Triple basepair changes within and adjacent to the conserved YY1 motif upstream of the U3 enhancer repeats of SL3-3 murine leukemia virus cause a small but significant shortening of latency of T-lymphoma induction (original) (raw)
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Journal of Virology
SL3-3 is a highly T-lymphomagenic murine retrovirus in which the transcriptional enhancer is a major oncogenic determinant. Here, we describe an SL3-3 enhancer variant that induced T-cell lymphomas in all inoculated mice with a shorter latency period than wild-type SL3-3. The enhancer repeat region of this variant contains two deletions encompassing the nuclear factor 1 binding sites in addition to an additional intact enhancer repeat element. Tumors induced by this variant were T-cell lymphomas, as indicated by T-cell receptor rearrangements, and contained the input provirus enhancer regions. The variant was the result of mutation of specific transcription factor binding sites in the viral enhancer, isolation of rare second-site enhancer variants from the resulting induced tumors, and subsequent restoration of the original first-site mutations of one such variant. We have termed this process assisted molecular evolution.
Molecular Therapy, 2002
Highly conserved enhancer sequences located in the upstream part of the long terminal repeat (LTR) of murine leukemia retroviruses (MLV) were reported to compromise viral gene expression in multipotent embryonic cells in vitro and to reduce the likelihood for maintenance of retroviral gene expression in hematopoietic cells in vivo. We show that deletion of these sequences (nucleotides +37 to +95) attenuates rather than increases the transcriptional activity of retroviral vectors in hematopoietic cells almost independently of the developmental lineage (erythroid, myeloid, or lymphoid). Expression rates of modified vectors were reduced by as much as 34-65%, although the strong enhancer array located in the direct repeat of the LTR was preserved. Sequence analysis and electrophoretic mobility shift assays revealed the presence of a highly conserved binding site for NFAT (nuclear factor of activated T cells) proteins that immediately neighbors a known binding site for the transcription factor Ying-Yang1 (YY1). Specific inactivation of the NFAT site reduced transgene expression in all cell types investigated and had a similar effect as the destruction of a neighboring SP1 motif. Combined destruction of individual motifs for NFAT, SP1, and E twenty-six transcription factors (ETS) resulted in a severe attenuation (by 40-60%) of the retroviral enhancer. These results provide novel clues for the manipulation of retrovirus replication and vector tropism.
Importance of a c-Myb binding site for lymphomagenesis by the retrovirus SL3-3
Journal of virology, 1997
All murine leukemia viruses (MuLVs) and related type C retroviruses contain a highly conserved binding site for the Ets family of transcription factors within the enhancer sequences in the viral long terminal repeats (LTRs). The T-cell lymphomagenic MuLV SL3-3 (SL3-3) also contains a c-Myb binding site adjacent to the Ets site. The presence of this Myb site distinguishes SL3 from most other MuLVs. We tested the importance of these two sites for the lymphomagenicity of SL3-3. Mutation of the Ets site had little effect on viral pathogenicity, as it only slightly extended the latency period to disease onset. In contrast, mutation of the Myb site strongly inhibited pathogenicity, as only a minority of the inoculated mice developed tumors in the two mouse strains that were tested. All tumors that were induced by either mutant appeared to be lymphomas, and no evidence for reversion of either mutation was detected. The effects of the Ets and Myb site mutations on transcriptional activity of the SL3 LTR were tested by inserting the viral enhancer sequences into a plasmid containing the promoter region of the c-myc gene linked to a reporter gene. Mutation the Myb site almost eliminated enhancer activity in T lymphocytes, while mutation of the Ets site had smaller effects. Thus, the effects of the enhancer mutations on transcriptional activity in T cells paralleled their effects on viral lymphomagenicity. The absence of the c-Myb site in the LTR enhancer of the weakly lymphomagenic MuLV, Akv, likely contributes to the low pathogenicity of this virus relative to SL3-3. However, Moloney MuLV also lacks the Myb site in its LTR, although it induces T-cell lymphomas with a potency similar to that of SL3-3. Thus, it appears that SL3-3 and Moloney MuLV evolved genetic determinants of T-cell lymphomagenicity that are, at least in part, distinct.
Virology, 2006
In a small sample of 57 retrovirus integration sites (RISs) isolated from 23 end-stage lymphomas induced in NMRI mice by the Blymphotropic Akv wt or an enhancer mutant hereof, Akv1-99, we identified 14 novel RISs and defined 9 novel CISs (common insertion sites). Moreover, when comparing with RISs from tumors induced by the T-lymphomagenic SL3-3, we observed that SL3-3 targets RefSeq promoter regions with a significantly higher frequency than Akv/Akv1-99 and in an orientation-dependent way. Altogether, our results strongly emphasize the importance of host genetic background and virus type for retroviral insertion mutagenesis screens and suggest that different types of MLV may favor specific genomic regions and orientations in order exert optimal effect on target gene expression during lymphoma induction and development.
Journal of Virology
Murine retrovirus SL3-3 is highly T lymphomagenic. Its pathogenic properties are determined by the transcriptional enhancer of the U3 repeat region which shows preferential activity in T cells. Within the U3 repeats, the major determinant of T-cell specificity has been mapped to binding sites for the AML1 transcription factor family (also known as the core binding factor [CBF], polyomavirus enhancer binding protein 2 [PEBP2], and SL3-3 enhancer factor 1 [SEF-1]). SL3-3 viruses with AML1 site mutations have lost a major determinant of T-cell-specific enhancer function but have been found to retain a lymphomagenic potential, although disease induction is slower than for the SL3-3 wild type. To compare the specificities and mechanisms of disease induction of wild-type and mutant viruses, we have examined lymphomas induced by mutant viruses harboring transversions of three consecutive base pairs critical to AML1 site function (B. Hallberg, J. Schmidt, A. Luz, F. S. Pedersen, and T. Grun...
Replication and pathogenicity of primer binding site mutants of SL3-3 murine leukemia viruses
Journal of virology, 1999
Retroviral reverse transcription is primed by a cellular tRNA molecule annealed to an 18-bp primer binding site sequence. The sequence of the primer binding site coincides with that of a negatively acting cis element that mediates transcriptional silencing of murine leukemia virus (MLV) in undifferentiated embryonic cells. In this study we test whether SL3-3 MLV can replicate stably using tRNA primers other than the cognate tRNAPro and analyze the effect of altering the primer binding site sequence to match the 3' end of tRNA1Gln, tRNA3Lys, or tRNA1,2Arg in a mouse pathogenicity model. Contrary to findings from cell culture studies of primer binding site-modified human immunodeficiency virus type 1 and avian retroviruses, our findings were that SL3-3 MLV may stably and efficiently replicate with tRNA primers other than tRNAPro. Although lymphoma induction of the SL3-3 Lys3 mutant was significantly delayed relative to that of the wild-type virus, molecular tumor analysis indicate...
Journal of Virology, 1998
Transcriptional enhancers within the long terminal repeats of murine leukemia viruses are major determinants of the pathogenic properties of these viruses. Mutations were introduced into the adjacent binding sites for three transcription factors within the enhancer of the T-cell-lymphomagenic virus SL3-3. The sites that were tested were, in 5′-to-3′ order, a binding site for core binding factor (CBF) called core II, a binding site for c-Myb, a site that binds members of the Ets family of factors, and a second CBF binding site called core I. Mutation of each site individually reduced transcriptional activity in T lymphocytes. However, mutation of the Myb and core I binding sites had larger effects than mutation of the Ets or core II site. The relative effects on transcription in T cells paralleled the effects of the same mutations on viral lymphomagenicity, consistent with the idea that the role of these sequences in viral lymphomagenicity is indeed to regulate transcription in T cel...
Journal of Virology, 1991
Sequences within the R components of the long terminal repeats (LTRs) of several retroviruses are known to be involved at various steps in expression of the viral genomes. A series of experiments was performed to test whether sequences within the R regions of the murine leukemia viruses Akv and SL3-3 affect viral expression. By using plasmid clones of the viral LTRs linked to a reporter gene, deletion of the R region was found to decrease expression to variable extents in a series of mammalian cell lines, with the largest effects being detected in murine fibroblasts. R-region sequences from the human immunodeficiency virus type 1 LTR or a random sequence were unable to substitute for the murine leukemia virus sequences. Transcripts from the R-region-deleted templates were initiated at the proper site in the LTR, but their levels were decreased at least 10-fold. Nuclear run-on assays showed that the decrease caused by the R-region deletions was due, in part, to an effect on RNA polym...
Journal of Virology, 1998
In addition to their role in reverse transcription, the R-region sequences of some retroviruses affect viral transcription. The first 28 nucleotides of the R region within the long terminal repeat (LTR) of the murine type C retrovirus SL3 were predicted to form a stem-loop structure. We tested whether this structure affected the transcriptional activity of the viral LTR. Mutations that altered either side of the stem and thus disrupted base pairing were generated. These decreased the level of expression of a reporter gene under the control of viral LTR sequences about 5-fold in transient expression assays and 10-fold in cells stably transformed with the LTR-reporter plasmids. We also generated a compensatory mutant in which both the ascending and descending sides of the stem were mutated such that the nucleotide sequence was different but the predicted secondary structure was maintained. Most of the activity of the wild-type SL3 element was restored in this mutant. Thus, the stem-lo...