Deletional analysis within the long terminal repeat (LTR) of Moloney murine leukemia virus (M-MuLV) was performed. By molecular cloning, deletions were made in the vicinity of the XbaI site at -150 base pairs (bp) in the U3 region, between the tandemly repeated enhancers and the TATA box. The effects of the deletions on LTR function were measured in two ways. First, deleted LTRs were fused to the bacterial chloramphenicol acetyltransferase gene and used in transient expression assays. Second, infectious M-MuLVs were generated by transfection of M-MuLV proviruses containing the deleted LTRs, and the relative infectivity of the mutant viruses was assessed by XC-syncytial assay. Most of the deleted LTRs examined showed relatively high promoter activity in the transient chloramphenicol acetyltransferase assays, with values ranging from 20 to 50% of the wild-type M-MuLV LTR. Thus, the sequences between the enhancers and the TATA box were not absolutely required for transient expression. However, infectivity of viruses carrying the same deleted LTRs showed more pronounced effects. Deletion of sequences from -195 to -174 bp reduced infectivity 20-to 100-fold. Deletion of sequences within the region from -174 to -122 bp did not affect infectivity, indicating that this region is dispensable. On the other hand, deletion of sequences from -150 to -40 bp reduced infectivity from 5 to 6 logs, although the magnitude of the reduction partly may have reflected threshold envelope protein requirements for positive XC assays. The reduced infectivity did not appear to result from a failure of proviral DNA synthesis or integration by the mutant. Thus, the infectivity measurements identified three functional domains in the region between the enhancers and the TATA box.Retroviruses generate viral DNA with long terminal repeats (LTRs) during reverse transcription (reviewed in references 21 and 22). The LTRs are essential for viral replication and expression because they contain the signals for viral DNA integration, transcription initiation, and RNA cleavage and polyadenylation. In particular, the U3 portion of the LTR contains canonical eucaryotic promoter signals (TATA and CCAAT homologies at approximately -30 and -80 base pairs [bp], respectively) as well as transcriptional enhancers ( . approximately -340 to -180 bp) (10,20). As such, the retroviral LTR is an excellent model system for studying normal eucaryotic gene expression.In the experiments reported here, sequences in the U3 portion of the Moloney murine leukemia virus (M-MuLV) LTR necessary for LTR function were investigated by deletional analysis. We previously demonstrated the requirement for the enhancers (12), so emphasis was placed on the sequences between the enhancers and the TATA box. The activities of the deleted LTRs in transient expression assays were assessed, similar to experiments reported by others (8, 10). In addition, we extended the analyses by generating infectious M-MuLVs containing the deleted LTRs.

Cells of the hemopoietic system arise by proliferation and differentiation of progenitor cells. This process begins with multipotential stem ceUls which can self-renew and also undergo progressive differentiation to progenitor cells committed to particular lineages, ultimately yielding mature blood cells (D. Metcalf and M. A. S. Moore, Haematopoietic Cells, 1971). Early commitment of lymphoid progenitors is generally believed to separate the lymphoid lineage from the myeloid and erythroid lineages, whose progenitors are separated late in differentiation (Metcalf and Moore, 1971). We recently developed a derivative of Moloney murine leukemia virus (M-MuLV) in which the enhancer sequences from simian virus 40 were substituted into the M-MuLV long terminal repeat. This recombinant virus (AMo+SV M-MuLV) induces pre-B and B lymphoid leukemia with long latency after inoculation of 2-day-old NIH Swiss mice (R. Hanecak, P. K. Pattengale, and H. Fan, J. Virol. 62:2427Virol. 62: -2436Virol. 62: , 1988 (Fig. 1B) demonstrated lymphoblastic lymphoma with small-to-intermediate lymphoid cells with monomorphic nuclei, scant cytoplasm, and primitive, dispersed nuclear chromatin. Positive immunoperoxidase staining of tumor tissue sections with the monoclonal antibody RA3-6B2 specific for the B220 antigen (a B-celllineage-specific marker [14]) indicated that the lymphoid tumor was B cell derived (data not shown). In addition, molecular analyses of high-molecular-weight DNA prepared from the tumor showed both immunoglobulin heavy-and light-chain gene rearrangements (8).The AMo+SV M-MuLV-induced lymphoblastic lymphoma described above was established in culture by finely mincing the leukemic cervical lymph node and plating the tissue in Dulbecco modified Eagle medium supplemented with 15% fetal calf serum, 4.0 mM L-glutamine, nonessential amino acids (GIBCO), and 5 x 10' M 2-mercaptoethanol. induced lymphoma (Fig. 1B), the 85-2M cells exhibited a morphology typical of macrophages on cytocentrifuge preparations (Fig. 1A). The cells demonstrated conspicuous vacuolated cytoplasm with eccentric nuclei and were irregular in shape.The 85-2M cells were also tested for the presence of surface antigens found associated with lymphoid cells or myeloid cells. 85-2M cell suspensions (1.5 x 106 cells) were incubated separately at 4°C with antibodies directed against Thyl.2 or B220 (T-and B-cell surface markers, respectively [5,12]) and also with antibodies against Mac-i or Mac-2, surface markers characteristic of myeloid cells and not generally found associated with lymphoid cells (9, 16). Monoclonal antibodies directed against B220, Mac-i, and Mac-2 antigens were not directly conjugated to fluorescein isothiocyanate and therefore were indirectly labeled by a second reaction with goat anti-rat fluorescein isothiocyanate-conjugated second antibody. Unstained 85-2M cells and 85-2M cells incubated with the second antibody alone were used to control for nonspecific fluorescence. The results of flow cytometry are shown in Fig. 2. The 85-2M cells, whil...