennent, J. M. Yuhas, N. K. Clapp, B. L. Batzing, and M. J. Snodgrass Autogenous FIG. 4. Autoradiogram showing synt:hesis of RFV immunity to endogenous RNA tumor virus antigens in viral DNAs in SC-1 cells, RFM/3T3 cellls, Ils,andSME and cells. At 6, 12, and 30 h after RFV inc Res. mice with a low natural incidence of lymphoma. Cancer )culation at a 32: Haran-Ghera, N., and A. Peled Induction of leuke- extracted mia in mice by irradiation and radiation leukemia virus multiplicity of infection of 0.5, the cells wvere for unintegrated viral DNAs by using a modified Hirt variants. Adv. Cancer Res. 30: extraction procedure. DNA preparationis were sepa- 9. Hartley, J. W., N. K. Wolford, L. J. Old, and W. P. Rowe. rated by electrophoresis in a 0.7% agarc)se gel, trans A new class of murine leukemia virus associated ferred to diazobenzyloxymethyl paper, and hybridized with development of spontaneous lymphoma. Proc. Natl. with 32P-labeled viral copy DNA. The pc)sitions of the Acad. Sci. U.S.A. 74: linear duplex form (III), closed circula r supercoiled 10. Hopkins, N., J. Schindler, and R. Hynes Six NBduplex forms (I), and open circular duple, * viral DNA are indicated. HindIII-gen forms (11 I tropic murine leukemia viruses derived from a formsa pigof virus of BALB/c have altered p30. J. Virol. 21: B-tropic terated phage 11. Hsu, I.-C., and W. K. Yang DNA transfection of DNA fragments were used as molecular weight mark- ecotropic murine leukemia viruses in mouse cell cultures. ers for linear DNA duplexes. Each larne represents Cancer Res. 37: DNA samples from cells in one 100-mm dish (1.58 x 12. IhIe, J. N., J. C. Lee, L. Enjuanes, I. Circurel, I. Horak, 106 SC-1 cells, 1.73 x 106 RFM/3T3 cells, and 1.66 x and L. Pepersack Chronic immune stimulation as a 106 SME cells at the time of infection; ce 11,11 comparable in the 30-h postinfection per growth was possible mechanism in C-type viral leukemogenesis, p. t ws In M. Essex, G. Todaro, and H. zur Hausen diography was for 15 h. rod). Autora- (ed.), Cold Spring Harbor conferences on cell proliferation: viruses in naturally occurring cancer, vol. 7. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 13. Jenkins, V. K., and A. C. Upton Cell-free transmission of radiogenic myeloid leukemia in the mouse. Cancer expressed or induced virus to targe t t not appear to occur to any signific cells does 14. Res. 23: Kaplan, H. S Interaction between radiation Sant and extent. viruses in the induction of murine thymic lymphomas and Although our results do not preclude some other lymphatic leukemia, p In J. F. Duplan (ed.), Radia- may be tion-induced leukemogenesis and related viruses. North- mechanisms through which viral genes etiologically involved, such as ( cytogenetic Holland Publishing Co., Amsterdam. events represented by generations oi ftrisomy 15. Kaplan, H. S., A. Decleve, M. Lieberman, and S. Ftrisomy Man. 15 teuil-brutlag Multistage recombinational origin of (5, 32) or an immune response eff*ect (7, 12), leukemogenic viruses during thymic lymphoma developlly suitable ment in mice exposed to physical and chemical carcino- RFM/Un mice may be an especia model in which to test potential iritragenomic gens, p In M. A. Rich and P. Furmanski (ed.), recombination or transposition of spoecific retro- York. Biological carcinogenesis. Marcel Dekker, Inc., New viral genes (26). 16. Kennel, S. J., and R. W. Tennant Analysis of proteins of mouse sarcoma pseudotype viruses: typespecific radioimmunoassays for ecotropic virus p30's. J. Virol. 30: Virus Cancer 17. Mayer, A., M. L. Duran-Reynals, and F. Lilly Fv-1 ler Interagency regulation of lymphoma development and of thymic ecoof Health and tropic and xenotropic MuLV expression in mice of the Energy, under AKR/J x RF/J cross. Cell 15: ie Corp. 18. Mayer, A., F. D. Struuck, M. L. Duran-Reynals, and

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