Free and integrated recombinant murine leukemia virus DNAs appear in preleukemic thymuses of AKR/J mice.

We studied the appearance and structure of murine leukemia viral genomes in preleukemic AKR/J mice by Southern hybridization. Up to an average of one to two copies per thymocyte of unintegrated murine leukemia virus DNA appears in the thymuses of preleukemic mice beginning at 4 to 5 months of age and disappears in leukemic thymuses. The free viral genomes are absent in the spleens, livers, and brains of preleukemic mice. Using a series of ecotropic and nonecotropic murine leukemia virus hybridization probes, we showed that the unintegrated viral genomes are structurally analogous to those of recombinant mink cell focus-forming viruses that appear as proviruses in leukemic AKR thymocytes, suggesting that these free viral DNAs are the direct precursors to the leukemia-specific proviruses. The mosaic of ecotropic and nonecotropic sequences within these unintegrated viral DNAs varies from one preleukemic thymus to another but often appears structurally homogeneous within individual thymuses, indicating that often each thymus was being infected by a unique mink cell focus-forming virus. Analysis of high-molecular-weight DNA shows that recombinant proviruses reside in the chromosomal DNA of thymocytes within the preleukemic thymus, with the number rising to an average of several copies per thymocyte, but we do not detect any preferred integration sites. These results suggest that, in general, before the development of thymic leukemias in AKR mice there is a massive infection by a unique mink cell focus-forming virus which then integrates into many different sites of individual thymocytes, one of which grows out to become a tumor.     

Frequent isolation of ecotropic murine leukemia virus after x-ray irradiation of C57BL/6 mice and establishment of producer lymphoid cell lines from radiation-induced lymphomas

Fractionated whole-body X irradiation of C57BL/Ka mice leads to the development of thymic leukemia in 90% of the treated animals at an average age of 6 months. Using a sensitive high-density cocultivation procedure, we were able to demonstrate the presence of ecotropic murine leukemia virus (MuLV) from 1 month post-irradiation up to leukemia development. These viruses are not specific to any one particular organ, but can be found in at least two of the three lymphoreticular tissues studied, namely, spleen, thymus, and bone marrow. Host range studies on the isolated viruses showed that both N- and B-tropic MuLV could be isolated early after irradiation. However, as mice reached an age where leukemias develop, only the B-tropic MuLV could be recovered. We have established cell lines from primary radiation-induced tumors that are being maintained in continuous culture: except one cell line, all are virus producers. The results clearly indicate that X irradiation induces ecotropic MuLV in C57BL/Ka mice and suggest that B-tropic MuLV might be involved in the disease process.     

Spontaneous and induced leukemias of myeloid origin in recombinant inbred BXH mice

BXH-2 recombinant inbred (RI) mice produce high titers of B-ecotropic murine leukemia virus beginning early in life and have a high incidence of non-T-cell leukemias that occur before 1 year of age. The leukemias that develop are in some cases associated with hind limb paralysis. In addition, a dualtropic mink cell focus-forming virus has been isolated from leukemic cells of BXH-2 mice. Immunological and cytochemical characterization of the BXH-2 leukemias showed that they are of the myeloid lineage. To assess the oncogenicity of the BXH-2 viruses, newborn mice of several BXH RI strains were inoculated at birth with biologically cloned B-ecotropic or mink cell focus-forming murine leukemia virus. These studies demonstrated that the B-ecotropic virus can induce myeloid leukemias in other BXH RI strains, whereas the dualtropic mink cell focus-forming isolates were nononcogenic in the strains tested. DNA-DNA reassociation analysis indicated that the organotropism of the B-ecotropic murine leukemia virus is confined to lymphoid tissues. Southern analysis of tumor DNAs showed that there was amplification of ecotropic virus-specific sequences in BXH-2 myeloid tumors and in all leukemias induced in other BXH RI strains by inoculation of the BXH-2 B-ecotropic virus. Although B-ecotropic virus is expressed in central nervous tissues of paralyzed BXH-2 mice, we were unable to induce the disorder in several BXH RI strains inoculated intracranially at birth with either the B-ecotropic or dualtropic virus. These results suggest that the paralysis that occurs in BXH-2 mice is due to the infiltration of leukemic cells into the central nervous system.     

Genetic control of retroviral disease in aging wild mice

Different populations of wild mice (Mus musculus domesticus) in Los Angeles and Ventura Counties were observed over their lifespan in captivity for expression of infectious murine leukemia virus (MuLV) and murine mammary tumor virus (MMTV) and for the occurrence of cancer and other diseases. In most populations of feral mice these indigenous retroviruses were infrequently expressed and cancer seldom occurred until later in life (>2 years old). MMTV was found in the milk of about 50% of wild mice, but was associated with only a low incidence (>1%) of breast cancer after one year of age. By contrast, in several populations, most notably at a squab farm near Lake Casitas (LC), infectious MuLV acquired at birth via milk was highly prevalent, and the infected mice were prone to leukemia and a lower motor neuron paralytic disease after one year of age. These two diseases were both caused by the same infectious (ecotropic)strain of MuLV and were the principal cause of premature death in these aging LC mice. A dominant gene called FV-4^R restricting the infection with ecotropic MuLV was found segregating in LC mice. Mice inheriting this FV-4^R allele were resistant to the ecotropic MuLV associated lymphoma and paralysis. The FV-4^R allele represents a defective endogenous MuLV provirus DNA segment that expresses an ecotropic MuLV envelope-related glycoprotein (gp70) on the cell surface. This FV-4^R encoded gp70 presumably occupies the receptor for ecotropic MuLV and blocks entry of the virus. The FV-4^R gene was probably acquired by the naturally occurring crossbreeding of LC feral mice with another species of feral mice (Mus castaneus) from Southeast Asia. The FV-4^R gp70 does not block entry of the amphotropic MuLV that uses a separate cell surface receptor. Therefore LC mice continued to be susceptible to the highly prevalent but weakly lymphogenic and nonparalytogenic amphotropic strain of MuLV. The study points out the potential of feral populations to reveal genes associated with specific disease resistance.     

Activation of retrovirus in transgenic mice: association with development of olfactory neuroblastoma.

A line of transgenic mice that express the human adenovirus type 12 E1A and E1B genes under the regulatory control of the mouse mammary tumor virus long terminal repeat was studied. Mice from this line develop olfactory neuroblastomas at approximately 6 months of age. Large numbers of type C retrovirus (ecotropic murine leukemia virus) particles were found in the tumor rosettes. No similar examples of virus activation were identified in tumors from other transgenic experiments. Examination of spontaneous olfactory neuroblastomas from three domestic cats also demonstrated retrovirus in tumor rosettes.     

Analysis of neoplasms induced by Cas-Br-M MuLV tumor extracts

Cas-Br-M is an ecotropic murine leukemia virus isolated from wild mice that induces a wide spectrum of hematopoietic neoplasms, including T and B cell lymphomas, myelogenous leukemias, and erythroleukemias. The purpose of this study was to determine if the induction of neoplasms belonging to multiple lineages was due to the ecotropic virus itself or to the generation of cell lineage-specific recombinant viruses. The results demonstrate that in some instances (two of 12 tumor extracts tested), recombinant viruses can be recovered from primary Cas-Br-M-induced tumors that will induce lymphomas of single lineages in mice inoculated as newborns. One of these viruses is a recombinant mink cell focus-forming virus that induces T cell lymphomas, and the other is a replication-defective, fibroblast-transforming virus that induces early B lineage lymphomas in mice. Histologic and flow microfluorometric cell surface antigen analyses of primary and in vitro adapted tumors are presented in support of a modified scheme of hematopoietic cell development.     

Expression of murine leukemia viruses in the highly lymphomatous BXH-2 recombinant inbred mouse strain

Among 12 recombinant inbred strains of mice derived from crossing two strains, C57BL/6J and C3H/HeJ, which have a low incidence of neoplastic disease, one strain (BXH-2) has been found to have a high incidence of lymphoma, of non-T-cell origin, at an early age. The BXH-2 strain carries the Fv-1b allele and spontaneously expresses a B-tropic murine leukemia virus beginning at as early as 10 days of gestation and continuing throughout their life. No significant differences in ecotropic virus titers were observed at any age tested (16 to 17 days of gestation through 7 months), whereas xenotropic virus was first detected in lymphoid tissues of 2-month-old mice and virus titers increased with age. Dual tropic virus(es), which induced cytopathic changes on mink lung cells, was isolated from BXH-2 lymphomatous tissues. Unlike AKR mink lung focus-forming virus (N-tropic recombinant), BXH-2 dual tropic virus is B tropic and induces cytopathic changes in mouse fibroblast cultures as well. The BXH-2 mouse provides a model system for studying the role of replication-competent viruses in spontaneously occurring leukemias of non-T-cell lineage and neurological disease.     

Abrogation of radiation leukemia virus-induced lymphomagenesis by antisera to thymotropic but not to ecotropic or dual-tropic viruses.

Leukemia induction by culture-grown thymotropic radiation leukemia virus or by tumor-derived virus present in cell-free tumor extracts was abrogated by incubation of either virus with anti-thymotropoc virus serum, but not by antiserum raised against ecotropic or dual-tropic (mink cell focus-inducing type) viruses that were isolated from radiation leukemia virus-induced thymic leukemias. Thus, virus similar or identical to the cultured thymotropic leukemogenic species may also be the major biologically active principle in tumor-derived extracts, even though the latter also contain viruses of the dual-tropic, mink cell focus-inducing type class.     

Somatically acquired recombinant murine leukemia proviruses in thymic leukemias of AKR/J mice.

We have probed the structure and arrangement of murine leukemia virus genomes in eight spontaneous AKR thymic leukemias by Southern hybridization with one ecotropic pol and four ecotropic env probes. These probes revealed many (in 2 cases over 15) somatically acquired proviruses that had undergone complex patterns of recombination. The large majority were not deleted and were structurally analogous to the oncogenic mink cell focus-inducing murine leukemia viruses isolated from AKR tumors in that the amino-terminal p15E-coding region derived from ecotropic AKR murine leukemia virus sequences, whereas certain gp70-coding sequences were nonecotropic. Nevertheless, we observed a few proviruses which did not appear to be gp70 recombinants; however, these proviruses were in general clearly recombinant within the p15E-coding sequences. Although the proviral recombination patterns were quite variable, in general the large majority of recombinant proviruses within each tumor appeared structurally identical, indicating that they originate from a common parent. Each tumor contained a unique pattern of provirus integrations; densitometer tracings of the Southern hybridizations indicated that many of the integrated proviruses were present at one copy per cell, suggesting that the tumors derive from a single cell which contained multiple integrated copies of a unique recombinant virus structurally similar to the mink cell focus-inducing viruses.     

Oncogenicity of AKR endogenous leukemia viruses.

Four biologically distinct groups of endogenous murine leukemia virus (MuLV) have been isolated from AKR mice. These viruses included (i) ecotopic XC+ MuLV that occur in high titer in normal tissues and serum of AKR mice throughout their life span, (ii) ecotropic XC- MuLV that are produced in high titers by leukemia cells, (iii) xenotropic MuLV that are readily demonstrable only in aged mice, and (iv) polytropic MuLV thatarise in the thymuses of aged mice as a consequence of genetic recombination between ecotropic and xenotropic MuLV. Virus of each of these biological classes were assayed in AKR mice for their ability to accelerate the occurrence of spontaneous leukemia. Certain isolates of ecotropic XC- MuLV and polytropic MuLV were found to have high oncogenic activity. These viruses induced 100% leukemias within 90 days of inoculation. In contrast, ecotropic XC+ MuLV that were obtained from AKR embryo fibroblasts and xenotropic MuLV that were obtained from the lymphoid tissues of aged AKR mice did not demonstrate oncogenic activity. These findings demonstrate fundamental differences between XC- and XC+ ecotropic MuLV that are found in leukemic and normal tissues, respectively. Furthermore, these findings point to the role of ecotropic XC- and polytropic MuLV in the spontaneous leukemogenesis of AKR mice.     

Leukemia induction by a new strain of Friend mink cell focus-inducing virus: synergistic effect of Friend ecotropic murine leukemia virus.

A new strain of Friend recombinant mink cell focus-inducing retrovirus, FMCF -1-E, was found to induce leukemias in NFS and IRW mice. Although the isolate was obtained from a stock of FMCF -1 ( Troxler et al., J. Exp. Med. 148:639-653, 1978), FMCF -1-E was distinguishable from FMCF -1 by oligonucleotide fingerprinting and antigenic analysis, using monoclonal antibodies. These analyses suggested that FMCF -1-E is a distinct FMCF isolate rather than a simple variant of FMCF -1. After neonatal inoculation, the latency for leukemia induction was 3 to 8 months. A similar long latency was also seen when Friend murine leukemia virus 57 was inoculated into adult (6-week-old) IRW mice. However, sequential inoculation of FMCF -1-E at birth followed by Friend murine leukemia 57 at 6 weeks of age led to a shortened latency period (2.5 to 4 months). Only neonatal inoculation of Friend murine leukemia virus 57 was able to induce a more rapid appearance of leukemia. The leukemia cell type in the majority of cases, regardless of virus inoculation protocol, was erythroid, but occasional myeloid, lymphoid, and mixed leukemias were also observed. In contrast to NFS and IRW mice, BALB/c mice were resistant to leukemia induction by FMCF -1-E and also showed some transient resistance to leukemia induction by Friend murine leukemia virus 57.     

Lymphomas and high-level expression of murine leukemia viruses in CFW mice

Historically, Swiss Webster mice of the CFW subline, both inbred and random-bred stocks, have been considered to have a low spontaneous occurrence of hematopoietic system tumors, and previous reports of infectious expression of murine leukemia viruses (MuLVs) have been rare and unremarkable. In marked contrast, in the present study of CFW mice from one source observed by two laboratories over a 2-year period, nearly 60% developed tumors, 85% of which were lymphomas, the majority of B-cell origin. All tumors tested expressed ecotropic MuLVs, and most expressed mink cell focus-inducing (MCF) MuLVs. Among normal mice of weanling to advanced age, over one-half were positive for ecotropic virus in tail or lymphoid tissues, and MCF virus was frequently present in lymphoid tissue, less often in tail. Patterns of ecotropic proviral integration indicated that natural infection occurred by both genetic and exogenous routes. Lymphomas were induced in NIH Swiss mice infected as neonates with tissue culture-propagated MuLVs isolated from normal and tumor tissue of CFW mice.     

Evidence the pp60src, the product of the Rous sarcoma virus src gene, undergoes autophosphorylation.

F/St mice are unique in producing high levels of both ecotropic and xenotropic murine leukemia virus. The high ecotropic virus phenotype is determined by three or more V (virus-inducing) loci. A single locus for inducibility of xenotropic murine leukemia virus was mapped to chromosome 1 close to, but possibly not allelic to, Bxv-1. Although the high ecotropic virus phenotype is phenotypically dominant, the high xenotropic virus phenotype was recessive in all crosses tested. Suppression of xenotropic murine leukemia virus is governed by a single gene which is not linked to the xenotropic V locus.     

Wild mouse ecotropic murine leukemia virus infection of inbred mice: dual-tropic virus expression precedes the onset of paralysis and lymphoma.

NFS/N mice inoculated at birth with an ecotropic murine leukemia virus (Cas-Br-MuLV) obtained from wild mice developed hind limb paralysis beginning at 7 weeks of age and nonthymic lymphomas beginning at more than 20 weeks of age. Studies of 1- to 7-week-old Cas-Br-M MuLV-infected mice showed the following: (i) a marked increase in nonecotropic MuLV-related antigens on spleen cells but not thymocytes beginning at 2 weeks; (ii) the appearance of dual-tropic mink cell focus-forming (MCF) MuLV-related gp70 in spleen but not thymus or brain cells at 4 weeks; and (iii) the isolation of infectious MCF MuLV from spleen cells of 7-week-old mice. A role for MCF MuLV in Cas-Br-M MuLV-induced nonthymic lymphomas is indicated by these studies, and a role for recombinant MuLV in neurological disease is considered.     

Characterization of infectious oncornaviruses from MOPC-460 plasmacytomas: their relation to A-type particles.

MOPC-460 mouse plasmacytoma cells produce intracellular A-type particles and extracellular oncornavirus-like particles ("myeloma-associated virus," abbreviated MAV). The genomes of these two particles are closely related. During attempts to establish infections with MOPC-460 extracellular particles, we isolated ecotropic and xenotropic infectious forms of murine leukemia virus. We have investigated the relation of these isolates to A-type particles and to MAV by nucleic acid hybridization. Using complementary DNA probes prepared from the two isolates, we found that these infectious murine leukemia viruses differ from A-type particles and from MAV. Moreover, we found that MAV is the predominant extracellular component: the ecotropic and xenotropic forms of murine leukemia virus were present at only low levels (less than 5%) in MAV preparations. Neither the SC-1 cells infected with ectropic murine leukemia virus nor the mink cells infected with xenotropic murine leukemia virus showed any A-type particles in their cytoplasm when examined by electron microscopy. Our inability to demonstrate infection by the A-type particle-related component, MAV, suggests that these may be defective.     

Comparison of the restriction endonuclease maps of unintegrated proviral DNAs from four xenotropic murine leukemia viruses.

From purified linear and superhelical DNAs, the restriction endonuclease maps of four xenotropic murine leukemia virus DNAs from NFS, NZB, BALB/c, and AKR mice were determined with ten restriction endonucleases. Each xenotropic proviral DNA was found to be a unique restriction endonuclease map, with differences in the gag, pol, env, and terminal repeated sequence regions. However, type-specific SacI and EcoRI sites in the env region were identical in all four xenotropic murine leukemia virus DNAs and were not found in ecotropic murine leukemia virus DNA. Comparison of the xenotropic murine leukemia virus DNA maps with maps of ecotropic murine leukemia virus DNA showed that the pol and terminal repeated sequence regions were highly conserved. Other similarities in ecotropic and some xenotropic viral DNAs suggest common origins.     

Identification of a spleen focus-forming virus in erythroleukemic mice infected with a wild-mouse ecotropic murine leukemia virus

An NFS/N mouse inoculated at birth with an ecotropic murine leukemia virus (MuLV) obtained from wild mice (Cas-Br-M MuLV) developed a lymphoma after 18 weeks. An extract prepared from the lymphomatous spleen was inoculated into newborn NFS/N mice, and these mice developed erythroleukemia within 9 weeks. Spleens from the erythroleukemic mice contained ecotropic and mink cell focus-inducing (MCF) MuLVs; however, when these viruses were biologically cloned and reinoculated into newborn NFS/N mice, no erythroleukemia was induced. In contrast, cell-free extracts prepared from the erythroleukemic spleens induced erythroleukemia within 5 weeks. Analysis of cell-free extracts prepared from the erythroleukemic spleens showed that they contained a viral species that induced splenomegaly and spleen focus formation in adult mice, with susceptibility controlled by alleles at the Fv-2 locus. The spleen focus-forming virus coded for a 50,000-dalton protein precipitated by antibodies specific to MCF virus gp70. RNA blot hybridization studies showed the genomic viral RNA to be 7.5 kilobases and to hybridize strongly to a xenotropic or MCF envelope-specific probe but not to hybridize with an ecotropic virus envelope-specific probe. The virus described here appears to be the fourth independent isolate of a MuLV with spleen focus-forming activity.     

Role of mink cell focus-inducing virus in leukemias induced by Friend ecotropic virus

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-Eco-inoculated 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-Eco-induced 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.     

Leukemogenicity and cell transformation mechanisms in vitro by Gross murine leukemia virus: analysis of virus subpopulations.

The leukemogenic activity of Gross murine leukemia virus adapted to rats was tested in W/Fu rats and NIH/Swiss mice. All animals infected with this virus developed thymic and nonthymic T-cell leukemia with a short latency period. It was observed that cell-free extracts from thymic lymphoma tissue of mice and rats, induced by either Gross murine leukemia virus or Gross murine leukemia virus adapted to rats, consisted of both small-plaque-forming and large-plaque-forming viruses, as determined by the XC plaque test. MCF-type virus was found in these virus complexes. Transformed cell foci were induced in SC-1 cell layers by double infection of the cloned MCF-type virus and an ecotropic virus. SC-1 cells containing transformed cell foci were shown to be tumorigenic upon inoculation into nude mice. The formation of transformed cell foci in mink lung cells was also observed after double infection with the cloned MCF-type virus and a xenotropic virus. The possible mechanism of leukemogenesis by endogenous viruses is discussed.     

Identification of virus found in mouse lymphomas induced by HIX murine oncornavirus.

Lymphomas induced by pure HIX murine leukemia virus in two mouse strains contained large amounts of virus with amphotropic properties. Analysis of tumor derived virus purified by limiting dilution techniques indicated that its interference and neutralization spectra were essentially identical to parental HIX virus and different from eco- and xenotropic viruses. Examination of virus progeny from many individual foci induced by virus derived directly from lymphomas indicated that each infectious unit contained only HIX virus. No evidence of ecotropic virus presence was observed.