Multiparameter analyses of spontaneous nonthymic lymphomas occurring in NFS/N mice congenic for ecotropic murine leukemia viruses.

Mouse strains congenic for ecotropic retrovirus genes have a much higher frequency of spontaneous lymphomas than the background NFS/N strain. In this study, most of these lymphomas have been identified as B-cell in origin by morphologic features, identification of immunoglobulin class, and cell-surface antigens. The classification suggested by Pattengale and Taylor proved to be applicable to the lymphomas studied. Most were of large follicular center cells and are considered typical of the type formerly designated as "reticulum cell sarcoma, type B." Many lymphomas contained a large proportion of nonneoplastic cells which partially obscured their neoplastic component. The role of ecotropic murine leukemia viruses as etiologic agents for B-cell lymphomas remains equivocal. However, because the only difference between the NFS/N and congenic mice is the expression of viruses in the latter, it appears that these viruses are somehow involved in induction of B-cell lymphomas.

At least four viral genes contribute to the leukemogenicity of murine retrovirus MCF 247 in AKR mice.

Nucleotide sequences encoding gp70, Prp15E, and the U3 region of the long terminal repeat (LTR) distinguish mink cell focus-forming (MCF) retroviruses that can induce leukemia in AKR mice from closely related MCF and ecotropic murine retroviruses that are nonleukemogenic in all inbred mouse strains tested (Lung et al., Cold Spring Harbor Symp. Quant. Biol. 44:1269-1274, 1979; Lung et al., J. Virol. 45:275-290, 1983). We used a set of recombinants constructed in vitro from molecular clones of leukemogenic MCF 247 and nonleukemogenic ecotropic Akv to separate and thereby directly test the role of these genetic elements in disease induction. Leukemogenicity tests of recombinants in AKR mice show that introduction of fragments containing either an MCF LTR or MCF gp70 coding sequences can confer only a very low incidence of disease induction on Akv virus, whereas an MCF type Prp15E alone is completely ineffective. Recombinants with an MCF 247 LTR in combination with MCF Prp15E are moderately oncogenic, whereas those with an MCF 247 LTR plus MCF gp70 coding segment are quite highly leukemogenic. Mice infected with the latter virus show a substantial increase in latent period of disease induction relative to MCF 247; this delay can be reduced when Prp15E, and hence the entire 3' half of the genome, is from MCF 247. Surprisingly, sequences in the 5' half of the genome can also contribute to disease induction. We found a good correlation between oncogenicity and recovery of MCF viruses from thymocytes of injected mice, with early recovery and high titers of MCF in the thymus being correlated with high oncogenicity. This correlation held for recombinants with either an MCF or ecotropic type gp70. Together, these results (i) demonstrate that at least four genes contribute to the oncogenicity of MCF viruses in AKR mice and (ii) suggest that recombinants with only some of the necessary MCF type genes induce leukemia because they recombine to generate complete MCF genomes. Although neither Akv nor MCF 247 is leukemogenic in NFS mice, recombinant viruses whose gp70 gene was derived from Akv but whose LTRs were derived from MCF 247 induced a low incidence of leukemia in this mouse strain.

Spontaneous tumors of NFS mice congenic for ecotropic murine leukemia virus induction loci.

NFS/N mice congenic for ecotropic murine leukemia virus (MuLV) induction loci from AKR and C58 mice ("NFS V-congenics") were evaluated for the development of spontaneous neoplasms in comparison to such development in virus-negative NFS/N mice. Congenic mice developed thymic lymphomas, whereas NFS/N did not. However, the frequency of thymic lymphomas was reduced, and the latent period for their development was prolonged in NFS V-congenics as compared to that in AKR/N or C58/Lw mice. In addition, the frequencies of nonthymic lymphomas and myelogenous leukemias were increased more than threefold in the congenics over NFS/N. The increased frequencies of hematopoietic neoplasms in congenic animals were related to early expression of high systemic levels of ecotropic MuLV.

Genes affecting mink cell focus-inducing (MCF) murine leukemia virus infection and spontaneous lymphoma in AKR F1 hybrids.

An assessment of the importance of mink cell focus-inducing (MCF)-type recombinant murine leukemia viruses in spontaneous thymic lymphomagenesis and of the genetic factors affecting its occurrence was carried out with F1 hybrids between AKR and various other inbred strains. There was generally close agreement between the frequency of detection of MCF virus, of thymocyte antigenic amplification in the preleukemic period, and of spontaneous lymphoma. Also, hybrid combinations with moderate to high spontaneous lymphoma were uniformly susceptible to lymphoma induction by neonatal inoculation of MCF 247 virus, while lower leukemic hybrids were at least partially resistant to the induced disease. At least four resistance genes can be identified as affecting the disease in the various hybrids: Fv-1, Rmcf, an unidentified gene carried by the C57 series of mice and SJL, and an unidentified minor gene carried by several other strains.

Deformed whiskers in mice infected with certain exogenous murine leukemia viruses.

Mice infected at birth with replication competent Friend, Moloney, CasBr-M, C2S-M, and 1504-A murine leukemia viruses developed abnormalities of the vibrissae consisting of erratic curvature, shortening, and loss. A number of other virus strains, as well as endogenous AKR-type ecotropic virus and AKR-type, mink cell focus-inducing (MCF) viruses, did not produce these abnormalities. In mice with erythroid and myeloid leukemia, the perivibrissal sinus is the site of extramedullary hematopoiesis, but this did not appear to be the basis of the deformities. Genetic evidence indicated that newly arisen MCF-type recombinant viruses are involved in the pathogenesis of the abnormalities, at least with some of the virus systems studied.

Role for the 3' end of the genome in determining disease specificity of Friend and Moloney murine leukemia viruses.

To probe the genetic basis of disease specificity of nondefective murine type C viruses, we are constructing recombinants in vitro between molecular clones of Friend murine leukemia virus (Fr-MuLV) and Moloney murine leukemia virus (Mo-MuLV). Fr-MuLV induces erythroleukemias when injected into newborn NFS mice, whereas Mo-MuLV almost invariably induces T-cell lymphomas. We find that a recombinant whose genome is derived primarily from Fr-MuLV but which has 621 nucleotides of Mo-MuLV information at its 3' end induces almost exclusively thymic lymphomas. The sequences derived from Mo-MuLV include 99 nucleotides encoding the carboxyl terminus of Prp15E, the origin of DNA +-strand synthesis, all of the U3 region, and 36 nucleotides of the R portion of the long terminal repeat. When the segment of Mo-MuLV was removed and replaced with the comparable segment from Fr-MuLV, the virus was again erythroblastosis-inducing. These results, in conjunction with studies from other laboratories [Laimins, L. A., Khoury, G., Gorman, C., Howard, B. & Gruss, P. (1982) Proc. Natl. Acad. Sci. USA 79, 6453-6457], suggest that transcriptional signals in U3 may determine tissue tropism and hence influence disease specificity ("targeting") of murine leukemia viruses.

Mouse immunoglobulin antibodies require complement for neutralization of mouse retroviruses.

The addition of guinea pig complement was found to enhance the neutralizing capacity of mouse antibodies directed against the endogenous ecotropic murine leukemia viruses. The same immune sera, when tested without complement, had low or negligible neutralizing capacities, regardless of whether freshly harvested, unfrozen virus was used to preserve virus infectivity. Antibodies in high titers were found in sera from NFS congenic mice carrying the mouse leukemia virus inducing locus Akv-2. These mouse antibodies were type specific and failed to neutralize either Friend or Moloney leukemia virus. The mouse serum immunoglobulin fraction containing the complement-dependent antibodies was tentatively identified as immunoglobulin M.

Large RNase T1-resistant oligonucleotides encoding p15E and the U3 region of the long terminal repeat distinguish two biological classes of mink cell focus-forming type C viruses of inbred mice.

We used T1 oligonucleotide maps, in conjunction with available nucleotide sequences of appropriate C-type viruses, to identify regions of the viral genome that distinguish two biological classes of mink cell focus-forming (MCF) viruses described previously by Cloyd et al. (J. Exp. Med. 151:542-522, 1980). We found that leukemogenic MCF viruses from thymus differed from non-leukemogenic MCFs isolated from nonthymic neoplasms in nucleotide sequences encoding Prp15E and the U3 portion of the long terminal repeat (LTR). The thymic isolates possessed recombinant Prp15E genes, with the 5' to mid portion derived from their ecotropic parents and the extreme 3' portion invariably derived from their nonecotropic parents. These viruses probably derived the entire U3 portion of their LTRs from their nonecotropic parents. The nonthymic MCFs appeared to inherit their entire Prp15E coding region from their nonecotropic parents. We failed to detect consistent differences in gp70-coding sequences between the two groups of MCFs, but this may simply reflect limitations of the data. The studies presented here, in conjunction with studies from a number of labs indicating a role for MCF gp70 in leukemogenesis, indicate that three genetic elements, gp70, p15E, and the U3 portion of the LTR, may all play a role in determining the leukemogenic phenotype of type C viruses of high-leukemic inbred mice.

Organization and stability of endogenous xenotropic murine leukemia virus proviral DNA in mouse genomes.

In a series of blot hybridization experiments, using a xenotropic envelope probe and restriction enzymes known to cut xenotropic proviral DNA a single time (EcoRI) or not at all (HindIII), we have studied the organization and relationship of endogenous xenotropic env-related sequences in various mouse strains. Multiple copies (18 to 28) of xenotropic env-reactive fragments were found in all mouse DNAs after digestion with either HindIII or EcoRI, and the majority of fragments were of sizes compatible with their origin from full-length proviral DNA. Five HindIII and five EcoRI restriction fragments were common to all inbred mouse DNAs tested. In addition, each strain exhibited unique characteristic xenotropic env-reactive bands; these bands were remarkably stable during many years of inbreeding. The cleavage patterns characteristic of each strain were also useful for showing genealogical relatedness among the various inbred mice.

Cloning of endogenous murine leukemia virus-related sequences from chromosomal DNA of BALB/c and AKR/J mice: identification of an env progenitor of AKR-247 mink cell focus-forming proviral DNA.

Recombinant phages containing murine leukemia virus (MuLV)-reactive DNA sequences were isolated after screening of a BALB/c mouse embryo DNA library and from shotgun cloning of EcoRI-restricted AKR/J mouse liver DNA. Twelve different clones were isolated which contained incomplete MuLV proviral DNA sequences extending various distances from either the 5' or 3' long terminal repeat (LTR) into the viral genome. Restriction maps indicated that the endogenous MuLV DNAs were related to xenotropic MuLVs, but they shared several unique restriction sites among themselves which were not present in known MuLV proviral DNAs. Analyses of internal restriction fragments of the endogenous LTRs suggested the existence of at least two size classes, both of which were larger than the LTRs of known ecotropic, xenotropic, or mink cell focus-forming (MCF) MuLV proviruses. Five of the six cloned endogenous MuLV proviral DNAs which contained envelope (env) DNA sequences annealed to a xenotropic MuLV env-specific DNA probe; in addition, four of these five also hybridized to an ecotropic MuLV-specific env DNA probe. Cloned MCF 247 proviral DNA also contained such dual-reactive env sequences. One of the dual-reactive cloned endogenous MuLV DNAs contained an env region that was indistinguishable by AluI and HpaII digestion from the analogous segment in MCF 247 proviral DNA and may therefore represent a progenitor for the env gene of this recombinant MuLV. In addition, the endogenous MuLV DNAs were highly related by AluI cleavage to the Moloney MuLV provirus in the gag and pol regions.

Variation in the number of copies and in the genomic organization of ecotropic murine leukemia virus proviral sequences in sublines of AKR mice.

DNAs isolated from individual mice of four AKR sublines (AKR/J, AKR/N, AKR/Cum, and AKR/Boy) were examined by hybridization of electrophoretically separated restriction enzyme fragments to a 500-base pair, 32P-labeled probe specific for env sequences of ecotropic murine leukemia virus. Variation in the number of proviral DNA copies and in their genomic organization, as reflected by the location of restriction enzyme sites in flanking cellular sequences, was observed both between and within AKR sublines. Evidence is presented for the continual acquisition of new proviruses in the four sublines studied. The ecotropic proviral DNA copies present in the four AKR sublines can be related to their genealogy; each subline contains two or three copies of proviral DNA in common with other sublines and from one to six unique ecotropic proviruses. Overall, a new copy appears about every 12 generations of inbreeding. Some of the unique proviral DNA copies contain internal alterations, as reflected by restriction enzyme maps that differ from those of prototype ecotropic proviruses.

Internal organization of endogenous proviral DNAs of xenotropic murine leukemia viruses.

The internal organization of endogenous xenotropic murine leukemia virus proviruses was determined in a series of blot hybridization experiments in which DNA from several different inbred mouse strains, digested with restriction enzymes known to cleave xenotropic proviral DNAs at least twice, was annealed to generalized murine leukemia virus or xenotropic env-specific DNA probes. Comigrating bands of variable intensity which hybridized to the xenotropic env probe were identified in all inbred mouse DNA preparations. At least seven classes of endogenous xenotropic proviral DNA with respect to SacI cleavage maps were detected in mouse DNA. Two of the seven classes were indistinguishable from proviruses associated with known infectious xenotropic murine leukemia viruses. These results are consistent with the existence of related but organizationally distinct families of endogenous xenotropic proviral DNA that are present in different relative abundances in mouse genomic DNA.

Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA.

Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA.

Genetic mapping of ecotropic murine leukemia virus-inducing loci in six inbred strains.

Mendelian segregation analysis was used to map chromosomal genes for the induction of endogenous N- and B-tropic ecotropic retroviruses (V loci) in high and low leukemic mouse strains. Patterns of virus expression were determined for mice of various inbred strains, congenic lines carrying single V loci, and the linkage testing stocks used in mapping studies. Segregation analysis resulted in the genetic mapping of V loci from six inbred strains to five mouse chromosomes. The V locus of A/J was mapped to chromosome 5 and shown to be allelic with that of BALB/cJ and C3H/HeJ (Cv); this suggests that Cv-represents a stable ancestral V locus present in Bagg albino stocks before the separation of inbred lines. The single, poorly inducible V locus of C57BL/10J and one of the four high virus loci of C58/Lw were mapped to the same region of chromosome 8 and may represent an allelic pair with different patterns of expression. An N-tropic V locus of the SEA/GnJ mouse was mapped to chromosome 9, and one of the three V loci of C3H/FgLw was mapped to chromosome 7. The endogenous B-tropic virus of B10.BR/SgLi was mapped to chromosome 11. These studies provide further evidence that endogenous ecotropic V loci are present at different chromosomal sites in unrelated mouse strains and emphasize the role of germ line reinfections in the generation of this diversity.

Cloning and characterization of an envelope-specific probe from xenotropic murine leukemia proviral DNA.

An 8.9-kilobase EcoRI restriction fragment was cloned from mink cells chronically infected with NFS-Th-1 xenotropic murine leukemia virus by using a lambda phage host vector system. After its transfer into pBR322, the EcoRI DNA insert was characterized and found to contain 6.7 kilobases of proviral DNA sequences and 2.2 kilobases of mink cellular DNA flanking the 5' end of the viral genome. A 500-base pair fragment which was located at the 3' terminus of the cloned DNA insert and which mapped to the env region of xenotropic proviral DNA was subcloned into pBR322. This xenotropic envelope proviral DNA segment did not hybridize to ecotropic murine leukemia proviruses but did anneal to representative alpha and beta xenotropic and seven different mink cell focus-inducing proviral DNAs. The cloned xenotropic envelope-specific probe was also used in blot hybridization experiments to analyze the arrangement of related sequences in preparations of different mouse liver DNAs.

Genetic study of lymphoma induction by AKR mink cell focus-inducing virus in AKR x NFS crosses.

The mink cell focus-inducing (MCF)-247 virus, originally isolated from an AKR thymoma, is lymphomagenic in AKR mice but not in the ecotropic virus-negative NFS mouse strain. Analysis of sensitivity to lymphoma-induction by AKR-247 MCF virus in genetic hybrids between these two strains showed that F1 mice inoculated as sucklings were uniformly sensitive, whereas those inoculated as weanlings were generally resistant. In NFS backcross mice inoculated as sucklings, inheritance and expression of endogenous ecotropic virus from AKR was an essential correlate of replication of MCF virus and subsequent development of lymphoma. However, one-third of the mice expressing ecotropic virus and replicating the inoculated MCF virus did not develop lymphoma. The results suggested that an additional gene that influenced development of lymphoma may be involved, and that mice inheriting both virus-inducing loci from AKR were more susceptible than those inheriting only one. These findings indicate that the causal role of ecotropic virus infection in spontaneous thymomagenesis in AKR mice involves not only the generation of leukemogenic MCF viruses but also the establishment of permissiveness for their growth.

Close similarity between endogenous ecotropic virus of Mus musculus molossinus and AKR virus.

By using seven different restriction endonucleases, the cleavage patterns of the unintegrated provioral DNA from an ecotropic murine leukemia virus isolated from Mus musculus molossinus were found to be identical to those of AKR virus. An AKR [3H]DNA probe can be completely saturated with M. musculus molossinus and M. musculus castaneus DNAs, although the arrangement of viral sequences in M. musculus molossinus DNA differed from that of AKR virus. These studies indicate that an AKR-type ecotropic virus is present in some wild Asiatic mice.

Genetic mapping of the ecotropic virus-inducing locus Akv-2 of the AKR mouse.

A combination of somatic cell hybridization and standard mendelian breeding techniques was used to map the AKR ecotropic virus inducibility locus Akv-2 to the centromeric end of chromosome 16. This assignment of Akv-2 further emphasizes the endogenous ecotropic retroviruses are inserted at multiple sites in mouse chromosomes.