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

Abstract

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.

cis-acting elements that mediate the negative regulation of Moloney murine leukemia virus in mouse early embryos

“…The main conclusion from these experiments is that the RBS/NRE, which has been shown to mediate the silencing of MoMuLV in undifferentiated EC cells (1,6,(17)(18)(19)(20)29), is also involved in the repression of MoMuLV template expression in early mouse embryos. Our observation that the B2 point mutation in the RBS/NRE relieves the block to expression from an active promoter in two-cell embryos as it does in EC cells strongly argues for identical mechanisms of repression in both cases.…”

“…A restriction on productive infection is also observed in undifferentiated murine embryonal carcinoma (EC) cells and in embryonal stem cell lines. Detailed studies have shown that the block to transcription from the MoMuLV long terminal repeat (LTR) in these cells is the effect of two independent mechanisms: (i) the inability of enhancer elements to activate transcription (16), imputed in part to the absence of the cognate transcription factors (24), and (ii) the presence of cisacting negatively regulating sequences (1,6,8,17,18,26). A point mutation in the 5Ј noncoding region, initially found in the B2 provirus, was shown to permit virus expression in F9 EC cells (1).…”

“…The core of this element overlaps the tRNA Pro primer binding site (PBS) for 17 of its 18 base pairs (15), but its role as a repressor is independent of its function in reverse transcription (9,21). Subsequent experiments indicated that this region is the site of binding on proviral DNA for a putative cellular repressor (6,(18)(19)(20)(21)29) named factor A by Petersen et al (20) and NRE binding factor by Loh et al (19). This factor is more abundant in EC cells than in differentiated cells, and mutations in the RBS/NRE abolish both the repressor function and the binding to the protein (19,30).…”

Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells

“…The NCR sequence has been shown to bind a cellular factor, thereby mediating transcriptional repression (13). Another well-defined inhibitory element is located at the primer binding site (PBS) of the MoMuLV leader region (12,24,39,42). The sequence from the MoMuLV PBS acts by binding a cellular factor which inhibits RNA transcription (22,25,30).…”

Feasibility of ex vivo gene therapy for neurological disorders using the new retroviral vector GCDNsap packaged in the vesicular stomatitis virus G protein

“…Especially, Moloney murine leukemia virus (MoMLV)-based retroviral vectors that have been most commonly used in gene therapy fields are very susceptible to de novo methylation in immature cells such as embryonic stem (ES) cells or embryonal carcinoma (EC) as well as somatic hematopoietic stem cells (Linney et al 1984;Tsukiyama et al 1989), resulting in shut off/silencing of the transgene expression in vivo. Intensive molecular analysis has revealed that the shut off/silencing of the transgene expression often observed in MoMLV-infected cells is attributed to the viral structure characterized by the longterminal repeat (LTR) with the negative control region (NCR) and the primer binding site that contain target sequences for the repressor binding protein and the negative transcriptional factors, respectively (Feuer et al 1989;Flanagan et al 1989). The presence of fetal calf serum (FSC) in the retroviral suspensions is also a critical problem because it prematurely induces the differentiation of neurospheres into neurons that have little, if any, proliferative activity.…”