Role of Mink Cell Focus-Inducing Virus in Leukemias Induced by Friend Ecotropic Virus

Transcription

1 JOURNAL OF VIROLOGY, June 1984, p X/84/ $02.00/0 Copyright 1984, American Society for Microbiology Vol. 50, No. 3 Role of Mink Cell Focus-Inducing Virus in Leukemias Induced by Friend Ecotropic Virus JONATHAN SILVER Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland Received 29 November 1983/Accepted 12 March 1984 Recombinant viruses have been implicated in the pathogenesis of murine leukemias induced by a variety of long-latency retroviruses. Neonatal mice of several strains were inoculated with Friend ecotropic virus (F-Eco) and analyzed for the presence of mink cell focus-inducing (MCF) virus or DNA restriction enzyme fragments which were specific for Friend MCF virus (F-MCF). MCF virus was detected within 2 weeks of inoculation in NFS/N mice and at about 2 months after inoculation in BALB/c mice. Both of these strains developed erythroblastosis after inoculation with F-Eco. In contrast, MCF virus was not detected in F-Ecoinoculated C57BL mice. These mice were resistant to erythroblastosis but developed lymphoma or myelogenous leukemia or both at about 5 months after inoculation. Thus, although MCF viruses were associated with F-Eco erythroblastosis in NFS/N and BALB/c mice, they were not necessary for F-Ecoinduced lymphoid or myeloid leukemias in C57BL mice. To investigate the association between resistance to erythroblastosis and absence of MCF virus, C57BL mice were inoculated with pseudotypic mixtures of F-Eco plus F-MCF; MCF virus replicated well in these mice, but the mice remained resistant to erythroblastosis. Furthermore, in genetic crosses between C57BL and NFS/N or BALB/c, some mice inherited resistance to F-Eco erythroblastosis without inheriting the C57BL resistance to the generation of MCF viruses. These results indicate that C57BL mice carry a gene for resistance to F-Eco erythroblastosis which is distinct from the C57BL genes which interfere with the generation of MCF viruses. Laboratory mice inherit a large number of DNA sequences related to murine leukemia viruses (MuLVs) (5, 24). When such mice are infected with ecotropic (mouse-tropic) MuLVs, recombination with endogenous virus-like sequences generates viruses with altered host ranges. These viruses were first recognized because they caused foci on mink cells (mink cell focus-inducing [MCF] viruses) (11). Several lines of evidence suggest that MCF viruses are pathogenic intermediates in at least some ecotropic virusinduced leukemias. First, MCF viruses are found in the thymuses of AKR mice a few months before they develop thymic lymphoma (11) and in preleukemic tissues of mice infected with a variety of exogenous MuLVs (10, 13, 26). Second, exogenous infection with MCF viruses can lead to development of lymphoma (7) or erythroleukemia (27). Third, a gene, Rmcff, which interferes with replication of MCF but not ecotropic viruses (12), induces resistance to AKR lymphoma and Friend helper virus erythroblastosis (19; J. W. Hartley and W. P. Rowe, unpublished data). Friend helper virus is a highly leukemogenic ecotropic virus (here designated F-Eco) which causes erythroblastosis in some strains of mice and lymphoma or myelogenous leukemia in other strains of mice (21). This communication describes studies of the role of MCF virus in F-Eco-induced leukemias and lymphomas. The results indicate that MCF viruses may be involved in erythroblastosis induced by F- Eco, but not in F-Eco-induced lymphoma or myelogenous leukemia in C57BL mice. Futhermore, C57BL mice appear to carry a gene for resistance to F-Eco erythroblastosis which is distinct from C57BL genes which interfere with the generation of MCF viruses. MATERIALS AND METHODS Mice. NFS/N, BALB/cAnn, C57BL/6N, C57BL10.D2, and C3H/HeN- mice were obtained from the Small Animal Section, National Institutes of Health. C57BL/6. C-H-7b and 872 CXB-H mice were obtained from the Jackson Laboratories, Bar Harbor, Maine. Mice were bred in this laboratory and inoculated at 0 to 2 days after birth. Virus. Biologically cloned NB tropic F-Eco was a gift from Janet W. Hartley, National Institutes of Health. F-MCF virus was cloned by Akinori Ishimoto in this laboratory by limiting dilution from a pool of in vivo-passaged Friend virus complex. Pseudotypes were prepared by infecting NFSIN mouse embryo cells with a mixture of F-Eco and F-MCF. MCF infectious center assay. Test cells (104 to 107) were plated as infectious centers on SC-1 cells. Three days later, cultures were UV irradiated and overlaid with CCL64 mink lung cells. Cultures were scored for MCF foci after 6 to 9 days; if no foci were detected, cultures were passaged once and scored for MCF foci 1 week later (8). Hematopathology. Diagnoses were based on hematocrit, gross pathology, Wright-Giemsa stains of blood, spleen and lymph node imprints, and histological sections of fixed tissues (21). Erythroleukemia was diagnosed in mice with: (i) severe anemia (hematocrit of less than 30%, usually -20%), (ii) hepatosplenomegaly, (iii) normal or smaller-than-normal thymus and lymph nodes, (iv) Wright-Giemsa stains of blood and spleen showing cells with characteristics of erythroblasts (deeply basophilic cytoplasm, condensed nuclear chromatin, heterogeneity in cell size), and (v) histology showing blasts infiltrating hepatic sinusoids, expanding splenic red pulp, and compressing splenic white pulp. Lymphoma was diagnosed in mice with: (i) grossly enlarged thymus or lymph nodes or both, (ii) Wright-Geimsa stains showing cells with characteristics of lymphoblasts (cytoplasm less basophilic, chromatin less condensed, cells more homogeneous in size as compared with erythroblasts), and (iii) histology showing blasts destroying the normal architecture of lymphoid organs. Mice with lymphoma usually had hematocrits of greater than 30% and splenomegaly with expanded splenic white pulp. When the liver was infiltrated

2 VOL. 50, 1984 ROLE OF MCF VIRUS IN LEUKEMIAS INDUCED BY F-ECO 873 TABLE 1. Effect of mouse genotype on generation and growth of F-MCF virus Inoculated with Days after No. positive for Mouse strain virus: inoculation Diagnosis MCF/no. tested' NFS/N F-Eco Preleukemic 9/ Erythroblastosis 8/8 BALB/c F-Eco Preleukemic 0/ Erythroblastosis 5/8 C57BLb F-Eco Preleukemic 0/ Lymphoma 0/ Myeloid Leukemia 0/9 BALB/c x C57BL/6 F-Eco Preleukemic 0/6C d Myeloid Leukemia 0/2 C57BL/6 F-Eco + F-MCFC Preleukemic 10/ Myeloid Leukemia 2/2 a Spleen cells were tested for MCF virus by the infectious-center assay (see text). b Includes 19 C57BL/6 mice, 3 C57BL/6.C-H-7b (Fv-2s) mice, and 2 C57BL/10.D2 mice. c Both spleen and thymus cells were tested for MCF virus in these mice. d Tested after establishment as transplantable tumors. e Ten mice were inoculated with an in vitro pseudotypic mixture of F-Eco and F-MCF, and six mice were inoculated with a spleen extract containing F-Eco and F-MCF from an NFS/N mouse with F-Eco-induced erythroblastosis. with lymphoblasts, the malignant cells infiltrated periportal areas much more prominently than in cases of erythroblastosis. Myelogenous leukemia was diagnosed in mice with (i) grossly enlarged, frequently greenish lymph nodes, (ii) splenomegaly, (iii) Wright-Giemsa stains showing predominantly immature myeloid cells, and (iv) histology showing immature myeloid cells infiltrating lymph nodes and expanding the splenic red pulp. A diagnosis of myelogenous leukemia was not made if there were signs of infection (which could lead to reactive myeloid hyperplasia). Blot hybridization. DNA was extracted from neoplastic organs (3), digested with EcoRI, electrophoresed on 0.6% agarose, transferred to nitrocellulose paper (23), hybridized to a 32P-labeled xenotropic virus envelope-specific probe (2) by a dextran-sulfate procedure (25), washed at 50 C in 0.lx SSC (SSC, 0.15 M NaCl plus M sodium citrate), and fluorographed with Kodak XR-5 film and an intensifying screen. The 32P-labeled probe was generously provided by Malcolm Martin, National Institutes of Health. RESULTS Mouse strain differences in generation of MCF virus. MCF virus was detected by the infectious-center assay in essentially all F-Eco-inoculated NFS/N mice which were 2 weeks of age or older (Table 1). Two NFS/N mice tested on day 5 were negative for MCF virus, and one NFS/N mouse tested on day 13 was negative for MCF virus in the spleen but positive for MCF virus in the bone marrow. The titer of MCF virus in NFS/N mice was usually >102 focus-forming units (FFU) per 107 spleen cells (12 of 18 mice), with occasional mice registering 10 to 102 FFU/107 spleen cells (5 of 18 mice). In contrast, MCF virus was not detected in F- Eco-inoculated BALB/c mice until they had developed erythroleukemia (Table 1), and not all mice had MCF virus in the spleen. In two of three leukemic BALB/c mice in which MCF virus was not detected in the spleen, MCF virus was detected in the thymus, even though the spleen but not the thymus was grossly infiltrated with leukemia cells. The titer of MCF virus in BALB/c mice with erythroblastosis was usually slightly lower than in NFS/N mice: four of eight BALB/c mice had 10 to 102 FFU/107 spleen cells, and only one BALB/c mouse registered >102 FFU/107 spleen cells. The earlier and more uniform detection of MCF virus in NFS/N than in BALB/c mice correlates with a shorter latency for development of erythroleukemia in NFS/N mice (a median latency of 62 days [range, 45 to 96 days] in 42 NFS/N mice compared with a median latency of 84 days [range, 45 to 220 days] in 55 BALB/c mice). MCF virus was not detected in any of 24 F-Eco-inoculated C57BL mice, even late after infection when the mice were frankly leukemic (Table 1). C57BL mice replicate this strain of F-Eco virus well (21) but are resistant to erythroblastosis (20) and develop lymphoma or myelogenous leukemia or both after a latent period of several months (a median latency of 160 days [range of 54 to 259 days] in 56 C57BL/6 mice) (21). MCF virus was also not detected in (BALB/c x C57BL/6)Fl mice (Table 1). These mice, like C57BL/6 mice, develop lymphoma and myelogenous leukemia but not erythroblastosis several months after neonatal inoculation with F-Eco (21). Detection of recombinant virus by blot hybridization. Because of the possibility that MCF virus might be missed in the infectious-center assay, another method of detecting recombinant virus was developed. MCF viruses (9), including F-MCF (26), are recombinants between ecotropic virus and endogenous MuLV-related sequences, with envelope region sequences derived from the nonecotropic parent. A hallmark of MCF viruses is the presence of an EcoRI site at -7.0 kilobases (kb) in the nonecotropic sequences (4). F- Eco has a single EcoRI site at -3.2 kb (18). MCF viruses derived from F-Eco may be expected to contain two EcoRI sites, one at -3.2 kb (from the ecotropic parent) and one at -7.0 kb (from the endogenous sequences). These two EcoRI sites have been found in two cloned F-MCF viruses (1, 17). We anticipated that the 3.8-kb fragment bounded by these Eco RI sites would react with a xenotropic virus envelope probe which detects nonecotropic sequences to the left of the EcoRI site in other MCF viruses (15). As illustrated in Fig. 1 and summarized in Tables 2 and 4, a 3.8-kb xenotropic

3 874 SILVER J. VIROL. A l - C F-ECO o-l As F-MCF 1 kb pro be Eco RI B a b cd 13.2 _ 4.4E0. a 1.9 ** 4w 4b 0 O FIG. 1. (A) Schematic representation of F-Eco and F-MCF proviruises showing EcoRI sites and region in F-MCF presumed to be derived from endogenous, MuLV-related sequences (1, 17, 18). The precise boundaries of the endogenous MuLV-related sequence in F-MCF were not determined. The probe was pxenv, derived from BglII-EcoRI envelope sequences from an infectious xenotropic virus (2). (B) Blot hybridization with the pxenv probe. Lanes a and d, size markers with size in kb indicated on left. Lane b, mink cell DNA. Lane c, DNA from mink tells chronically infected with F-MCF. Lane 1, liver DNA from control (uninfected) C57BL/6 mouse. Lane m, liver DNA from control (uninfected) BALB/c mouse. Lane n, tumor DNA from BALB/c erythroleukemia. Lanes e to k, tumor DNA from individual BALB/c x (BALB/c x C57BL/6) first-backcross mice with F-Eco-induced erythroleukemia. Lane o, tumor DNA from BALB/c x (BALB/c x C57BL/6) first-backcross mouse with F-Eco induced lymphoma. Lanes p to v, tumor DNA from individual C57BL mice with F-Eco-induced lymphoma (lane q) or myelogenous leukemia (lanes p and r to v). Lane w, tumor DNA from BALB/c x (BALB/c x C57BL/6) first-backcross mouse with myelogenous leukemia. The arrow indicates the position of the 3.8-kb internal EcoRI fragment from F-MCF virus. The other bands resulted from xenotropic envelope-related sequences inherited by BALB/c and C57BL/6 mice (14). envelope-reactive fragment was detected in biologically cloned F-MCF (Fig. ib, lane c) and in spleen DNA from a variety of mice with F-Eco erythroblastosis (Fig. ib, lanes e to k and n) but not in uninfected mice (Fig. ib, lanes and m) or in F-Eco-induced lymphomas or myeloid leukemias from C57BL and (BALB/c x C57BL/6)Fi mice (Fig. ib, lanes p to v). These blot hybridization results provide additional evidence that C57BL mice infected with F-Eco do not contain recombinant viruses similar to those in mice with F- Eco-itduced erythroblastosis. Occasional mice with F-Eco-induced lymphoma displayed non-germ line EcoRI fragments slightly smaller than 3.8 kb (Fig. ib, lane o), and some mice with F-Eco erythroblastosis contained more than one new EcoRI fragment (Fig. ib, lane i). These bands could be derived from recombinant viruses with deletions, rearrangements, or additional EcoRI sites. On rare occasions, F-Eco induced lymphoma rather than erythroblastosis in NFS/N mice. It is interesting that of two such F-Eco-induced lymphomas in NFS/N mice, one was negative for MCF virus by the blot hybridization assay, whereas the other had a single novel 2-kb EcoRI fragment (not shown). C57BL/6 resistance to F-Eco erythroblastosis is not due solely to resistance to generation of F-MCF virus. Two TABLE 2. Presence of a F-MCF-specific 3.8-kb EcoRI fragment in F-Eco-induced leukemias No. of mice with F- Mouse strain Type of MCF-specific 3.8-kb leukemia' EcoRI fragment/no. tested NFS E 3/3 NFS/N x C3H/HeN E 2/2 BALB/c E 3/3 CXB-H E 1/1 NFS/N L 0/2 C57BL L 0/2 C57BL M 0/7 BALB/c x C57BL M 0/7 a E, Erythroblastosis; L, lymphoma; M, myeloid leukemia.

4 VOL. 50, 1984 ROLE OF MCF VIRUS IN LEUKEMIAS INDUCED BY F-ECO 875 TABLE 3. Source of virus Leukemias induced by tumor extracts and in vitro pseudotypic mixtures of F-Eco plus F-MCF Mouse No. with disease/no. inoculated strain inoculated Erythroblastosis Lymphoma or inoculated ~~~~~~~~~~myeloid leukemia F-Eco + F-MCF in vitro pseudotypic mixture C57BL/6 0/13 10/13a BALB/c mouse with erythroblastosis C57BL/6 0/6 5/6b NFS/N mouse with erythroblastosis C57BL/6 1/13 11/13cd NFS/N mouse with erythroblastosis NFS/N 9/9 0/9 C57BL/6 lymphoma BALB/c 22/22 0/22 a Two mice died without diagnosis, and one mouse with skin ulcerations was killed on day 206. b One mouse with skin ulcerations was killed on day 185. c One mouse had lymphoma plus erythroblastosis (see text). d Two mice were killed while healthy (for virus assay) on days 219 and 234. experiments were done to determine whether the C57BL/6 resistance to F-Eco erythroblastosis could be attributed to resistance to generation of F-MCF viruses. First, neonatal C57BL/6 mice were inoculated with pseudotypic mixtures of F-Eco and F-MCF and followed for development of erythroblastosis. The pseudotypic mixtures were made from biologically cloned F-Eco and F-MCF, as well as spleen homogenates from NFS/N and BALB/c mice with F-Eco-induced erythroleukemias (which contained F-Eco plus F-MCF). MCF virus was easily detected in C57BL/6 mice inoculated with these pseudotypic mixtures, in contrast to C57BL/6 mice inoculated with F-Eco alone (Table 1). The titer of MCF viruses in these mice was usually in the range of 10 to 102 FFU/107 spleen cells (8 of 14 mice) and occasionally higher (4 of 14 mice). This was comparable to the titer of MCF virus in the spleens of BALB/c mice with F-Ecoinduced erythroblastosis. Nevertheless, these doubly-infected C57BL mice were resistant to erythroblastosis (Table 3). Of such mice, 26 of 32 developed lymphoma or myelogenous leukemia after latent periods of 60 to 260 days (median, 140 days), comparable to C57BL mice infected with F-Eco alone (21). One mouse with lymphoma was unusual in that it had severe anemia and erythroblasts in the peripheral blood. It is unclear whether this mouse had virus-induced erythroblastosis in addition to lymphoma or just erythroid hyperplasia secondary to severe anemia. Despite this one, possibly exceptional, mouse, the data indicate that C57BL mice were substantially resistant to erythroblastosis even in the presence of high titers of F-MCF virus. In a second series of experiments, progeny of genetic crosses derived from C57BL/6 mice were examined for correlation between F-MCF virus and development of F-Eco erythroblastosis. NFS/N x (NFS/N x C57BL/6) first-backcross mice which had been inoculated with F-Eco as neonates underwent hemi-splenectomy at 4 to 5 weeks of age to test for MCF virus by the infectious-center assay. Almost all backcross mice had evidence of F-MCF, even though nearly one-half the mice failed to develop erythroblastosis (Table 4). The titer of MCF virus was about the same in backcross mice developing erythroblastosis (geometric mean, -500 FFU/107 spleen cells; n = 12 mice) as in backcrossed mice developing lymphoma or myeloid leukemia (geometric mean -600 FFU/107 spleen cells, n = 8 mice). Similar results were obtained in BALB/c x (BALB/c x C57BL/6) first-backcross mice inoculated as neonates with F-Eco and tested for F- MCF by blot hybridization after they had developed leukemia. Almost all of these mice had evidence of F-MCF, even though one-half of the mice had lymphoma or myelogenous leukemia (Table 4). Backcross mice which do not develop erythroblastosis have inherited a C57BL/6 gene for resistance to erythroblastosis (21). Thus, these experiments show that backcross mice could inherit resistance to F-Eco erythroblastosis without inheriting resistance to the generation of F-MCF virus. Lack of evidence for lymphomagenic recombinant viruses in F-Eco-inoculated C57BL/6 mice. In an additional attempt to identify leukemogenic recombinant viruses in F-Eco-inoculated C57BL/6 mice, BALB/c mice were inoculated with an extract from a C57BL/6 F-Eco-induced lymphoma. If the extract contained a lymphomagenic recombinant, some of the BALB/c mice might be expected to develop lymphoma. None of the 22 BALB/c recipients developed lymphoma, and all succumbed to erythroblastosis (Table 3). Since more than one-half of these BALB/c mice survived beyond the minimum latency for F-Eco induction of lymphoma in C57BL/6 mice (55 days), the lack of lymphoma in these BALB/c mice argues against a lymphomagenic recombinant in F-Eco-induced C57BL/6 lymphoma. DISCUSSION These experiments confirmed a recent report that MCF virus is not detected by infectious center assay in F-Ecoinoculated C57BL mice (6). The results reported here extend this observation by using a blot hybridization assay which does not require in vitro growth of virus or induction of cytopathic effect on mink cells, and which therefore may detect a wider class of recombinant viruses. It should be noted that the blot hybridization assay has quantitative and qualitative limitations. For example, it would not detect F- MCF at less than about one-tenth of its concentration in F- Eco-induced erythroblastosis (Fig. 1B, lanes e to k and n). Also, it would not detect recombinant viruses which do not retain the proximal EcoRI site from the ecotropic parent or pick up xenotropic envelope-reactive sequences to the left of TABLE 4. Detection of F-MCF in crosses with C57BL/6 Cross Type of No. positive leukemia' for F-MCF/ no. tested NFS/N x (NFS/N x C57BL/6) E 12/12b L 7/8b BALB/c x (BALB/c x C57BL/6) E 10/10C L or M 10/11C a E, Erythroblastosis; L, lymphoma; M, myeloid leukemia. b MCF virus was detected by infectious-center assay on spleen cells obtained by splenic biopsy in mice 4 to 5 weeks of age. c MCF virus was detected by blot hybridization assay on DNA from spleen cells obtained when the mice were killed.

5 876 SILVER a new EcoRI site. Recombinant viruses with extensive substitutions in the proximal part of F-Eco do exist (T. Cloyd, personal communication). Despite these limitations, the blot hybridization assay confirmed the striking difference between C57BL mice and mice of several strains susceptible to F-Eco erythroblastosis in the amount or type of recombinant virus generated. In other systems, it has been found that ecotropic viruses give rise to different recombinants in different strains of mice. Thus, the MCF viruses induced by F-Eco in NFS and AKR mice are distinguishable by oligonucleotide mapping (T. Cloyd and L. Evans, personal communication), and MCF viruses isolated from AKR mice differ in tissue tropism, pathogenicity (7), and oligonucleotide maps (16) from MCF viruses generated by the AKR ecotropic virus in NFS congenic mice. These mouse strain effects on recombinant viruses generated by a given ecotropic virus could be related to the extensive polymorphism of endogenous MuLV-related sequences (14). However, absence of MCF virus in (BALB/c x C57BL/6)Fj mice cannot be attributed to lack of the appropriate sequences for recombination, since all BALB/c sequences are present in these mice. An important biological question is whether differences in recombinant viruses induced in different mouse strains are responsible for the different types of disease induced by F- Eco virus. The data reported here, and other experiments (20), indicate a correlation between presence of F-MCF virus and erythroblastosis, suggesting that F-MCF is pathogenic in F-Eco erythroblastosis. This hypothesis is attractive for two reasons. First, a virus which is very closely related to F-MCF, the spleen focus-forming virus (26) of Friend virus complex, causes acute erythroblastosis. Second, the Rmcff gene, which restricts replication of F-MCF virus, interferes with the development of erythroblastosis (W. P. Rowe and J. W. Hartley, unpublished data). On the other hand, it should be kept in mind that only a few mouse strains have been examined for correlation between F-MCF and erythroblastosis. The correlations observed to date could be coincidental. In a search for mice with erythroblastosis which may be negative for F-MCF, we are examining F-Ecoinoculated (NFS/N x C57BL/6)F1 mice. Most of these mice are negative for MCF virus at 4 to 5 weeks and succumb to lymphoma, but about 25% develop erythroblastosis. It will be interesting to determine whether these mice with erythroblastosis are positive for F-MCF when they are grossly leukemic. Whether or not F-MCF is necessary for F-Eco-induced erythroblastosis, it seems clear that C57BL mice carry a gene for resistance to F-Eco erythroblastosis which acts by a mechanism other than inhibition of F-MCF. Thus, C57BL/6 mice inoculated with pseudotypic mixtures of F-Eco and F- MCF replicate F-MCF but do not develop erythroblastosis, and progeny of crosses with C57BL/6 mice may inherit resistance to F-Eco erythroblastosis without inheriting resistance to generation of F-MCF. The C57BL gene determining resistance to development of F-Eco erythroblastosis is distinct from other Friend virus-resistance loci, including Fv-J and Fv-2 (21, 22.). F-Eco induces MCF virus in almost all backcross mice from C57BL to BALB/c or NFS/N, whereas (C57BL x BALB/c)F1 and (C57BL x NFSIN)F1 mice remain (mostly) MCF negative (Tables 1, 2, and 4 and unpublished data). This suggests that several dominant C57BL genes act in concert to prevent the appearance of MCF virus. The results reported here are relevant to earlier studies which showed that C57BL/6 mice spontaneously express J. VIROL. MCF-related antigen (20). It was hypothesized that expression of MCF-related antigen caused resistance to erythroblastosis by interfering with replication of MCF virus. The current studies indicated that C57BL/6 mice were not resistant to the replication of F-MCF when they were inoculated as neonates with a pseudotypic mixture of F-Eco plus F- MCF. Lack of resistance to replication of F-MCF is consistent with observations that C57BL/6 mice carry the Rmcfs allele (12). MCF viruses may be pathogenic in some leukemias, such as AKR lymphoma or F-Eco erythroblastosis, but not in others, such as F-Eco-induced lymphoma and myeloid leukemia in C57BL/6 mice. Circumstantial evidence suggests that Gross Passage A virus and the highly leukemogenic ecotropic recombinant SL3 also do not require MCF intermediates for pathogenesis, since the Rmcff allele does not interfere with leukemogenesis by these viruses (W. P. Rowe and J. W. Hartley, personal communication). The fact that F-Eco induces lymphoid and myelogenous leukemias in C57BL mice without the generation of F-MCF has practical implications. Some of these leukemias have only a few integrated copies of F-Eco as judged by blot hybridization (unpublished data). The apparent absence of recombinant virus insertions makes feasible the cloning of all virus-cell junction fragments in these leukemias in an effort to determine whether the region of virus integration is correlated with the type of leukemia. Such experiments are in progress. ACKNOWLEDGMENTS I thank J. Chandler for expert technical assistance, J. W. Hartley, M. A. Martin, and C. A. Kozak for helpful comments, and S. Grove for preparing this manuscript. LITERATURE CITED 1. Adachi, A., K. Sakai, N. Kitamura, S. Nakanishi, 0. Niwa, M. Matsuyama, and A. Ishimoto Characterization of the env gene and long terminal repeat of molecularly cloned Friend mink cell focus-inducing virus DNA. J. Virol. 50: Buckler, C. E., M. D. Hoggan, H. W. Chan, J. F. Sears, A. S. Khan, J. L. Moore, J. W. Hartley, W. P. Rowe, and M. A. Martin Cloning and characterization of an envelopespecific probe from xenotropic murine leukemia proviral DNA. J. Virol. 41: Chan, H. W., T. Bryan, J. L. Moore, S. P. Staal, W. P. Rowe, and M. A. Martin Identification of ecotropic proviral sequences in inbred strains of mice with a cloned subgenomic DNA fragment. Proc. Natl. Acad. Sci. U.S.A. 77: Chattopadhyay, S. K., M. R. Lander, S. Grupton, E. Rands, and D. Lowy Origin of mink cytopathic focus-forming (MCF) viruses: comparison with ecotropic and xenotropic murine leukemia virus genomes. Virology 113: Chattopadhyay, S. K., D. R. Lowy, N. M. Teich, A. S. Levine, and W. P. Rowe Quantitative and qualitative studies of AKR-type murine leukemia virus sequences in mouse DNA. Cold Spring Harbor Symp. Quant. Biol. 39: Chesebro, B., J. L. Pointer, K. Wehrley, and J. Nishio Effect of murine host genotype on MCF virus expression, latency and leukemia cell type of leukemia induced by Friend murine leukemia helper virus. Virology 128: Cloyd, M. W., J. W. Hartley, and W. P. Rowe Lymphomagenicity of recombinant mink cell focus-inducing murine leukemia viruses. J. Exp. Med. 151: Cloyd, M. W., J. W. Hartley, and W. P. Rowe Genetic study of lymphoma induction by AKR mink cell focus inducing virus in AKR x NFS crosses. J. Exp. Med. 154: Elder, J. H., J. W. Gautsch, F. C. Jensen, R. A. Lerner, J. W. Hartley, and W. P. Rowe Biochemical evidence that MCF murine leukemia viruses are envelope (env) gene recombinants. Proc. Natl. Acad. Sci. U.S.A. 74:

6 VOL. 50, 1984 ROLE OF MCF VIRUS IN LEUKEMIAS INDUCED BY F-ECO Fischinger, P. J., A. E. Franke, J. H. Elder, R. A. Lerner, J. N. Ihie, and D. P. Bologesi Biological, immunological and biochemical evidence that HIX virus is a recombinant between Moloney leukemia virus and a murine xenotropic C-type virus. Virology 90: Hartley, J. W., N. K. Wolford, L. J. Old, and W. P. Rowe A new class of murine leukemia virus associated with development of spontaneous lymphomas. Proc. Natl. Acad. Sci. U.S.A. 74: Hartley, J. W., R. A. Yetter, and H. C. Morse III A mouse gene on chromosome 5 that restricts infectivity of mink cell focus-forming recombinant murine leukemia viruses. J. Exp. Med. 158: Hoffman, P. M., W. F. Davidson, S. R. Ruscetti, T. M. Chused, and H. C. Morse III Wild mouse ecotropic murine leukemia virus infection of inbred mice: dual-tropic virus expression precedes the onset of paralysis and lymphoma. J. Virol. 39: Hoggan, M. D., C. E. Buckler, J. F. Sears, W. P. Rowe, and M. A. Martin Organization and stability of endogenous xenotropic murine leukemia virus proviral DNA in mouse genomes. J. Virol. 45: Khan, A. S., R. Repaske, C. F. Garon, H. W. Chan, W. P. Rowe, and M. A. Martin Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA. J. Virol. 41: Lung, M. L., J. W. Hartley, W. P. Rowe, and N. H. Hopkins Large RNase T,-resistant oligonucleotides encoding plse and the U3 region of the long terminal repeat distinguish two biological classes of mink cell focus-forming type C viruses of inbred mice. J. Virol. 45: Oliff, A., L. Collins, and C. Mirenda Molecular cloning of Friend mink cell focus-inducing virus: identification of mink cell focus-inducing virus-like messages in normal and transformed cells. J. Virol. 48: Oliff, A. I., G. L. Hager, E. H. Chan, E. M. Scolnick, H. W. Chan, and D. R. Lowy Transfection of a molecularly cloned Friend murine leukemia virus DNA yields a highly leukemogenic helper-independent type C virus. J. Virol. 33: Rowe, W. P., and J. W. Hartley. Genes affecting mink cell focus-inducing (MCF) murine leukemia virus infection and spontaneous lymphoma in AKR Fl hybrid. J. Exp. Med. 158: Ruscetti, S., N. Davis, J. Field, and A. Oliff Friend murine leukemia virus-induced leukemia is associated with the formation of mink cell focus-inducing viruses and is blocked in mice expressing endogenous mink cell focus-inducing xenotropic viral envelope genes. J. Exp. Med. 154: Silver, J. E., and T. N. Fredrickson A new gene that controls the type of leukemia induced by Friend murine leukemia virus. J. Exp. Med. 158: Silver, J. E., and T. N. Fredrickson Susceptibility to Friend helper virus leukemias in CXB recombinant inbred mice. J. Exp. Med. 158: Southern, E. M Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 38: Steffen, D., S. Bird, W. P. Rowe, and R. A. Weinberg Identification of DNA fragments carrying ecotropic proviruses of AKR mice. Proc. Natl. Acad. Sci. U.S.A. 76: Thomas, P. S Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. U.S.A. 77: TroxIer, D. H., E. Yuan, D. Linemeyer, S. Ruscetti, and E. M. Scolnick Helper-independent mink cell focus-inducing strains of Friend murine type C virus: potential relationship to the origin of replication defective spleen focus-forming virus. J. Exp. Med. 148: Van Griensven, L. J., and M. Vogt Rauscher "Mink cell focus-inducing" (MCF) virus causes erythroleukemia in mice: its isolation and properties. Virology 101: