Escape from in vivo restriction of Moloney mink cell focus-inducing viruses driven by the Mo+PyF101 long terminal repeat (LTR) by LTR alterations

Transcriptional enhancer sequences within the long terminal repeats (LTRs) of murine leukemia viruses are the primary genetic determinants of the tissue specificity and potency of the oncogenic potential of these retroviruses. SL3-3 (SL3) is a murine leukemia virus that induces T-cell lymphomas. The LTR enhancer of this virus contains two binding sites for the transcription factor CBF (also called AML1 and PEBP2) that flank binding sites for c-Myb and the Ets family of factors. Using cotransfection assays in P19 cells, we report here that CBF and c-Myb cooperatively stimulate transcription from the SL3 LTR. By itself, c-Myb had no stimulatory effect on transcription. However, when cotransfected with a cDNA encoding one form of the ␣ subunit of CBF called CBF␣2-451, a level of transactivation higher than that seen with CBF␣2-451 alone was detected. The negative regulatory domain near the carboxyl terminus of c-Myb did not affect this activity. Electrophoretic mobility shift assays indicated that CBF and c-Myb bind to DNA independently. Therefore, it appears that the cooperative stimulation of transcription by these factors occurs at a step in the process of transcription after the two factors are bound to the enhancer. Sequences near the carboxyl terminus of CBF␣2-451 were important for cooperativity with c-Myb, consistent with previous reports that this region contains an activation domain. However, CBF␣2-451 failed to activate transcription from a version of the SL3 LTR in which the enhancer was replaced with five tandem CBF-binding sites. Thus, it appears that transcriptional activation of the SL3 enhancer by CBF requires that an appropriate heterologous transcription factor be bound to a neighboring site in the regulatory sequences.

Section: Discussionmentioning

confidence: 99%

Transcriptional activation of a retrovirus enhancer by CBF (AML1) requires a second factor: evidence for cooperativity with c-Myb

Zaiman

Lenz

1996

“…Alternatively, deletion of polyomavirus enhancer sequences and the adjacent XbaI site could have converted the MCF virus-diagnostic fragment to viral-cellular junction fragments of indeterminate size. In subsequent studies, we observed both classes of LTR alterations (2,4). The BamHI-ClaI restriction digestion used in these experiments is not influenced by alterations in the MoϩPyF101 M-MuLV LTR.…”

Section: Discussionmentioning

confidence: 71%

“…DNA was extracted from single-cell suspensions or cell lines by a modification of standard methods (8) and suspended in 10 mM Tris-0.1 mM EDTA (pH 8). Restriction endonuclease digestion of high-molecular-weight DNA, gel electrophoresis, transfer, and hybridization were performed as previously described (2,4,6). DNA fragments used as radioactive probes for Southern blot analyses included the 199-bp PyF101 enhancer-containing PvuII-4 fragment inserted at the XbaI site of MoϩPyF101 M-MuLV as previously described (23); the 620-bp BamHI-EcoRI fragment from the 5Ј env region of the M-MCF recombinant that is reactive with the M-MCF recombinant and endogenous polytropic and modified polytropic viruses (6), and the 1.1-kb BamHI-ClaI fragment from the 3Ј env region of M-MuLV that is reactive with both ecotropic M-MuLV and polytropic M-MCF recombinants (4).…”

Section: Methodsmentioning

confidence: 99%

Appearance of Mink Cell Focus-Inducing Recombinants during In Vivo Infection by Moloney Murine Leukemia Virus (M-MuLV) or the Mo+PyF101 M-MuLV Enhancer Variant: Implications for Sites of Generation and Roles in Leukemogenesis

Lander

Chesebro

Fan

1999

One hallmark of murine leukemia virus (MuLV) leukemogenesis in mice is the appearance of env gene recombinants known as mink cell focus-inducing (MCF) viruses. The site(s) of MCF recombinant generation in the animal during Moloney MuLV (M-MuLV) infection is unknown, and the exact roles of MCF viruses in disease induction remain unclear. Previous comparative studies between M-MuLV and an enhancer variant, Mo+PyF101 MuLV, suggested that MCF generation or early propagation might take place in the bone marrow under conditions of efficient leukemogenesis. Moreover, M-MuLV induces disease efficiently following both intraperitoneal (i.p.) and subcutaneous (s.c.) inoculation but leukemogenicity by Mo+PyF101 M-MuLV is efficient following i.p. inoculation but attenuated upon s.c. inoculation. Time course studies of MCF recombinant appearance in the bone marrow, spleen, and thymus of wild-type and Mo+PyF101 M-MuLV i.p.- and s.c.-inoculated mice were carried out by performing focal immunofluorescence assays. Both the route of inoculation and the presence of the PyF101 enhancer sequences affected the patterns of MCF generation or early propagation. The bone marrow was a likely site of MCF recombinant generation and/or early propagation following i.p. inoculation of M-MuLV. On the other hand, when the same virus was inoculated s.c., the primary site of MCF generation appeared to be the thymus. Also, when Mo+PyF101 M-MuLV was inoculated i.p., MCF generation appeared to occur primarily in the thymus. The time course studies indicated that MCF recombinants are not involved in preleukemic changes such as splenic hyperplasia. On the other hand, MCFs were detected in tumors from Mo+PyF101 M-MuLV s.c.-inoculated mice even though they were largely undetectable at preleukemic times. These results support a role for MCF recombinants late in disease induction.

“…The amplification of the LVb-core region has been documented in proviruses of T-lymphoid tumors induced by several other MuLVs or FeLVs (3,5,14,33,35,44,49). In a prior study by Chen and Yoshimura, proviral insertions adjacent to c-myc that had duplications in the MCF 13 enhancers invariably included the core and LVb binding sites (12).…”

Section: Discussionmentioning

confidence: 94%

“…3). However, we and others have detected enhancer alterations in end-stage tumors induced by various MuLV and feline leukemia virus (FeLV) recombinants (3,5,14,33,35,44,49). Thus, a more detailed PCR analysis with SRS-specific oligonucleotide primers was used to search for modifications in the SRS sequences ( Fig.…”

Section: Generation Of ⌬Mo؉srs M-mulvmentioning

confidence: 99%

Tandemization of a Subregion of the Enhancer Sequences from SRS 19-6 Murine Leukemia Virus Associated with T-Lymphoid but Not Other Leukemias

Granger

Bundy

Fan

1999

Most simple retroviruses induce tumors of a single cell type when infected into susceptible hosts. The SRS 19-6 murine leukemia virus (MuLV), which originated in mainland China, induces leukemias of multiple cellular origins. Indeed, infected mice often harbor more than one tumor type. Since the enhancers of many MuLVs are major determinants of tumor specificity, we tested the role of the SRS 19-6 MuLV enhancers in its broad disease specificity. The enhancer elements of the Moloney MuLV (M-MuLV) were replaced by the 170-bp enhancers of SRS 19-6 MuLV, yielding the recombinants ΔMo+SRS+ and ΔMo+SRS− M-MuLV. M-MuLV normally induces T-lymphoid tumors in all infected mice. Surprisingly, when neonatal mice were inoculated with ΔMo+SRS+ or ΔMo+SRS−M-MuLV, all tumors were of T-lymphoid origin, typical of M-MuLV rather than SRS 19-6 MuLV. Thus, the SRS 19-6 MuLV enhancers did not confer the broad disease specificity of SRS 19-6 MuLV to M-MuLV. However, all tumors contained ΔMo+SRS M-MuLV proviruses with common enhancer alterations. These alterations consisted of tandem multimerization of a subregion of the SRS 19-6 enhancers, encompassing the conserved LVb and core sites and adjacent sequences. Moreover, when tumors induced by the parental SRS 19-6 MuLV were analyzed, most of the T-lymphoid tumors had similar enhancer alterations in the same region whereas tumors of other lineages retained the parental SRS 19-6 MuLV enhancers. These results emphasize the importance of a subregion of the SRS 19-6 MuLV enhancer in induction of T-cell lymphoma. The relevant sequences were consistent with crucial sequences for T-cell lymphomagenesis identified for other MuLVs such as M-MuLV and SL3-3 MuLV. These results also suggest that other regions of the SRS 19-6 MuLV genome contribute to its broad leukemogenic spectrum.