Murine leukemia virus long terminal repeat sequences can enhance gene activity in a cell-type-specific manner.

Abstract

We tested the ability of sequences in the long terminal repeat (LTR) of a mink cell focus-forming (MCF) murine leukemia virus to function as an enhancer in a cell-type-specific manner. In a stable transformation assay, the MCF or Akv LTR and the simian virus 40 enhancer had similar activities in murine fibroblasts. In contrast, the MCF LTR had a significantly greater activity in murine T lymphoid cells than did either the simian virus 40 enhancer or the Akv LTR.One of the regulatory signals for transcription of eucaryotic genes is cis-acting enhancer elements that can augment the transcriptional activity of different genes (for review, see references 14 and 18). Enhancer elements were first identified in the simian virus 40 (SV40) genome but have subsequently been found in other viruses, including retroviruses (17,23,24), and have recently been identified for cellular genes (1,13,29). Unique characteristics of these enhancer elements include their ability to function in an orientationindependent manner and over relatively long distances (2,12,25). Enhancer activity can also occur in a tissue-or species-specific manner (10, 21). Enhancers have been identified in the U3 region of the long terminal repeats (LTRs) of retroviruses (17,19,20). More recently, the LTRs of murine leukemia viruses (MLVs) have been implicated in the specific type of neoplasia caused by these viruses (5, 9, 22, 28), and it is possible that the sequences resembling enhancers are responsible for this specificity (4).We have examined the LTR of a mink cell focus-forming (MCF) MLV ) to study the role that these terminal repeated sequences play in the specificity of tumor formation by MCF viruses. Lymphomagenic MCF MLVs have been implicated in the development of thymic lymphomas in strain AKR mice because of their ability to accelerate the onset of tumorigenesis (6,27 (Fig. 1, in brackets) in the U3 region. From a comparison of the nucleotide sequence of the LTRs of MCF-13 and Akv (Fig. 1), it seems clear that the greatest differences are present in the U3 region. This is a region in the LTR where duplicated sequences resembling enhancer elements have been identified for other retroviruses, and these sequences also appear to be present in the case of MCF-13 and Akv (Fig. 1). We wished to determine whether the repeated sequences for MCF-13 and Akv could also * Corresponding author. function as enhancer elements and whether this activity could occur in a cell-type-specific manner. Tissue-specific differences in the activities of these enhancerlike elements could help to account for the different pathogenic properties of these two viruses.To test this idea, we compared the LTRs of MCF-13 and Akv, along with the prototypical SV40 enhancer, in plasmid constructs containing each enhancer and the selectable gene conferring neomycin resistance (Neor) in murine cells in culture. A clone generated by Southern and Berg (33) which contains the SV40 origin of replication was used to test the SV40 enhancer (Fig. 2). The SV40 sequences that are present in thi...

In vitro transfection of fresh thymocytes and T cells shows subset-specific expression of viral promoters.

“…Elucidation of such regulatory interactions will be needed to explain the broad range of T-cell types in which these LTRs are active. In contrast to our earlier observation that the MCF LTR is transcriptionally more active than the Akv LTR in T-cell lines (62), both LTRs were equally active in PNA+ thymocytes. It is thought that the MCF but not the Akv virus infects and transforms a cycling subset of thymic cortical cells, i.e., the cortical blast cell (12,43).…”

Different activities of viral enhancer elements before and after stable integration of transfected DNAs.

“…1) resulted in an RNA-to-DNA ratio which was 6-to 13-fold greater than the ratios of the other enhancer-containing plasmids. These results corroborated our previous observations with the same clones for which we used a stable transformation assay instead (19). Transient assays of RNA levels have also been successfully used by other laboratories to demonstrate the cell-typespecific activity of other enhancer elements (1)(2)(3)5).…”

“…A parental virus of the MCF MLVs, known as Akv MLV, does not possess this neoplastic property. By using a stable transformation assay dependent on the expression of a gene conferring neomycin resistance (Neo%, we previously demonstrated that the MCF-13 enhancer has a significantly greater activity than the Akv enhancer in murine T lymphoid cells (19). Although other cis-acting signals within the LTR may contribute to this activity, we have attributed this effect to enhancerlike sequences because we obtained similar results with the LTR in either orientation.…”

“…Enhancer activity in various tissues has been observed for sequences which reside in the long terminal repeats (LTRs) of a number of different murine leukemia viruses (MLVs). It is thought that the tissue-specific activities of MLV enhancer elements contribute to the specific tropism and transforming properties of these viruses (3,16,19).…”

Differential Cell Killing by Lymphomagenic Murine Leukemia Viruses Occurs Independently of p53 Activation and Mitochondrial Damage

“…Calculation of the copy number per cell of linear unintegrated viral DNA (UVD) was made by comparing band intensities measured from Southern blots using the ImageQuant software (Molecular Probes, Eugene, Oreg.) against a standard curve plotted from the intensities of known amounts of a purified plasmid DNA fragment comprising the complete MCF13 MLV genome (46). To obtain the copy number per cell, we divided the UVD value by the total cell number.…”