Moloney murine leukaemia virus (M-MuLV) infection of embryonal carcinoma (EC) cells results in the integration of proviral DNA into the host cell genome, but not in virus production. One suggested explanation for the lack of viral gene expression in EC cells has been methylation of the integrated viral DNA. However, subsequent reports indicated that integration of the M-MuLV DNA occurs soon after infection, but that viral DNA methylation occurs considerably later. Nevertheless, viral gene expression is not observed even at early times. One possible explanation is that certain M-MuLV regulatory sequences do not function in EC cells. We now present evidence which supports this hypothesis.

Retroviruses contain three genes for replication: gag, pol, and env, which encode polyprotein precursors for the internal capsid proteins, reverse transcriptase, and envelope glycoprotein, respectively (1). Murine leukemia virus (MuLV) differs from most other retroviruses in that it encodes two different pathways for gag gene expression (2). These two pathways begin with two independent translation products, gPr80gag and Pr65ag. Pr65gag is processed by proteolytic cleavage to yield the internal capsid proteins of the virus particle and is analogous to the gag polyprotein precursors of other retroviruses (3-5). gPr8Ogg contains the amino acid sequences of Pr65gag as well as 4-6 kilodaltons (kDa) of amino-terminal protein (6, 7). The additional amino-terminal peptides result in glycosylation of gPr8O'ag during translation (8, 9). gPr8 gag is processed by the further addition of carbohydrate and exported to the cell surface where it appears as a glycoprotein of 95 kDa (8-11). It may be also be released into the extracellular medium as cleavage products of 55 and 40 kDa (8,12). Glycosylated gag products are not incorporated into MuLV virions, but they associate with the extracellular matrix (13).While the function of Pr65gag is known, the role of gPr809ae in MuLV infection is not known. Glycosylated gag might provide some function required for viral replication, or it might play an accessory role. We previously isolated mutants of Moloney MuLV (M-MuLV)-infected mouse fibroblasts that did not express gag proteins at the cell surface, and they were deficient for virus production. However, these mutants were cellular, not viral, in nature and they produced normal amounts of gPr8g'ag intracellularly (14). To obtain a more definitive answer, we constructed two mutants of M-MuLV at the recombinant DNA level and recovered virus by transfection. The constructions. and characterizations of the mutant viruses are described here.MATERIALS AND METHODS Cells and Viruses. All cells were grown in Dulbecco-modified Eagle's medium/10% calf serum; Mouse NIH-3T3 cells were described previously (15), as were M-MuLV-infected NIH-3T3 cells (clone A9) (16).The UV-XC assay for MuLV was carried out as described (17). Assay of viral reverse transcriptase by the addition of exogenous poly(rA):oligo(dT) template-primer (16) and banding of virus in sucrose density gradients (18) was carried out as described.Recombinant DNA Cloning. A phage recombinant DNA clones (A-MLV clones 48, 61, and 63) carrying integrated copies of M-MuLV provirus were described previously (19). pMSV-1 is a plasmid clone of unintegrated Moloney murine sarcoma virus (M-MSV) DNA permuted about the unique HindIII site (20). pSLT is a subclone of pMSV-1 deleted from the Sal I site in M-MSV DNA to the Sal I site in the pBR322.vector (see Fig. 2) and was kindly provided by Inder Verma. P90(Abl), a plasmid clone of unintegrated Abelson MuLV (Ab-MuLV) (P90 strain) DNA, was kindly provided by Owen Witte. Plasmid vector pBR328 has been described by Soberon et al. (21).Restriction ...

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The effects of rearrangement and insertion of sequences in the Moloney murine leukemia virus (M-MuLV) long terminal repeat (LTR) were investigated. The alterations were made by recombinant DNA manipulations on a plasmid subclone containing an M-MuLV LTR. Promoter activity of altered LTRs was measured by fusion to the bacterial chloramphenicol acetyltransferase gene, followed by transient expression assay in NIH 3T3 cells. M-MuLV proviral organizations containing the altered LTRs were also generated, and infectious virus was recovered by transfection. Infectivity of the resulting virus was quantified by XC plaque assay, and pathogenicity was determined by inoculating neonatal NIH Swiss mice. Inversion of sequences in the U3 region containing the tandemly repeated enhancer sequences (-150 to-353 base pairs [bp]) reduced promoter activity approximately fivefold in the transient-expression assays. Infectious virus containing the inverted sequences (Mo-M-MuLV) showed a 20-fold reduction in relative infectivity compared with wild-type M-MuLV, but the virus still induced thymus-derived Iymphoblastic lymphoma or leukemia in mice, with essentially the same kinetics as for wild-type M-MuLV. We previously derived an M-MuLV which carried inserted enhancer sequences from the F101 strain of polyomavirus (Mo+PyF101 M-MuLV) and showed that this virus is nonleukemogenic. In Mo+PyF101 M-MuLV, the PyFlOl sequences were inserted between the M-MuLV promoter and the M-MuLV enhancers (at-150 bp). A new LTR was generated in which the PyFlOl sequences were inserted to the 5' side of the M-MuLV enhancers (at-353 bp, PyF1O1+Mo M-MuLV). The PyFl1l+Mo LTR exhibited promoter activity similar (40 to 50%) to that of wild-type M-MuLV, and infectious PyF101+Mo M-MuLV had high infectivity on NIH 3T3 cells (50% of wild type). In contrast to the nonleukemogenic Mo+PyF101 M-MuLV, PyF101+Mo M-MuLV induced leukemia with kinetics similar to that of wild-type M-MuLV. Thus, the position of the PyFlOl sequences relative to the M-MuLV LTR affected the biological behavior of the molecular construct. Furthermore, PyFlOI+Mo M-MuLV induced a different spectrum of neoplastic disease. In comparison with wild-type M-MuLV, which induces a characteristic thymus-derived lymphoblastic lymphoma with extremely high frequency, PyF101+Mo M-MuLV was capable of inducing both acute myeloid leukemia or thymus-derived lymphoblastic lymphoma, or both. Tumor DNA from both the PyFlOl+Moand MoM -MuLV-inoculated animals contained recombinant proviruses with LTRs that differed from the initially inoculated virus. Retroviruses synthesize viral DNA via reverse transcriptase during infection (40, 41). A linear double-stranded viral DNA copy of the virion RNA is made which contains terminally repeated sequences, the long terminal repeats (LTRs). Viral DNA integration into the host chromosomal DNA yields the provirus, and the LTRs provide the regulatory signals for initiation and termination of viral transcription. These signals, located in the U3 region of the LTR, include promoter elem...