Rearrangements and insertions in the Moloney murine leukemia virus long terminal repeat alter biological properties in vivo and in vitro.

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 lymphoblastic 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 PyF101 sequences were inserted between the M-MuLV promoter and the M-MuLV enhancers (at -150 bp). A new LTR was generated in which the PyF101 sequences were inserted to the 5' side of the M-MuLV enhancers (at -353 bp, PyF101 + Mo M-MuLV). The PyF101 + 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 PyF101 sequences relative to the M-MuLV LTR affected the biological behavior of the molecular construct. Furthermore, PyF101 + 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 PyF101 + Mo- and Mo- M-MuLV-inoculated animals contained recombinant proviruses with LTRs that differed from the initially inoculated virus.     

Recombinant mink cell focus-inducing virus and long terminal repeat alterations accompany the increased leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus after intraperitoneal inoculation.

We recently showed that different routes of inoculation affect the leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV). Intraperitoneal (i.p.) inoculation of neonatal mice with Mo+PyF101 M-MuLV greatly enhanced its leukemogenicity compared with subcutaneous (s.c.) inoculation. We previously also suggested that the leukemogenicity defect of Mo+PyF101 M-MuLV when inoculated s.c. may result from the inability of this virus to form env gene recombinant (mink cell focus-inducing [MCF]) virus. In this study, virus present in end-stage tumors and in preleukemic animals inoculated i.p. by Mo+PyF101 M-MuLV was characterized. In contrast to s.c. inoculation, all tumors from i.p.-inoculated mice contained high levels of recombinant MCF virus. Furthermore, Southern blot analyses demonstrated that the majority of the tumors contained altered Mo+PyF101 M-MuLV long terminal repeats. The U3 regions from several tumors with altered long terminal repeats were cloned by PCR amplification. Sequence analyses indicated that the M-MuLV 75-bp tandem repeat in the enhancer region was triplicated. This amplification was also previously observed in mice infected s.c. with a pseudotypic mixture of Mo+PyF101 M-MuLV and Mo+PyF101 MCF virus. The enhancer triplication was an early event, and it occurred within 2 weeks postinfection. Recombinant MCF viruses were not detected by Southern blot analyses until 4 weeks postinfection. Thus, the M-MuLV enhancer triplication event was initially important for efficient propagation of ecotropic Mo+PyF101 M-MuLV. The increased leukemogenicity following i.p. inoculation could be explained if the triplication enhances Mo+PyF101 M-MuLV replication in the bone marrow and bone marrow infection is required for recombinant MCF virus formation.     

Generation and characterization of a recombinant Moloney murine leukemia virus containing the v-myc oncogene of avian MC29 virus: in vitro transformation and in vivo pathogenesis.

A new retrovirus consisting of the v-myc oncogene sequences of avian MC29 virus inserted into the genome of Moloney murine leukemia virus (M-MuLV) was generated. This was accomplished by constructing a recombinant DNA clone containing the desired organization, introducing the recombinant DNA into mouse NIH 3T3 cells, and superinfecting the cells with replication-competent M-MuLV. The construction was designed so that an M-MuLV gag-myc fusion protein would be produced. The resulting virus, M-MuLV(myc), morphologically transformed uninfected NIH 3T3 cells. Stocks of M-MuLV(myc)-M-MuLV were infected into secondary mouse embryo cultures. M-MuLV(myc) induced striking growth and proliferation of hematopoietic cells. These cells were of the myeloid lineage by morphology, phagocytic properties, and surface staining with Mac-1 and Mac-2 monoclonal antibodies. They resembled mature macrophages, although they displayed minor properties of immaturity. The myeloid cells were transformed in comparison with uninfected myeloid cells since they were less adherent and had unlimited proliferative capacity and reduced growth factor requirements. The transformed myeloid cells with proliferative potential were actually myeloid progenitors which apparently underwent terminal differentiation to macrophages. It was possible to derive a permanent line of factor-independent macrophages from M-MuLV(myc)-transformed myeloid cells. M-MuLV(myc) also immortalized and morphologically transformed mouse embryo fibroblasts. These in vitro properties closely resembled the biological activity of MC29 virus in avian cells and suggested that the nature of the v-myc oncogene was an important determinant in transformation specificity. Neonatal NIH Swiss mice inoculated intraperitoneally with M-MuLV(myc)-M-MuLV only developed lymphoblastic lymphoma characteristic of the M-MuLV helper alone, and no acute fibrosarcomas or myeloid tumors resulted. In light of the strong myeloid transformation observed in vitro, the absence of acute in vivo myeloid disease was noteworthy. Interestingly, when a derivative of M-MuLV(myc) carried by a nonpathogenic amphotropic MuLV helper was inoculated, T lymphomas developed with long latency. Molecular hybridization confirmed that these tumors contained M-MuLV(myc).     

The leukemogenic potential of an enhancer variant of Moloney murine leukemia virus varies with the route of inoculation.

We previously showed that the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV) is poorly leukemogenic when inoculated subcutaneously (s.c.) into neonatal mice. We recently found that intraperitoneal (i.p.) inoculation of neonatal mice with the same virus significantly enhanced its leukemogenicity. In this study, infections of neonatal mice by the two different routes of inoculation were compared. We studied replication of the virus in vivo to identify critical preleukemic events. These would be observed in mice inoculated i.p. by Mo+PyF101 M-MuLV but not when inoculation was s.c. Infectious center assays indicated that regardless of the route of inoculation, Mo+PyF101 M-MuLV showed delayed infection of the thymus compared with wild-type M-MuLV. On the other hand, i.p.-inoculated mice showed more rapid appearance of infectious centers in the bone marrow than did s.c.-inoculated animals. Thus, the enhanced leukemogenicity of i.p. inoculation correlated with efficient early infection of the bone marrow and not with early infection of the thymus. These results suggest a role for bone marrow infection for efficient leukemogenesis in Mo+PyF101 M-MuLV-infected mice. Consistent with this notion, if bone marrow infection was decreased by injecting 10- to 12-day-old animals i.p., leukemogenicity resembled that of s.c. inoculation. Thus, two cell types that are critical for the induction of efficient leukemia were implicated. One cell delivers virus from the site of s.c. inoculation (the skin) to the bone marrow and is apparently restricted for Mo+PyF101 M-MuLV replication. The second cell is in the bone marrow, and its early infection is required for efficient leukemogenesis.     

Preleukemic hematopoietic hyperplasia induced by Moloney murine leukemia virus is an indirect consequence of viral infection.

We previously showed that neonatal mice inoculated with Moloney murine leukemia virus (M-MuLV) exhibit a preleukemic state characterized by splenomegaly and increased numbers of hematopoietic progenitors. An M-MuLV variant with greatly reduced leukemogenic potential, Mo+PyF101 M-MuLV, does not generally induce this preleukemic state. In order to investigate the mechanism involved in M-MuLV induction of preleukemic hyperplasia, we tested the CFU-mixed myeloid and erythroid (CFUmix) from M-MuLV- and Mo+PyF101 M-MuLV-inoculated mice for the presence of virus by antibody staining and for the release of infectious virus. The majority of CFUmix colonies from both M-MuLV- and Mo+PyF101 M-MuLV-inoculated mice contained infectious virus even though M-MuLV-inoculated mice showed elevated levels of CFUmix while the Mo+PyF101 M-MuLV-inoculated mice did not. This indicates that direct infection of hematopoietic progenitors was not sufficient to induce hyperplasia. Rather, hematopoietic hyperplasia may result indirectly from infection of some other cell type.     

Appearance of Mink Cell Focus-Inducing Recombinants during In Vivo Infection by Moloney Murine Leukemia Virus (M-MuLV) or the Mo+PyF101 M-MuLV Enhancer Variant: Implications for Sites of Generation and Roles in Leukemogenesis

One hallmark of murine leukemia virus (MuLV) leukemogenesis in mice is the appearance of env gene recombinants known as mink cell focus-inducing (MCF) viruses. The site(s) of MCF recombinant generation in the animal during Moloney MuLV (M-MuLV) infection is unknown, and the exact roles of MCF viruses in disease induction remain unclear. Previous comparative studies between M-MuLV and an enhancer variant, Mo+PyF101 MuLV, suggested that MCF generation or early propagation might take place in the bone marrow under conditions of efficient leukemogenesis. Moreover, M-MuLV induces disease efficiently following both intraperitoneal (i.p.) and subcutaneous (s.c.) inoculation but leukemogenicity by Mo+PyF101 M-MuLV is efficient following i.p. inoculation but attenuated upon s. c. inoculation. Time course studies of MCF recombinant appearance in the bone marrow, spleen, and thymus of wild-type and Mo+PyF101 M-MuLV i.p.- and s.c.-inoculated mice were carried out by performing focal immunofluorescence assays. Both the route of inoculation and the presence of the PyF101 enhancer sequences affected the patterns of MCF generation or early propagation. The bone marrow was a likely site of MCF recombinant generation and/or early propagation following i.p. inoculation of M-MuLV. On the other hand, when the same virus was inoculated s.c., the primary site of MCF generation appeared to be the thymus. Also, when Mo+PyF101 M-MuLV was inoculated i.p., MCF generation appeared to occur primarily in the thymus. The time course studies indicated that MCF recombinants are not involved in preleukemic changes such as splenic hyperplasia. On the other hand, MCFs were detected in tumors from Mo+PyF101 M-MuLV s. c.-inoculated mice even though they were largely undetectable at preleukemic times. These results support a role for MCF recombinants late in disease induction.     

Effects of nonleukemogenic and wild-type Moloney murine leukemia virus on lymphoid cells in vivo: identification of a preleukemic shift in thymocyte subpopulations.

Infection of mice with Moloney murine leukemia virus (M-MuLV) as well as with a nonpathogenic variant, Mo+PyF101 M-MuLV, was studied. Mo+PyF101 M-MuLV differs from wild-type M-MuLV by the addition of enhancer sequences from polyomavirus in the long terminal repeat. Previous experiments indicated that Mo+PyF101 establishes infection in animals, even though it does not induce disease. In vivo infection studies with particular attention to the thymus were performed, since the thymus is the target organ for M-MuLV leukemogenesis. Mice inoculated at birth with wild-type M-MuLV developed maximal levels of thymic infection by 2 to 3 weeks. Animals inoculated with Mo+PyF101 M-MuLV showed considerably less thymic infection at early times (2 to 4 weeks); nevertheless, by 5 to 6 weeks infection equivalent to wild-type M-MuLV-inoculated animals developed. Therefore the nonpathogenicity of Mo+PyF101 M-MuLV did not simply reflect a lack of thymotropism. Furthermore, thymic infection by itself may not be sufficient to induce leukemia. The relative deficit of Mo+PyF101 M-MuLV thymic infection at early versus late times did not reflect a change in the nature of the cells in the thymus, since in vitro infection of primary thymocytes from 2- and 6-week-old animals was equally efficient. One possible explanation is that infected thymocytes normally arise from progenitor cells which were infected in the bone marrow or spleen, and the cells restricted for Mo+PyF101 M-MuLV are located in those organs. Comparison of wild-type and Mo+PyF101 M-MuLV also allowed identification of important preleukemic changes in the thymus of wild-type M-MuLV-inoculated mice. Flow cytometry with monoclonal antibodies specific for thymocyte subpopulations was used. Staining of cells for Thy-1 or Thy-1.2 antigens indicated a shift toward low or negative cells. A concomitant increase in cells positive for antigen Pgp-1 was also observed. This is consistent with an increase in the relative frequency of immature blastlike cells. Importantly, thymuses from mice inoculated with Mo+PyF101 M-MuLV did not show these shifts in thymocyte subpopulations.     

Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment

All blood cell lineages derive from a common hematopoietic stem cell (HSC). The current model implicates that the first lineage commitment step of adult pluripotent HSCs results in a strict separation into common lymphoid and common myeloid precursors. We present evidence for a population of cells which, although sustaining a high proliferative and combined lympho-myeloid differentiation potential, have lost the ability to adopt erythroid and megakaryocyte lineage fates. Cells in the Lin-Sca-1+c-kit+ HSC compartment coexpressing high levels of the tyrosine kinase receptor Flt3 sustain granulocyte, monocyte, and B and T cell potentials but in contrast to Lin-Sca-1+c-kit+Flt3- HSCs fail to produce significant erythroid and megakaryocytic progeny. This distinct lineage restriction site is accompanied by downregulation of genes for regulators of erythroid and megakaryocyte development. In agreement with representing a lymphoid primed progenitor, Lin-Sca-1+c-kit+CD34+Flt3+ cells display upregulated IL-7 receptor gene expression. Based on these observations, we propose a revised road map for adult blood lineage development.     

Deletion of a GC-rich region flanking the enhancer element within the long terminal repeat sequences alters the disease specificity of Moloney murine leukemia virus.

Moloney murine leukemia virus (M-MuLV) is a replication-competent retrovirus which induces T-lymphoblastic lymphoma 2 to 4 months after inoculation. Enhancer sequences in the U3 region of the M-MuLV long terminal repeat, primarily the 75-bp tandem repeats, strongly influence the disease specificity and latency of M-MuLV. We investigated the role of GC-rich sequences downstream of the tandem repeats in the disease specificity of M-MuLV. A recombinant M-MuLV lacking 23 bases of a GC-rich sequence (-174 to -151), Delta 27A M-MuLV, was tested for pathogenesis in neonatal NIH Swiss mice. Delta 27A M-MuLV induced disease with a longer latency than did M-MuLV (7 versus 3 months) in greater than 85% of inoculated mice. More interestingly, this virus showed an expanded repertoire of hematopoietic diseases. Molecular analyses and histopathologic examinations indicated that while 39% of mice inoculated with Delta 27A M-MuLV developed T-cell lymphoblastic lymphoma typical of wild-type M-MuLV, the majority developed acute myeloid leukemia, erythroleukemia, or B-cell lymphoma. Viral DNA corresponding to Delta 27A M-MuLV was detectable in most of the tumors analyzed. These findings indicate that the GC-rich region significantly influences the disease specificity and latency of M-MuLV.     

Moloney Murine Leukemia Virus Infects Cells of the Developing Hair Follicle after Neonatal Subcutaneous Inoculation in Mice

The nature of Moloney murine leukemia virus (M-MuLV) infection after a subcutaneous (s.c.) inoculation was studied. We have previously shown that an enhancer variant of M-MuLV, Mo+PyF101 M-MuLV, is poorly leukemogenic when used to inoculate mice s.c., but not when inoculated intraperitoneally. This attenuation of leukemogenesis correlated with an inability of Mo+PyF101 M-MuLV to establish infection in the bone marrow of mice at early times postinfection. These results suggested that a cell type(s) is infected in the skin by wild-type but not Mo+PyF101 M-MuLV after s.c. inoculation and that this infection is important for the delivery of infection to the bone marrow, as well as for efficient leukemogenesis. To determine the nature of the cell types infected by M-MuLV and Mo+PyF101 M-MuLV in the skin after a s.c. inoculation, immunohistochemistry with an anti-M-MuLV CA antibody was performed. Cells of developing hair follicles, specifically cells of the outer root sheath (ORS), were extensively infected by M-MuLV after s.c. inoculation. The Mo+PyF101 M-MuLV variant also infected cells of the ORS but the level of infection was lower. By Western blot analysis, the level of infection in skin by Mo+PyF101 M-MuLV was approximately 4- to 10-fold less than that of wild-type M-MuLV. Similar results were seen when a mouse keratinocyte line was infected in vitro with both viruses. Cells of the ORS are a primary target of infection in vivo, since a replication defective M-MuLV-based vector expressing β-galactosidase also infected these cells after a s.c. inoculation.     

Microtubules control nuclear shape and gene expression during early stages of hematopoietic differentiation

Hematopoietic stem and progenitor cells (HSPC) can differentiate into all hematopoietic lineages to support hematopoiesis. Cells from the myeloid and lymphoid lineages fulfill distinct functions with specific shapes and intra‐cellular architectures. The role of cytokines in the regulation of HSPC differentiation has been intensively studied but our understanding of the potential contribution of inner cell architecture is relatively poor. Here, we show that large invaginations are generated by microtubule constraints on the swelling nucleus of human HSPC during early commitment toward the myeloid lineage. These invaginations are associated with a local reduction of lamin B density, local loss of heterochromatin H3K9me3 and H3K27me3 marks, and changes in expression of specific hematopoietic genes. This establishes the role of microtubules in defining the unique lobulated nuclear shape observed in myeloid progenitor cells and suggests that this shape is important to establish the gene expression profile specific to this hematopoietic lineage. It opens new perspectives on the implications of microtubule‐generated forces, in the early commitment to the myeloid lineage.     

Lineage development of hematopoietic stem and progenitor cells

Hematopoietic stem cells have the potential to develop into multipotent and different lineage-restricted progenitor cells that subsequently generate all mature blood cell types. The classical model of hematopoietic lineage commitment proposes a first restriction point at which all multipotent hematopoietic progenitor cells become committed either to the lymphoid or to the myeloid development, respectively. Recently, this model has been challenged by the identification of murine as well as human hematopoietic progenitor cells with lymphoid differentiation capabilities that give rise to a restricted subset of the myeloid lineages. As the classical model does not include cells with such capacities, these findings suggest the existence of alternative developmental pathways that demand the existence of additional branches in the classical hematopoietic tree. Together with some phenotypic criteria that characterize different subsets of multipotent and lineage-restricted progenitor cells, we summarize these recent findings here.     

Spi-B can functionally replace PU.1 in myeloid but not lymphoid development

Mature macrophages, neutrophils and lymphoid cells do not develop in PU.1(-/-) mice. In contrast, mice lacking the highly related protein Spi-B generate all hematopoietic lineages but display a B-cell receptor signaling defect. These distinct phenotypes could result from functional differences between PU.1 and Spi-B or their unique temporal and tissue-specific expression (PU.1: myeloid and B cells; Spi-B: B cells only). To address this question, we introduced the Spi-B cDNA into the murine PU.1 locus by homologous recombination. In the absence of PU.1, Spi-B rescued macrophage and granulocyte development when assayed by in vitro differentiation of embryonic stem cells. Adherent, CD11b(+)/F4/80(+) cells capable of phagocytosis were detected in PU.1(Spi-B/Spi-B) embryoid bodies, and myeloid colonies were present in hematopoietic progenitor assays. Despite its ability to rescue myeloid differentiation, Spi-B did not rescue lymphoid development in a RAG-2(-/-) complementation assay. These results demonstrate an important difference between PU.1 and Spi-B. Careful comparison of these Ets factors will delineate important functional domains of PU.1 involved in lymphocyte lineage commitment and/or maturation.     

Escape from in vivo restriction of Moloney mink cell focus-inducing viruses driven by the Mo+PyF101 long terminal repeat (LTR) by LTR alterations.

Mo+PyF101 M-MuLV is a variant Moloney murine leukemia virus containing polyomavirus F101 enhancers inserted just downstream from the M-MuLV enhancers in the long terminal repeat (LTR). The protein coding sequences for this virus are identical to those of M-MuLV. Mo+PyF101 M-MuLV induces T-cell disease with a much lower incidence and longer latency than wild-type M-MuLV. We have previously shown that Mo+PyF101 M-MuLV is defective in preleukemic events induced by wild-type M-MuLV, including splenic hematopoietic hyperplasia, bone marrow depletion, and generation of recombinant mink cell focus-inducing viruses (MCFs). We also showed that an M-MCF virus driven by the Mo+PyF101 LTR is infectious in vitro but does not propagate in mice. However, in these experiments, when a pseudotypic mixture of Mo+PyF101 M-MuLV and Mo+PyF101 MCF was inoculated into newborn NIH Swiss mice, they died of T-cell leukemia at times almost equivalent to those induced by wild-type M-MuLV. Tumor DNAs from Mo+PyF101 M-MuLV-Mo+PyF101 MCF-inoculated mice were examined by Southern blot analysis. The predominant forms of Mo+PyF101 MCF proviruses in these tumors contained added sequences in the U3 region of the LTR. The U3 regions of representative tumor-derived variant Mo+PyF101 MCFs were cloned by polymerase chain reaction amplification, and sequencing indicated that they had acquired an additional copy of the M-MuLV 75-bp tandem repeat in the enhancer region. NIH 3T3 cell lines infected with altered viruses were obtained from representative Mo+PyF101 M-MuLV-Mo+PyF101 MCF-induced tumors, and mice were inoculated with the recovered viruses. Leukemogenicity was approximately equivalent to that in the original Mo+PyF101 M-MuLV-Mo+PyF101 MCF viral stock. Southern blot analysis on the resulting tumors now predominantly revealed loss of the polyomavirus sequences. These results suggest that the suppressive effects of the PyF101 sequences on M-MuLV-induced disease and potentially on MCF propagation were overcome in two ways: by triplication of the M-MuLV direct repeats and by loss of the polyomavirus sequences.     

Myeloid cell transformation by ras-containing murine sarcoma viruses.

BALB and Harvey murine sarcoma viruses contain ras transforming genes capable of altering the proliferation and differentiation of cells within the erythroid and lymphoid lineages (W. D. Hankins and E. M. Scolnick, Cell 26:91-97, 1981; J. H. Pierce and S. A. Aaronson, J. Exp. Med. 156:873-887, 1982; E. M. Scolnick et al., Mol. Cell. Biol. 1:68-74). The present studies demonstrate hematopoietic targets of ras-containing viruses within the myeloid lineage. Diffuse colonies were induced by BALB or Harvey marine sarcoma virus infection of murine bone marrow cells. Generally, these colonies were made up of relatively mature macrophages which exhibited increased self-renewal capacity but eventually underwent terminal differentiation in culture. Cells from one BALB murine sarcoma virus-induced colony displayed phenotypic markers of more immature myelomonocytic cells. This colony, designated BAMC1, readily established as a continuous cell line and was highly malignant in vivo. Exposure of these cells to 12-O-tetradecanoylphorbol-13-acetate led to the induction of a more mature myeloid phenotype, which was associated with decreased growth potential in vitro and in vivo. The effects of the inducing agent were not mediated by an alteration in the level of expression of the ras-coded p21 transforming protein. Our present findings extend the spectrum of targets whose growth is altered by ras-containing retroviruses to cells at several stages of differentiation within each of the major hematopoietic lineages.     

Generation of glucocorticoid-responsive Moloney murine leukemia virus by insertion of regulatory sequences from murine mammary tumor virus into the long terminal repeat.

The glucocorticoid-regulatory sequences from the murine mammary tumor virus long terminal repeat (MMTV LTR) were introduced into the LTR of Moloney murine leukemia virus (M-MuLV) by recombinant DNA techniques. The site of insertion was in the M-MuLV LTR U3 region at -150 base pairs with respect to the RNA cap site. Infectious M-MuLVs carrying the altered LTRs (Mo + MMTV M-MuLVs) were recovered by transfection of proviral clones into NIH-3T3 cells. The Mo + MMTV M-MuLVs were hormonally responsive in that infection was 3 logs more efficient when performed in the presence of dexamethasone, irrespective of the orientation of the inserted MMTV sequences. However, even in the presence of hormone, the Mo + MMTV M-MuLVs were less infectious than wild-type M-MuLV. In contrast to the large effect on infectivity, dexamethasone induced virus-specific RNA levels in chronically Mo + MMTV M-MuLV-infected cells only two- to fourfold. Fusion plasmids between the altered LTRs and the bacterial chloramphenicol acetyltransferase gene allowed the investigation of LTR promoter strength by the transient chloramphenicol acetyltransferase expression assay. The chloramphenicol acetyltransferase assays indicated that the insertion of MMTV sequences into the M-MuLV LTR reduced promoter activity in the absence of glucocorticoids but that promoter activity could be induced two- to fivefold by dexamethasone. The Mo + MMTV M-MuLVs were also tested for the possibility that viral DNA synthesis or integration during initial infection was enhanced by dexamethasone. However, no significant difference was detected between cultures infected in the presence or absence of hormone. The insertion of MMTV sequences into an M-MuLV LTR deleted of its enhancer sequences did not yield infectious virus or active promoters, even in the presence of dexamethasone.