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
