AKR ecotropic murine leukemia virus SL3-3 forms envelope gene recombinants in vivo.

The ecotropic AKR virus SL3-3 was injected into neonatal mice of the high-leukemia strains HRS/J and CWD/J and the low-leukemia strains CBA/J, SEA/J, and NIH Swiss. SL3-3 was highly leukemogenic in each strain, and 90% of the inoculated animals died by 6 months of age. T1 oligonucleotide fingerprint analysis of the genomic RNAs of viruses recovered from 9 of 13 leukemic animals revealed the presence of the SL3-3 virus and recombinant viruses with polytropic virus-related envelope gene sequences. Recombinant proviruses were detected by the Southern blot technique in the DNAs of 17 of 18 tumors. The pattern of substitutions within the envelope genes of the SL3-3 recombinant viruses appeared to be dependent on the strain of the animal. These observations indicate that the SL3-3 virus formed envelope gene recombinants in vivo in each of the strains that were studied. However, the role of these recombinants during leukemogenesis remains to be defined.     

Multiple steps are required for the induction of tumors by Abelson murine leukemia virus.

Helper virus-free Abelson murine leukemia virus (A-MuLV) was used to induce monoclonal pre-B-cell tumors in mice. The clonality, patterns of immunoglobulin heavy-chain gene rearrangement, tumorigenicity, and v-abl oncogene expression in individual preleukemic and leukemic colonies were compared. Our results indicate that A-MuLV preleukemic cells with low or undetectable tumorigenic potential give rise to leukemic cells with high tumorigenic potential by a process of subclone selection. The levels of v-abl oncogene product in preleukemic and leukemic cell populations were not significantly different. These results suggest that an additional event(s) unrelated to the level of the v-abl protein product is required for A-MuLV-transformed cells to become fully malignant.     

Multiple components are required for sequence recognition of the AP1 site in the gibbon ape leukemia virus enhancer.

At least two subunits contributed to the formation in vitro of a specific complex binding to the AP1 consensus sequence (TGAGTCA) in the gibbon ape leukemia virus (GALV) enhancer in MLA144 cells. This complex can be dissociated on a monomeric GALV oligonucleotide affinity column. One protein, termed the core protein, was retained on the oligonucleotide affinity column. The second protein flowed through the oligonucleotide affinity column and, when alone, did not bind to DNA; however, when present with the core protein, it bound strongly and very specifically to the GALV sequence. MLA144 cells contained only trace amounts of c-fos and c-jun by immunoblot analysis, suggesting that the proteins specifically binding to the GALV AP1 site were distinct from c-fos and c-jun. In addition to the major complex that recognized the GALV element, MLA144 cells contained a minor complex that is chromatographically different from and antigenically related to c-fos. The factor in the flowthrough complemented a human T-cell nuclear extract (Jurkat cell line), which, when alone, had no assayable complex that specifically bound to the GALV enhancer; this complementation gave rise to a specific complex similar to that seen in MLA144 cells. Together, these results suggest that the GALV enhancer can interact with multicomponent protein complexes in a cell-line-specific manner.     

Mutation of all Runx (AML1/core) sites in the enhancer of T-lymphomagenic SL3-3 murine leukemia virus unmasks a significant potential for myeloid leukemia induction and favors enhancer evolution toward induction of other disease patterns

SL3-3 murine leukemia virus is a potent inducer of T-lymphomas in mice. Using inbred NMRI mice, it was previously reported that a mutant of SL3-3 with all enhancer Runx (AML1/core) sites disrupted by 3-bp mutations (SL3-3dm) induces predominantly non-T-cell tumors with severely extended latency (S. Ethelberg, J. Lovmand, J. Schmidt, A. Luz, and F. S. Pedersen, J. Virol. 71:7273-7280, 1997). By use of three-color flow cytometry and molecular and histopathological analyses, we have now performed a detailed phenotypic characterization of SL3-3- and SL3-3dm-induced tumors in this mouse strain. All wild-type induced tumors had clonal T-cell receptor beta rearrangements, and the vast majority were CD3(+) CD4(+) CD8(-) T-lymphomas. Such a consistent phenotypic pattern is unusual for murine leukemia virus-induced T-lymphomas. The mutant virus induced malignancies of four distinct hematopoietic lineages: myeloid, T lymphoid, B lymphoid, and erythroid. The most common disease was myeloid leukemia with maturation. Thus, mutation of all Runx motifs in the enhancer of SL3-3 severely impedes viral T-lymphomagenicity and thereby discloses a considerable and formerly unappreciated potential of this virus for myeloid leukemia induction. Proviral enhancers with complex structural alterations (deletions, insertions, and/or duplications) were found in most SL3-3dm-induced T-lymphoid tumors and immature myeloid leukemias but not in any cases of myeloid leukemia with maturation, mature B-lymphoma, or erythroleukemia. Altogether, our results indicate that the SL3-3dm enhancer in itself promotes induction of myeloid leukemia with maturation but that structural changes may arise in vivo and redirect viral disease specificity to induction of T-lymphoid or immature myeloid leukemias, which typically develop with moderately shorter latencies.     

Change of tropism of SL3-2 murine leukemia virus, using random mutational libraries

SL3-2 is a polytropic murine leukemia virus with a limited species tropism. We cloned the envelope gene of this virus, inserted it into a bicistronic vector, and found that the envelope protein differs from other, similar envelope proteins that also utilize the polytropic receptor (Xpr1) in that it is severely impaired in mediating infection of human and mink cells. We found that two adjacent amino acid mutations (G212R and I213T), located in a previously functionally uncharacterized segment of the surface subunit, are responsible for the restricted tropism of the SL3-2 wild-type envelope. By selection from a two-codon library, several hydrophobic amino acids at these positions were found to enable the SL3-2 envelope to infect human TE 671 cells. In particular, an M212/V213 mutant had a titer at least 6 orders of magnitude higher than that of the wild-type envelope for human TE 671 cells and infected human, mink, and murine cells with equal efficiencies. Notably, these two amino acids are not found at homologous positions in known murine leukemia virus isolates. Functional analysis and library selection were done on the basis of sequence and tropism analyses of the SL3-2 envelope gene. Similar approaches may be valuable in the design and optimization of retroviral envelopes with altered tropisms for biotechnological purposes.     

Mutant analysis of protein interactions with a nuclear factor I binding site in the SL3-3 virus enhancer.

Nuclear factor I (NFI) is shown to be of importance for the activity of the enhancer element of a T-cell leukemogenic murine retrovirus, SL3-3, and for the regulation of this element by glucocorticoid. Each nucleotide of the binding site of the NFI proteins was mutated, and the effects of the mutations were quantitated with an electrophoretic mobility shift assay. Mutations in the inverted repeat of the binding site have symmetric effects which strongly support the notion that NFI proteins preferentially bind to dyad symmetry sites. Such binding sites were shown to be more than 100 fold stronger than the corresponding single binding sites. We find dyad symmetry sequences which are much stronger NFI binding sites than NFI sites identified in different genes and also stronger than previously proposed consensus binding sequences for NFI.     

Identification of the SL3-3 virus enhancer core as a T-lymphoma cell-specific element.

Transient expression assays were used to determine the sequences within the long terminal repeat (LTR) that define the high activity in T-lymphoma cells of the leukemogenic SL3-3 virus in comparison with that of the nonleukemogenic Akv virus. Each of these viruses contains sequences related to the consensus element, the enhancer core. The SL3-3 and Akv enhancer cores differ at a single base pair. Substitution of the Akv core element into the SL3-3 LTR decreased expression in T-lymphoma cells but not in other cell types. Likewise, substitution of the SL3-3 core sequence into the Akv LTR increased expression in T-lymphoma cells but not in other types of hematopoietic cells. These data indicate that the SL3-3 enhancer core sequence functions better than that of Akv in T-lymphoma cells, but in other hematopoietic cell types the two are approximately equivalent. Competition DNA-protein binding assays were used to assess what nuclear factors from T-lymphoma lines and non-T lines bound to the SL3-3 and Akv core elements. Factors were detected that bound specifically to either the SL3-3 or Akv core but not to the other. Another factor was detected that bound equally well to both. However, none of these factors was specific to T-lymphoma cells.     

Identification of mouse chromosomes required for murine leukemia virus replication.

The replication patterns of five ecotropic and two amphotropic strains of murine leukemia virus (MuLV) were studied by infecting 41 Chinese hamster x mounse hybrid primary clones segregating mouse (Mus musculus) chromosomes. Ecotropic and amphotropic strains replicated in mouse and some hybrid cells, but not in hamster cells, indicating that replication of exogenous virus requires dominantly expressed mouse cellular genes. The patterns of replication of the five ecotropic strains in hybrid clones were similar; the patterns of replication of the two amphotropic strains were also similar. When compared to each other, however, the replication patterns of ecotropic and amphotropic viruses were dissimilar, indicating that these two classes of MuLV require different mouse chromosomes for replication. Chromosome and isozyme analyses assigned a gene, Rec-1 (replication of ecotropic virus), to mouse chromosome 5 that is necessary and may be sufficient for ecotropic virus replication. Because of preferential retention of mouse chromosomes 15 and 17 in the hybrid clones, however, the possibility that these chromosomes carry genes that are necessary but not sufficient for ecotropic virus replication cannot be excluded. Similarly, the data indicate that mouse chromosome 8 (or possibly 19) carried a gene we have designated Ram-1 (replication of amphotropic virus) which is necessary and may be sufficient for amphotropic virus replication. Because chromosomes 8 and 19 tended to segregate together and two of the three clones excluding 19 have chromosome reaggrangements, we cannot exclude 19 as being independent of amphotropic virus replication. In addition, because of preferential retention, chromosomes 7, 12, 15, 16 and 17 cannot be excluded as being necessary but not sufficient. Hybrid cell genetic studies confirm the assignment of the Fv-1 locus to chromosome 4 previously made by sexual genetics. In addition, our results demonstrate that hybrid cells which have segregated mouse chromosome 4 but have retained 5 become permissive for replication of both N and B tropic strains of MuLV.     

Tagging the genome of the murine leukemia retrovirus SL3-3 by a bacterial lac operator sequence.

The bacterial lactose operator (lacO) was introduced into the PstI site of the long terminal repeat of the SL3-3 murine leukemia virus, generating a virus, SL3-3lacO, that can replicate in NIH3T3 cell cultures. DNA sequences harboring the lacO sequence might be recovered by molecular cloning in Escherichia coli lac+ lacZ+ using bacteriophage lambda or plasmid vectors. The high copy numbers of the lacO sequence titrate out the lac repressor, leading to the induction of the lac operon in the host. We show here that the lacO and the proviral sequences are carried stably together in the genomes of SL3-3lacO-infected cell cultures and in viral particles. This system is designed to facilitate studies on the provirus and the site of viral integration.     

Disruption of hematopoiesis and thymopoiesis in the early premalignant stages of infection with SL3-3 murine leukemia virus

A time course analysis of SL3-3 murine leukemia virus (SL3) infection in thymus and bone marrow of NIH/Swiss mice was performed to assess changes that occur during the early stages of progression to lymphoma. Virus was detectable in thymocytes, bone marrow, and spleen as early as 1 to 2 weeks postinoculation (p.i.). In bone marrow, virus infection was detected predominantly in immature myeloid or granulocytic cells. Flow cytometry revealed significant reductions of the Ter-119(+) and Mac-1(+) populations, and significant expansions of the Gr-1(+) and CD34(+) populations, between 2 and 4 weeks p.i. Analysis of colony-forming potential confirmed these findings. In the thymus, SL3 replication was associated with significant disruption in thymocyte subpopulation distribution between 4 and 7 weeks p.i. A significant thymic regression was observed just prior to the clonal outgrowth of tumor cells. Proviral long terminal repeats (LTRs) with increasing numbers of enhancer repeats were observed to accumulate exclusively in the thymus during the first 8 weeks p.i. Observations were compared to the early stages of infection with a virtually nonpathogenic SL3 mutant, termed SL3DeltaMyb5, which was shown by real-time PCR to be replication competent. Comparison of SL3 with SL3DeltaMyb5 implicated certain premalignant changes in tumorigenesis, including (i) increased proportions of Gr-1(+) and CD34(+) bone marrow progenitors, (ii) a significant increase in the proportion of CD4(-) CD8(-) thymocytes, (iii) thymic regression prior to tumor outgrowth, and (iv) accumulation of LTR enhancer variants. A model in which disrupted bone marrow hematopoiesis and thymopoiesis contribute to the development of lymphoma in the SL3-infected animal is discussed.     

Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor.

Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.