Duplication of U3 Sequences in the Long Terminal Repeat of Mink Cell Focus-Inducing Viruses Generates Redundancies of Transcription Factor Binding Sites Important for the Induction of Thymomas

Abstract

The ability of mink cell focus-inducing (MCF) viruses to induce thymomas is determined, in part, by transcriptional enhancers in the U3 region of their long terminal repeats (LTRs). To elucidate sequence motifs important for enhancer function in vivo, we injected newborn mice with MCF 1dr (supF), a weakly pathogenic, molecularly tagged (supF) MCF virus containing only one copy of a sequence that is present as two copies (known as the directly repeated [DR] sequence) in the U3 region of MCF 247 and analyzed LTRs from supF-tagged proviruses in two resulting thymomas. Tagged proviruses integrated upstream and in the reverse transcriptional orientation relative to c-myc provided the focus of our studies. These proviruses are thought to contribute to thymoma induction by enhancer-mediated deregulation of c-myc expression. The U3 region in a tagged LTR in one thymoma was cloned and sequenced. Relative to MCF 1dr (supF), the cloned U3 region contained an insertion of 140 bp derived predominantly from the DR sequence of the injected virus. The inserted sequence contains predicted binding sites for transcription factors known to regulate the U3 regions of various murine leukemia viruses. Similar constellations of binding sites were duplicated in two proviral LTRs integrated upstream from c-myc in a second thymoma. We replaced the U3 sequences in an infectious molecular clone of MCF 247 with the cloned proviral U3 sequences from the first thymoma and generated an infectious chimeric virus, MCF ProEn. When injected into neonatal AKR mice, MCF ProEn was more pathogenic than the parental virus, MCF 1dr (supF), as evidenced by the more rapid onset and higher incidence of thymomas. Molecular analyses of the resultant thymomas indicated that the U3 region of MCF ProEn was genetically stable. These data suggest that the arrangement and/or redundancy of transcription factor binding sites generated by specific U3 sequence duplications are important to the biological events mediated by MCF proviruses integrated near c-myc that contribute to transformation. AKR mice succumb to spontaneous thymomas between 6 and 12 months of age. The virological events leading to leukemia include the inheritance and replication of Akv, a nonleukemogenic, ecotropic murine leukemia virus (MuLV), and the sequential recombination between Akv and two other endogenous retrovirus sequences to produce leukemogenic mink cell focus-inducing (MCF) viruses (6,11,18,26). The first recombination event replaces the long terminal repeat (LTR) sequences in the ecotropic virus with those from the inducible xenotropic virus, Bxv-1, while the second recombination event replaces a portion of the ecotropic envelope (env) gene with polytropic env sequences (6,11,18,26). During the in vivo spread and replication of the recombinant virus containing both the LTR and env substitutions, sequences in the U3 region of the LTR are duplicated, creating the tandemly duplicated sequences commonly referred to as the directly repeated (DR) sequences (18,26). Both recombination events a...

Recombinant Origins of Pathogenic and Nonpathogenic Mouse Gammaretroviruses with Polytropic Host Range

“…Viral segments linked to pathogenicity: LTR. Previous studies had variously associated the oncogenic potential of MLVs with three different types of LTR variations: replacement of the Emv LTR with the Bxv1 LTR (25,26), duplication of the U3 LTR enhancer (11,59,60), and specific mutations in the transcription factor binding sites in the enhancer (61)(62)(63)(64). These changes are all found in our six lymphomagenic P-MLVs, and none are in the six nonlymphomagenic P-MLVs, but no single change characterizes all six pathogenic viruses ( Table 4).…”

The expressions of β-catenin, c-myc and cyclin D1 in thymoma induced by ionizing radiation

“…Under normal circumstances, c-myc gene does not show transcriptional activity or low levels of expression, while the gene is activated oncogene, the expression appears high levels, which makes the cell growth break away from the normal regulation, causes a high proliferation and malignant transformation. As the role of c-myc in the devepment thymoma, researchers did a lot of experiment and thought the increased expression promotes cell proliferation [9], ectopic cmyc gene expression plays an important role in the occurrence of thymoma induced MCF virus [10], c-myc genetic abnormalities caused by promoter demethylation leads to the occurrence of thymoma induced ionizing radiation [11]. In this study, the positive expression of c-myc showed in the nucleus and the cytoplasm in precancerous lesions 3 months after irradiation, it expressed significantly in lymphoma, which indicated that c-myc had been activated and showed high expression in the early stage of thymoma.…”