SL3-3 Murine Leukemia Virus Research Articles

Second-site proviral enhancer alterations in lymphomas induced by enhancer mutants of SL3-3 murine leukemia virus: negative effect of nuclear factor 1 binding site.

SL3-3 is a highly T-lymphomagenic murine retrovirus. Previously, mutation of binding sites in the U3 repeat region for the AML1 transcription factor family (also known as core binding factor [CBF], polyomavirus enhancer binding protein 2 [PEBP2], and SL3-3 enhancer factor 1 [SEF1]) were found to strongly reduce the pathogenicity of SL3-3 (B. Hallberg, J. Schmidt, A. Luz, F. S. Pedersen, and T. Grundström, J. Virol. 65:4177-4181, 1991). We have now examined the few cases in which tumors developed harboring proviruses that besides the AML1 (core) site mutations carried second-site alterations in their U3 repeat structures. In three distinct cases we observed the same type of alteration which involved deletions of regions known to contain binding sites for nuclear factor 1 (NF1) and the addition of extra enhancer repeat elements. In transient-expression experiments in T-lymphoid cells, these new U3 regions acted as stronger enhancers than the U3 regions of the original viruses. This suggests that the altered proviruses represent more-pathogenic variants selected for in the process of tumor formation. To analyze the proviral alterations, we generated a series of different enhancer-promoter reporter constructs. These constructs showed that the additional repeat elements are not critical for enhancer strength, whereas the NF1 sites down-regulate the level of transcription in T-lymphoid cells whether or not the AML1 (core) sites are functional. We therefore also tested SL3-3 viruses with mutated NF1 sites. These viruses have unimpaired pathogenic properties and thereby distinguish SL3-3 from Moloney murine leukemia virus.

Mapping of a major osteomagenic determinant of murine leukemia virus RFB-14 to non-long terminal repeat sequences.

Certain isolates of murine leukemia viruses (MuLVs) have, apart from a leukemogenic potential, the capability of inducing diseases of nonhematopoietic tissues in susceptible strains of mice. We have reported on the molecular cloning of a bone-tumorigenic virus, RFB-14 MuLV, which was found to induce benign bone tumors, osteomas, with 100% incidence in mice of the CBA/Ca strain (L. Pedersen, W. Behnisch, J. Schmidt, A. Luz, F. S. Pedersen, V. Erfle, and P. G. Strauss, J. Virol. 66:6186-6190, 1992). In order to analyze the bone tumor-inducing phenotype of RFB-14 MuLV, we have studied the pathogenic potential of recombinant viruses between RFB-14 and the nonosteomagenic, highly leukemogenic SL3-3 MuLV. The recombinants were constructed so as to reveal whether a major determinant of osteomagenicity maps to sequences within or outside the long terminal repeats (LTR). Our data show that a major determinant of the osteoma-inducing potential of RFB-14 MuLV maps to the non-LTR region of the genome. Furthermore, we demonstrate that a strong determinant of leukemogenicity is harbored by the non-LTR region of SL3-3 MuLV.

The effect of selection for high-level vector expression on the genetic and functional stability of a single transcript vector derived from a low-leukemogenic murine retrovirus.

Single-gene murine leukemia virus-based retroviral vectors carrying the G418-resistance gene (neo) under transcriptional control of the long terminal repeat were used to study the effect of selection on long-term vector expression in a murine lymphoid cell line, L691. We used two isogenic vectors carrying either a strong or a weak transcriptional enhancer from low-leukemogenic Akv and high-leukemogenic SL3-3 murine leukemia virus, respectively. Effects of G418 selection were studied at the level of vector-transduced cell populations and at the level of single-vector-transduced cell clones obtained without selection for vector expression. Selection for vector expression prior to isolation of cell clones changed the range of vector expression for the two populations of cell clones. Cell clones harboring the Akv enhancer, isolated without selection and then subjected to prolonged growth under selective conditions, exhibited no mutations in the enhancer region or major vector rearrangements although showing increased vector expression in some cases. Our results are discussed in terms of retrovirus-mediated gene transfer strategies employing selection for expression of a selective marker in single-gene or bicistronic vectors with a low- or nonleukemogenic virus-derived backbone.

Basic helix-loop-helix proteins in murine type C retrovirus transcriptional regulation

E boxes, recognition sequences for basic helix-loop-helix (bHLH) transcription factors, are detected in the enhancer and promoter regions of several murine type C retroviruses. Here we show that ALF1, a member of bHLH protein family of transcription factors, in vitro binds with differing affinities to distinct E-box sequences found in the U3 regulatory regions of Friend, Moloney, SL3-3, and Akv murine leukemia viruses (MLVs) as well as Friend spleen focus-forming virus (SFFV). In NIH 3T3 fibroblasts, ALF1 overexpression elevated transcription from the U3 region of Moloney MLV and the complete long terminal repeat regions of Friend SFFV, Akv MLV, and SL3-3 MLV but neither from the U3 region nor from the complete long terminal repeat of Friend MLV. Introduction of mutations in the Akv MLV E boxes showed the E-box cis elements to be required for the function of ALF1 as a transcription factor. ALF1 and the glucocorticoid receptor, with overlapping DNA binding sequences, did not act synergistically with respect to transcriptional trans activation of expression from the Akv MLV promoter-enhancer region. We conclude that ALF1 in vivo may be an important transcription regulator for Akv, SL3-3, and Moloney MLVs as well as for Friend SFFV.

Lack of correlation between basal expression levels and susceptibility to transcriptional shutdown among single-gene murine leukemia virus vector proviruses

Integrated retroviruses or retroviral vectors may be transcriptionally inactive although their promoter-enhancer regions are able to direct transcription in the host cell. We have used single-gene retroviral vectors with a long terminal repeat-directed neo marker gene to determine if the level of transcription relates to the susceptibility of a provirus to inactivation. We used two isogenic vectors, carrying long terminal repeats with a strong and a weak transcriptional enhancer derived from SL3-3 and Akv murine leukemia viruses, respectively. Nonselected cell clones of the murine lymphoid cell line L691 with single integrated vector proviruses exhibiting a 20-fold range of initial expression levels were studied. The basal expression level of a given cell clone with a single provirus did not show any pattern of correlation with the long-term stability of expression, as monitored for periods up to 150 days. Our results thus indicate that the inactivation mechanism operates independently of the initial transcriptional activity of the provirus.

Purification of SEF1 proteins binding to transcriptional enhancer elements active in T lymphocytes.

Binding sites for SL3-3 enhancer factor 1 (SEF1) are important for the transcriptional activity in T lymphocytes and the tumorigenicity of SL3-3 murine leukemia virus. SEF1 is also implicated in the activity of many other leukemia, lymphoma, and sarcoma virus enhancers, and enhancers of genes for T cell receptor-CD3 subunits. We have purified several proteins binding to SEF1 sites from bovine thymus using a five-step purification procedure. The proteins migrated as 19 distinct bands representing molecular masses from 23 kDa to about 200 kDa in SDS-polyacrylamide gel electrophoresis. Ten DNA binding proteins, with molecular masses between 23 and 67 kDa, could be isolated after separation by SDS-polyacrylamide gel electrophoresis. The DNA binding specificities of these proteins were similar and corresponded to that of the SEF1 binding activity in nuclear extracts. Each of these isolated SEF1 proteins also bound to the essential delta-E3 element of the human T cell receptor delta enhancer. Antibodies against one SEF1 protein only reacted with the protein used for immunization, which indicates a limited homology between at least some SEF1 proteins. We also present data suggesting that SEF1 proteins exist in multiple forms with differences in their DNA binding specificity, and that high affinity DNA binding of the SEF1 proteins requires protein phosphorylation.

The role of viral enhancer "core" motif-related sequences in regulating T cell receptor-gamma and -delta gene expression.

T cells express clonally distributed alpha beta or gamma delta Ag receptor heterodimers. Transcriptional enhancers for the genes of all four subunits are active in both gamma delta and alpha beta T cells, but are less active or inactive in other cells. Conserved sequence motifs are present in all four enhancers, suggesting that common transcription factors regulate TCR gene expression. One of these motifs in the gamma 3 site of the TCR-gamma enhancer is similar to motifs found in several other lymphoid-specific and viral enhancers. This conserved "core" sequence is present in the enhancers of Moloney and SL3-3 murine leukemia viruses, important for transcription in T cells and in determining disease specificity. Here we characterize the gamma 3 site of the gamma enhancer and a corresponding homologous site, delta E3, of the TCR-delta enhancer. Our results suggest that the core site is critical for activity of the 200-bp gamma enhancer fragment and of the gamma 3 and delta E3 sites. Furthermore, we identify a nuclear factor in human T cell lines that specifically binds the core region in these and several other core-containing enhancers. This factor may be identical to or related to a purified bovine nuclear core binding factor that binds the core region of the Moloney murine leukemia virus enhancer, gamma 3 and delta E3 sites, suggesting that similar proteins regulate the TCR-gamma, delta and Moloney murine leukemia virus enhancers. Other sequences in the gamma 3 site upstream of the core sequence are also critical for activity in T cells, suggesting that at least two different factors are required for functional activity of the gamma 3 site.

Generation and Pathogenicity of an NB-tropic SL3-3 Murine Leukemia Virus

A 428-base pair region of the gag gene of a molecular clone of the SL3-3 murine leukemia virus (MuLV) was replaced with allelic sequences that contain the NB-tropic determinants of the Moloney MuLV. SL3-3NB, the ecotropic virus derived from this modified clone, rapidly induced T-cell lymphoblastic lymphomas in the Fv-1 n mouse strains CWD and NIH Swiss and the Fv-1 b strain, B10.Br. By Southern blot assay, each of the CWD tumors and all but one of the B10.Br tumors that were tested contained recombinant proviruses. The envelope genes of the B10.Br recombinant viruses retained the SL3-3NB 5′ p15E (TM) gene sequences (type I env), whereas the CWD recombinants did not (type II env). The production of type I env recombinants by B10.Br mice is a characteristic that is shared with other mouse strains that express the H-2 k haplotype. The results indicate that the inserted Moloney virus gag sequences conferred NB-tropism to SL3-3NB and did not interfere with the expression of SL3-3 pathogenic determinants or the formation of recombinant viruses.

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, SL3-3 lacO, 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 λ 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 SL3-3 lacO-infected cell cultures and in viral particles. This system is designed to facilitate studies on the provirus and the site of viral integration.

SL3-3 enhancer factor 1 transcriptional activators are required for tumor formation by SL3-3 murine leukemia virus.

The transcriptional enhancers of retroviruses that lack an oncogene are important determinants of their oncogenicity. However, no specific cellular transcriptional activator has yet been found to determine the oncogenicity for any of these viruses. The SL3-3 enhancer factor 1 (SEF1) cellular transcriptional activators are expressed preferentially in T lymphocytes. In the SL3-3 murine leukemia virus enhancer, two different sequences can bind SEF1 activators. We show that mutation of the SEF1 binding sites disrupts the disease potential of SL3-3 murine leukemia virus, implying that SEF1 transcriptional activators are required for tumor induction by SL3-3. The SEF1 site mutations did not appear to affect the pathogenicity of SL3-3 by impairment of virus multiplication, but rather by a specific defect in the ability of neoplastic transformation.

Differences in activities of murine retroviral long terminal repeats in cytotoxic T lymphocytes and T-lymphoma cells.

Transcriptional activities of the long terminal repeats (LTRs) of various murine leukemia viruses were tested in the cytotoxic T-cell lines CTLL-1 and CTLL-2. In contrast to T-lymphoma cells, in which the LTRs of T-lymphomagenic virus SL3-3 and Moloney murine leukemia virus are more active than those of other viruses, transcriptional activity in these mature, interleukin-2-dependent cells is not correlated with the specificity of viral leukemogenicity. Several approaches were used to investigate the molecular basis for LTR activity differences in lymphoma cells and mature cytotoxic T cells. Deletion analysis of the Moloney virus LTR showed that the direct repeats associated with enhancer activity have, at most, a slight effect on expression in CTLL-1 cells, whereas they stimulate expression six- to eightfold in T-lymphoma cells. This suggests that the mature T-cell line lacks one or more factors present in T-lymphoma cells that function to augment transcription from the Moloney murine leukemia virus LTR. We also used recombinant viral LTRs to investigate the role of the enhancer core element of SL3-3 in CTLL-1 and CTLL-2 cells. A one-base-pair difference between the core sequences of SL3-3 and nonleukemogenic Akv virus, which is important for SL3-3 activity in T-lymphoma cells, had no effect in these cells. The inability to distinguish the single-base-pair difference in expression assays was correlated with the absence of binding of a cellular factor, S-CBF, to the SL3-3 enhancer core in extracts of CTLL-1 and CTLL-2 nuclei. These studies may have implications for identification of the target cells for viral leukemogenesis, as well as for tracing of changes in the transcriptional machinery during T-lymphocyte differentiation.

Suppression of retroviral propagation and disease by suramin in murine systems.

Retroviral propagation crucially depends on reverse transcriptase (RT). We have developed murine models to test the biological effectiveness of the RT inhibitor suramin. The drug was active in our assay system, which includes (i) inhibition of RT activity in the murine T-cell tropic virus SL3-3 and Rauscher murine leukemia virus (MuLV), (ii) inhibition of plaque formation in the XC plaque assay, (iii) inhibition of viral infection of cultured murine T cells, and (iv) inhibition of splenomegaly induced by Rauscher MuLV in BALB/c mice. Suramin decreases viral titers significantly, even if started 36 hr after infection. Viral titers and number of infected cells increased to control levels after removal of the drug. BALB/c mice treated i.v. with 40 mg of suramin per kg twice per week following infection with Rauscher MuLV showed a 35% decrease in splenomegaly. Suramin is an active antiretroviral agent whose effect on retroviral propagation is reversible. We conclude that it acts as a virustatic drug and that long-term administration of suramin will be necessary if it is used for experimental treatment of human retroviral illnesses such as the acquired immune deficiency syndrome.