Rearrangements and insertions in the Moloney murine leukemia virus long terminal repeat alter biological properties in vivo and in vitro

Self Cite

Transient expression assays were used to investigate the restriction of Moloney murine leukemia virus (MoMuLV) expression in undifferentiated mouse F9 embryonal carcinoma (EC) cells. We previously reported that the MoMuLV long terminal repeat (LTR) is inactive in undifferentiated F9EC cells due to inactivity of the tandemly repeated MoMuLV transcriptional enhancers. Others suggested that the inactivity was due to the presence of negative regulatory elements that interact with the MoMuLV tandem repeats. Two heterologous enhancer sequences that are active in undifferentiated F9 EC cells were inserted into the MoMuLV LTR: the B enhancers from the F101 variant of polyomavirus and a cellular enhancer sequence isolated from EC cells that we previously identified. The chimeric LTRs were then fused to the bacterial chloramphenicol acetyltransferase gene and tested for expression by transfection into F9 EC or NIH 3T3 cells. Insertion of these enhancers either upstream or downstream of the MoMuLV tandem repeats resulted in transcriptionally active LTRs in undifferentiated EC cells, which did not support the existence of negative regulatory elements interacting with the tandem repeats. In our previous MoMuLV enhancer deletion constructs, the GC-rich sequences downstream from the tandem repeats were also deleted, which might have contributed to the inactivity in EC cells. However, restoration of the GC-rich sequences did not yield an active LTR. The experiments also suggested that the EC cellular enhancer was preferentially active in undifferentiated EC cells and inactive in NIH 3T3 cells. The possibility of negative regulatory sequences in the vicinity of the MoMuLV primer-binding site was tested by inserting MoMuLV sequences from +30 to +419 base pairs into the LTR-chloramphenicol acetyltransferase gene constructs downstream of the transcriptional start site. Transient expression assays confirmed that these sequences reduced expression from functional LTRs in undifferentiated F9 EC cells but reduced expression significantly less in NIH 3T3 cells. Moreover, equivalent sequences from myeloproliferative sarcoma virus did not exhibit this effect. These results supported restriction of MoMuLV expression in undifferentiated F9 EC cells at two levels, inactivity of the MoMuLV enhancers and interaction of negative regulatory factors in the vicinity of the primer-binding site.

Section: Resultsmentioning

confidence: 99%

“…Molecular cloning procedures were performed by standard protocols (26). Construction of LTR-chloramphenicol acetyltransferase (CAT) gene fusion plasmids has been described previously (6,14,23). Isolation of the F9 EC cellular enhancer element has also been described previously (32).…”

Section: Methodsmentioning

confidence: 99%

Two blocks in Moloney murine leukemia virus expression in undifferentiated F9 embryonal carcinoma cells as determined by transient expression assays

Feuer

Taketo

Hanecak

et al. 1989

Self Cite

“…Detection of proviral DNA in tumors and molecular characterization. Genomic DNAs were harvested from the spleen, thymus, and lymph nodes of moribund leukemic mice as previously described (19). Restriction endonuclease digestion of genomic DNA (5 g), agarose gel electrophoresis (0.8% agarose), and transfer to GeneScreen Plus (New England Nuclear) were performed as described previously (19).…”

Section: Construction Of Recombinant Provirus and Production Of An Inmentioning

confidence: 99%

“…Genomic DNAs were harvested from the spleen, thymus, and lymph nodes of moribund leukemic mice as previously described (19). Restriction endonuclease digestion of genomic DNA (5 g), agarose gel electrophoresis (0.8% agarose), and transfer to GeneScreen Plus (New England Nuclear) were performed as described previously (19). Hybridization probes included the random primed SRS enhancer PCR fragment for the detection of input ⌬MoϩSRS ϩ and ⌬MoϩSRS Ϫ M-MuLVs, as well as DNA fragments containing c-myc, Pim-1, Pvt-1, T-cell receptor beta (TCR-␤), immunoglobulin heavy-chain (IgH), and Ig gene sequences for the molecular characterization of each tumor type as described previously (18).…”

Section: Construction Of Recombinant Provirus and Production Of An Inmentioning

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.

“…We previously studied the role of enhancer sequences in gene expression and pathogenesis for M-MuLV. By molecular cloning, substitutions and insertions with heterologous viral enhancers resulted in chimeric LTRs that showed altered transcriptional properties in transient expression assays (11,13,18,36). Infectious M-MuLVs containing these altered LTRs also showed altered biological properties in tissue culture and in inoculated mice (10,17,18).…”

mentioning

confidence: 99%

Substitution of murine transthyretin (prealbumin) regulatory sequences into the Moloney murine leukemia virus long terminal repeat yields infectious virus with altered biological properties

Feuer

Fan

1990

Self Cite

The effects of inserting cellular regulatory sequences from the murine transthyretin (TTR) gene into the Moloney murine leukemia virus (M-MuLV) long terminal repeat (LTR) were investigated. Transthyretin is expressed predominantly in the liver and choroid plexus in adult mice, and TTR upstream regulatory elements were previously shown to potentiate transcription in liver-derived cells. The effects of inserting the TTR distal enhancer and/or promoter-proximal sequences into an M-MuLV LTR lacking its enhancers were measured in three ways. (i) Chimeric LTRs were fused to the bacterial chloramphenicol acetyltransferase gene (cat) and tested for transient gene expression by transfection into liver-derived cells or NIH 3T3 fibroblasts. (ii) Infectious M-MuLV containing an altered LTR [AMo+TTR(PD) MuLVI was generated, and infectivity in culture on hepatocyte lines and NIH 3T3 cells was tested. (iii) Infection of AMo+TTR(PD) MuLV in vivo was tested by inoculating NFS/N mice and performing in situ hybridization of whole animal sections. Chimeric LTR-cat constructs showed higher levels of cat gene expression in liver-derived cell lines than in NIH 3T3 cells, indicating increased LTR activity in these cells. However, in vitro infection did not show significantly higher infectivity in hepatocytes for AMo+TTR(PD) M-MuLV than did wild-type M-MuLV. In vivo, AMo+TTR(PD) MuLV showed expression in the same tissues as with wild-type M-MuLV-inoculated mice, i.e., lymphoid organs and the intestines and, additionally, two novel sites not seen in wild-type M-MuLV-inoculated animals. Of 10 mice, 8 showed viral expression in the brain and 3 showed expression in the liver. Thus, insertion of TTR elements into the M-MuLV LTR altered LTR activity both in vitro and in vivo.