Frequent involvement of the fim-3 region in Friend murine leukemia virus-induced mouse myeloblastic leukemias.

fim-3 is a new proviral integration region involved in 23% (16 of 68) of Friend murine leukemia virus-induced myeloblastic leukemias. This region is distinct from 20 oncogenes and from putative oncogenes tested so far. Proviruses are integrated in a 16-kilobase region, always in the same orientation. No RNA expression of fim-3 was detected.     

Common proviral integration region on mouse chromosome 7 in lymphomas and myelogenous leukemias induced by Friend murine leukemia virus.

Friend murine leukemia virus (F-MuLV) induces a variety of hematopoietic neoplasms 2 to 12 months after inoculation into newborn mice. These neoplasms are clonal or oligoclonal and contain a small number of F-MuLV insertions in high-molecular-weight DNA. To investigate whether different tumors have proviral insertions in the same region, a provirus-cellular DNA junction fragment from an F-MuLV-induced myelogenous leukemia was cloned in lambda gtWES, and a portion of the flanking cellular DNA sequence was used in blot-hybridization studies of 34 additional F-MuLV-induced neoplasms. Three of these additional neoplasms (one myelogenous leukemia and two lymphomas) were found to have altered copies of the flanking cellular sequence. Restriction enzyme analysis of genomic DNA from these tumors revealed that in each case a proviral copy of F-MuLV had inserted into the same 1.5-kilobase region; all proviruses had the same orientation. Using mouse-Chinese hamster somatic cell hybrids, we mapped this common integration region, designated Fis-1, to mouse chromosome 7. Fis-1 is distinct from three oncogenes on mouse chromosome 7, Ha-ras, fes, and Int-2, based on restriction enzyme analysis and blot hybridization. Therefore, Fis-1 appears to be a novel sequence implicated in both lymphoid and myeloid leukemias induced by F-MuLV.     

Identification of a new common provirus integration site in gross passage A murine leukemia virus-induced mouse thymoma DNA.

The Gross passage A murine leukemia virus (MuLV) induced T-cell leukemia of clonal (or oligoclonal) origin in inoculated mice. To study the role of the integrated proviruses in these tumor cells, we cloned several newly integrated proviruses (with their flanking cellular sequences) from a single tumor in procaryotic vectors. With each of the five clones obtained, a probe was prepared from the cellular sequences flanking the provirus. With one such probe (SS8), we screened several Gross passage A MuLV-induced SIM.S mouse tumor DNAs and found that, in 11 of 40 tumors, a provirus was integrated into a common region designated Gin-1. A 26-kilobase-pair sequence of Gin-1 was cloned from two lambda libraries, and a restriction map was derived. All proviruses were integrated as a cluster in the same orientation within a 5-kilobase-pair region of Gin-1, and most of them had a recombinant structure of the mink cell focus-forming virus type. The frequency of Gin-1 occupancy by provirus was much lower in thymoma induced by other strains of MuLV in other mouse strains. Using somatic-cell hybrid DNAs, we mapped Gin-1 on mouse chromosome 19. Gin-1 was not homologous to 16 known oncogenes and was distinct from the other common regions for provirus integration previously described. Therefore, Gin-1 appears to represent a new common provirus integration region. The integration of a provirus within Gin-1 might be an important event leading to T-cell transformation, and the Gin-1 region might harbor sequences which are involved in tumor development.     

Rearrangements in the long terminal repeat of extra mouse mammary tumor proviruses in T-cell leukemias of mouse strain GR result in a novel enhancer-like structure.

Male GR mice develop T-cell leukemia at low frequency late in life. These leukemia cells invariably contain large amounts of mouse mammary tumor virus (MMTV) RNA and MMTV proteins and have extra MMTV proviruses integrated in their DNA. We show here that the extra MMTV proviruses are all derived from the endogenous MMTV provirus associated with the Mtv-2 locus and that the T-cell leukemias are clonal with respect to the acquired MMTV proviruses. The extra MMTV proviruses in six transplantable T-cell leukemia lines studied had rearranged, shortened long terminal repeats (LTRs); each T-cell leukemia, however, had a different LTR rearrangement within its extra MMTV provirus. The alteration within the extra LTRs of T-cell leukemia line 42 involved deletion of 453 nucleotides and generation of a tandem repeat region consisting of regions flanking the deletion. This alteration generated a sequence similar to the adenovirus enhancer core sequence. The viral RNAs in the T-cell leukemias contained corresponding alterations in their U3 regions. These results demonstrate that expression of MMTV in T-cell leukemias of GR mice may be the consequence of the generation of a novel enhancer, which could also stimulate expression of any adjacent cellular oncogene.     

Analysis of integrated proviral DNA sequences with an octadecanucleotide probe designed for specific identification of spleen focus-forming virus in the mouse genome.

The Friend spleen focus-forming virus (SFFV) is an envelope gene recombinant between the ecotropic Friend murine leukemia virus and the endogenous xenotropic mink cell focus-forming retroviral sequences. We synthesized an octadecanucleotide complementary to the 3' end of the SFFV env gene designed for discriminating the SFFV proviruses from the xenotropic mink cell focus-forming virus and ecotropic exogenous or endogenous viral sequences. Under appropriate hybridization conditions this probe allowed the identification, in addition to few endogenous DNA fragments, of multiple SFFV proviruses integrated in the genome of Friend malignant cells. Therefore this probe should be of interest in further characterizing the SFFV integration sites and possibly the SFFV ancestor gene.     

A tagged helper-free Friend virus causes clonal erythroblast immortality by specific proviral integration in the cellular genome.

A colinear molecular clone of the Lilly-Steeves polycythemia strain of Friend spleen focus-forming virus (SFFV) was modified by inserting a 215-base-pair tag of simian virus 40 DNA into its nonfunctional pol gene region. The DNA was then transfected into psi-2 packaging cells, and helper-free tagged SFFV was recovered in the culture medium. Injection of this helper-free virus into NIH/Swiss mice caused transient mild splenomegaly and formation of spleen foci at 9 to 10 days. Although the vast majority of infected erythroblast clones then differentiated and died out, rare cell clones that were present in only 20 to 30% of the mice grew extensively by 26 to 33 days to form transplantable leukemias. The clonality of these leukemias was established by Southern blot analysis of their DNAs by using several restriction endonucleases and the simian virus 40 tag as a hybridization probe. All transplantable leukemias lacked helper virus contamination and contained a single tagged SFFV provirus that expressed the mitogenic env gene product gp55. The SFFV proviruses in these leukemias also appeared to be integrated into a few tightly clustered sites in the cellular genome. Although the tagged SFFV caused polycythemia during the polyclonal early stage of erythroblastosis, growth of the helper-free clonal erythroleukemias caused severe anemia. These results suggest that a single SFFV can cause mitosis of erythroblasts, and that cell immortalization also occurs when the provirus integrates into a critical site in the host genome. We propose that mice with clonal-stage leukemia become anemic because the immortalizing proviral integrations interfere with the cellular commitment to differentiate.     

The integrated genome of murine leukemia virus

The Southern gel filter transfer technique has been used to characterize the integrated genome of Moloney murine leukemia virus (M-MuLV) and the genomes of the endogenous viruses of the mouse. Study of 10 clones of rat cell independently infected by M-MuLV indicates a minimum of 15 integration sites into which the M-MuLV provirus can be inserted. No common integration site is observed among these clones. Clones productively infected by M-MuLV acquire multiple proviruses, whereas infected cells unable to produce virus contain only one M-MuLV provirus. Once established, the integrated genomes are stable for at least two years after initial infection.The use of M-MuLV probe allows detection of a spectrum of Eco RI-cleaved mouse DNA fragments containing endogenous MuLV genomes. DNAs of different inbred laboratory mouse strains yield similar patterns of provirus with each strain showing minor characteristic differences. In some instances, mouse cells infected by M-MuLV reveal additional proviruses beyond those seen in the uninfected cell. DNAs from three different M-MuLV-induced thymomas indicate, as in rat cells, multiple possible integration sites.     

Organization, distribution, and stability of endogenous ecotropic murine leukemia virus DNA sequences in chromosomes of Mus musculus.

The endogenous ecotropic murine leukemia virus DNA content and integration sites were characterized for 54 inbred strains and substrains of mice by restriction enzyme digestion, Southern blotting, and hybridization with an ecotropic murine leukemia virus DNA-specific probe. More than 75% of these strains carried endogenous ecotropic proviruses which were located in at least 29 distinct integration sites in chromosomes of Mus musculus. Fourteen of these proviruses have been assigned specific locus designations. Most, but not all, of the endogenous ecotropic proviruses were structurally indistinguishable by this analysis from the prototype AKR ecotropic virus, and the distribution of these proviruses followed known relationships among the inbred strains and substrains of mice. These results suggest that, in general, viral DNA integration preceded the establishment of inbred mouse strains and that these integrations are relatively stable.     

Genetic analysis of myeloproliferative leukemia virus, a novel acute leukemogenic replication-defective retrovirus.

The myeloproliferative leukemia virus (MPLV) is a new acute leukemogenic, nonsarcomatogenic retroviral complex that is generated during the in vivo passage of a molecularly cloned Friend ecotropic helper virus. Examination of viral RNA expression in MPLV-producing cells revealed the presence of two distinct molecular species that hybridized with a long terminal repeat or an ecotropic env-specific probe but not with a xenotropic mink cell focus-forming virus env-specific probe derived from a spleen focus-forming virus: an 8.2-kilobase species corresponding to a full-length Friend murine leukemia virus (F-MuLV) and a deleted species with a genomic size of 7.4 kilobases. This deleted virus was biologically cloned by limiting dilutions and single cell cloning in Mus dunni fibroblasts. Three nonproducer clones with normal morphologies and containing one single integrated copy of the deleted virus were superinfected with F-MuLV, Moloney murine leukemia virus, Gross murine leukemia virus, mink cell focus-forming virus (HIX), or the amphotropic 1504 murine leukemia virus. All pseudotypes caused macroscopic and microscopic abnormalities in mice that were similar to those seen in the parental stock. A comparison of the physical maps of F-MuLV and MPLV, which was deduced from the restriction enzyme digests of unintegrated proviral DNAs, indicated that the MPLV-defective genome (i) is probably derived from F-MuLV, (ii) has conserved the F-MuLV gag and pol regions, and (iii) is deleted and rearranged in the env region in a manner that is clearly distinct from that of Friend or Rauscher spleen focus-forming viruses.     

Chromosome integration domain for bovine leukemia provirus in tumors.

The 3'-end host-virus junction fragments from two bovine leukemia virus (BLV)-induced lymphoid tumors (tumors 15-4 and 1351), each containing a single provirus, were used as probes to detect large restriction fragments flanking these proviruses. The DNAs from 28 other independent BLV-induced tumors were checked by Southern analysis of their restriction fragments for possible rearrangement due to the insertion of a BLV provirus in the cellular sequences corresponding to those flanking the proviruses in tumors 15-4 and 1351. In no case did proviral integration occur in cellular sequences corresponding to those implicated in the tumors of origin. According to the statistical analysis performed, if a preferential domain for BLV integration exists, it has a size of 1,304 kilobases when the probability of not observing an integration event in the cellular fragments considered in tumors 15-4 and 1351 is 0.50.     

Integration of spleen focus-forming virus proviruses in Friend tumor cells.

Using the Southern blot procedure, we studied the presumed spleen focus-forming virus (SFFV) provirus integration sites in the genome of the premalignant and the malignant cells isolated during the course of Friend erythroleukemia. Two restriction endonucleases, PstI and BamHI, discriminated the presumed integrated SFFV proviruses from the endogenous xenotropic-mink cell focus-forming viral sequences. No SFFV integration sites were detectable in the premalignant cells, suggesting a random integration of SFFV proviruses in the genome of these cells. In contrast, SFFV proviruses were detected at a single or very few sites in the genome of all malignant cells we analyzed. These results indicate that the event leading to the malignant transformation in acute Friend leukemia is clonal. In two of the six animals examined, tumors cells isolated from the spleens and the livers of individual mice showed identical SFFV integration patterns. This last result suggests that in some cases different tumors in a same leukemic animal could be derived from a unique clonal event.     

Genetic alterations by human papillomaviruses in oncogenesis.

The integration sites in the cellular genome of human papillomavirus are located in chromosomal regions always associated with oncogenes or other known tumor phenotypes. Two regions, 8q24 and 12q13, are common to several cases of cervical carcinoma and can have integrated more than one type of papillomavirus DNA. These two chromosomal regions contain several genes implicated in oncogenesis. These observations strongly imply that viral integration sites of DNA tumor viruses can be used as the access point to chromosomal regions where genes implicated in the tumor phenotype are located, a situation similar to that of non-transforming retroviruses.     

Different abilities of Friend murine leukemia virus (MuLV) and Moloney MuLV to induce promonocytic leukemia are due to determinants in both psi-gag-PR and env regions.

Moloney murine leukemia virus (M-MuLV) is capable of inducing promonocytic leukemia in 50% of adult BALB/c mice that have received peritoneal injections of pristane, but Friend MuLV strain 57 (F-MuLV) is nonleukemogenic under similar conditions. It was shown earlier that these differences could not be mapped to the U3 region of the virus long terminal repeat, indicating the probable influence of structural genes and/or R-U5 sequences. In this study, reciprocal chimeras containing exchanged structural genes and R-U5 sequences from these two closely related viruses were analyzed for differences in ability to induce disease. Results showed that two regions of F-MuLV, psi-gag-PR and env, when substituted for those of M-MuLV were dramatically disease attenuating. The 5'-most region, which is widely distributed, overlaps with the 5' end of the env intron and includes the RNA packaging region, psi, the entire gag coding region, and the viral protease coding region (PR) of pol. It was also found that reciprocal constructs having substitutions of both of these regions of M-MuLV in an F-MuLV background allowed full reestablishment of promonocytic leukemia. These leukemias were positive for c-myb rearrangements which are characteristic of M-MuLV-induced promonocytic leukemias. Neither region alone, however, was sufficient to produce disease with a greater incidence than 13%. Further studies demonstrated that the inability of viruses with psi, gag, PR, or env sequences from F-MuLV to induce leukemia in this model system was not due to their inability to replicate in hematopoietic tissue, to integrate into the c-myb locus early on after infection in vivo, or to express gag-myb mRNA characteristic of M-MuLV-induced preleukemic cells and acute leukemia.