Duplication of U3 sequences in the long terminal repeat of mink cell focus-inducing viruses generates redundancies of transcription factor binding sites important for the induction of thymomas.

The ability of mink cell focus-inducing (MCF) viruses to induce thymomas is determined, in part, by transcriptional enhancers in the U3 region of their long terminal repeats (LTRs). To elucidate sequence motifs important for enhancer function in vivo, we injected newborn mice with MCF 1dr (supF), a weakly pathogenic, molecularly tagged (supF) MCF virus containing only one copy of a sequence that is present as two copies (known as the directly repeated [DR] sequence) in the U3 region of MCF 247 and analyzed LTRs from supF-tagged proviruses in two resulting thymomas. Tagged proviruses integrated upstream and in the reverse transcriptional orientation relative to c-myc provided the focus of our studies. These proviruses are thought to contribute to thymoma induction by enhancer-mediated deregulation of c-myc expression. The U3 region in a tagged LTR in one thymoma was cloned and sequenced. Relative to MCF 1dr (supF), the cloned U3 region contained an insertion of 140 bp derived predominantly from the DR sequence of the injected virus. The inserted sequence contains predicted binding sites for transcription factors known to regulate the U3 regions of various murine leukemia viruses. Similar constellations of binding sites were duplicated in two proviral LTRs integrated upstream from c-myc in a second thymoma. We replaced the U3 sequences in an infectious molecular clone of MCF 247 with the cloned proviral U3 sequences from the first thymoma and generated an infectious chimeric virus, MCF ProEn. When injected into neonatal AKR mice, MCF ProEn was more pathogenic than the parental virus, MCF 1dr (supF), as evidenced by the more rapid onset and higher incidence of thymomas. Molecular analyses of the resultant thymomas indicated that the U3 region of MCF ProEn was genetically stable. These data suggest that the arrangement and/or redundancy of transcription factor binding sites generated by specific U3 sequence duplications are important to the biological events mediated by MCF proviruses integrated near c-myc that contribute to transformation.

High expression of transgenes mediated by hybrid retroviral vectors in hepatocytes: comparison of promoters from murine retroviruses in vitro and in vivo.

To achieve high transgene expression in the liver, we have compared the reporter gene expression among various murine retroviral long terminal repeats (LTRs) or leader sequences in vitro. Transient reporter gene expression assays revealed the highest gene expression by the polycythemic strain of spleen focus-forming virus (SFFVp) LTR in differentiated hepatocellular carcinoma cell lines, HuH-7 and PLC/PRF/5. However, remarkable difference was not observed among LTRs in other types of human liver tumor cell lines. Essentially the same results were obtained by infecting these cells with a series of retroviral vectors. Repression of transgene expression was observed by the leader sequences from Moloney murine leukemia virus (MoMLV), but not from mouse embryonic stem cell virus (MESV). Strengths of the promoters were further compared in murine hepatocytes in vivo. Although the proportions of genomic integration were almost the same, higher gene expression was observed by the FMEV-type vector, which contained the SFFVp LTR and the MESV leader, in comparison with that by the MoMLV-based vector. Thus, FMEV-type vectors may represent a novel type of vectors for human gene therapy with hepatocytes.

Analysis of the disease potential of a recombinant retrovirus containing Friend murine leukemia virus sequences and a unique long terminal repeat from feline leukemia virus.

We have molecularly cloned a feline leukemia virus (FeLV) (clone 33) from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV proviruses from other cats with AML, contains an unusual structure in its U3 region upstream of the enhancer (URE) consisting of three tandem direct repeats of 47 bp. To test the disease potential and specificity of this unique FeLV LTR, we replaced the U3 region of the LTR of the erythroleukemia-inducing Friend murine leukemia virus (F-MuLV) with that of FeLV clone 33. When the resulting virus, F33V, was injected into newborn mice, almost all of the mice eventually developed hematopoietic malignancies, with a significant percentage being in the myeloid lineage. This is in contrast to mice injected with an F-MuLV recombinant containing the U3 region of another FeLV that lacks repetitive URE sequences, none of which developed myeloid malignancies. Examination of tumor proviruses from F33V-infected mice failed to detect any changes in FeLV U3 sequences other than that in the URE. Like F-MuLV-infected mice, those infected with the F-MuLV/FeLV recombinants were able to generate and replicate mink cell focus-inducing viruses. Our studies are consistent with the idea that the presence of repetitive sequences upstream of the enhancer in the LTR of FeLV may favor the activation of this promoter in myeloid cells and contribute to the development of malignancies in this hematopoietic lineage.

Ikaros, a lymphoid-cell-specific transcription factor, contributes to the leukemogenic phenotype of a mink cell focus-inducing murine leukemia virus.

Mink cell focus-inducing (MCF) viruses induce T-cell lymphomas in AKR/J strain mice. MCF 247, the prototype of this group of nonacute murine leukemia viruses, transforms thymocytes, in part, by insertional mutagenesis and enhancer-mediated dysregulation of cellular proto-oncogenes. The unique 3' (U3) regions in the long terminal repeats of other murine leukemia viruses contain transcription factor binding sites known to be important for enhancer function and for the induction of T-cell lymphomas. Although transcription factor binding sites important for the biological properties of MCF 247 have not been identified, pathogenesis studies from our laboratory suggested to us that binding sites for Ikaros, a lymphoid-cell-restricted transcriptional regulator, affect the biological properties of MCF 247. In this report, we demonstrate that Ikaros binds to predicted sites in U3 sequences of MCF 247 and that site-directed mutations in these sites greatly diminish this binding in vitro. Consistent with these findings, ectopic expression of Ikaros in murine cells that do not normally express this protein significantly increases transcription from the viral promoter in transient gene expression assays. Moreover, site-directed mutations in specific Ikaros-binding sites reduce this activity in T-cell lines that express Ikaros endogenously. To determine whether the Ikaros-binding sites are functional in vivo, we inoculated newborn mice with a variant MCF virus containing a mutant Ikaros-binding site. The variant virus replicated in thymocytes less efficiently and induced lymphomas with a delayed onset compared to the wild-type virus. These data are consistent with the hypothesis that the Ikaros-binding sites in the U3 region of MCF 247 are functional and cooperate with other DNA elements for optimal enhancer function in vivo.

Role of the LTR region between the enhancer and promoter in mink cell focus-forming murine leukemia virus pathogenesis.

Long terminal repeat (LTR) sequences are important determinants of mink cell focus-forming (MCF) murine leukemia virus pathogenesis. These sequences include the enhancer and sequences between the enhancer and promoter (DEN). In a previous study we showed that a virus missing the DEN region in its LTR was severely attenuated in its ability to induce thymic lymphoma. In this study we observed that a virus with an LTR consisting of DEN but no enhancer sequences was pathogenic. We compared the pathogenicity of this DEN virus with other LTR mutant MCF13 viruses that contained a single enhancer (1R) or a single enhancer plus DEN (1R + DEN). All LTR mutant viruses generated thymic lymphoma, however, at a much lower incidence and with a longer latency compared with wild-type (WT) MCF13 virus. DEN virus replication in the thymus was the lowest compared with the 1R and 1R + DEN viruses. Viral replication in a different thymic subpopulation could not explain the decreased pathogenicity of the LTR mutant viruses compared with WT virus. However, lower levels of mutant virus replication in the thymus compared with WT during the preleukemic period may contribute to the attenuation of pathogenicity. The phenotype of tumors induced by the mutant viruses was similar and differed from tumors induced by WT virus by the presence of CD3(-)CD4(-)CD8(-) cells. Analysis of LTR sequences of infectious virus rescued from tumors induced by the 1R and 1R + DEN viruses showed that amplification of enhancer sequences had occurred during tumor development. The lack of DEN virus expression by tumor cells led us to propose that DEN sequences may play a role at an early step in tumorigenesis.

Lymphomas and high-level expression of murine leukemia viruses in CFW mice.

Historically, Swiss Webster mice of the CFW subline, both inbred and random-bred stocks, have been considered to have a low spontaneous occurrence of hematopoietic system tumors, and previous reports of infectious expression of murine leukemia viruses (MuLVs) have been rare and unremarkable. In marked contrast, in the present study of CFW mice from one source observed by two laboratories over a 2-year period, nearly 60% developed tumors, 85% of which were lymphomas, the majority of B-cell origin. All tumors tested expressed ecotropic MuLVs, and most expressed mink cell focus-inducing (MCF) MuLVs. Among normal mice of weanling to advanced age, over one-half were positive for ecotropic virus in tail or lymphoid tissues, and MCF virus was frequently present in lymphoid tissue, less often in tail. Patterns of ecotropic proviral integration indicated that natural infection occurred by both genetic and exogenous routes. Lymphomas were induced in NIH Swiss mice infected as neonates with tissue culture-propagated MuLVs isolated from normal and tumor tissue of CFW mice.

Retroviral vector-mediated gene expression in human CD34+CD38- cells expanded in vitro: cis elements of FMEV are superior to those of Mo-MuLV.

A novel murine stromal cell line, HESS-M28, was established, which supports the expansion of human CD34+CD38- cells more than 300-fold in vitro in the presence of human IL-3 and SCF. These cells were used in an attempt to evaluate cis-acting elements of retroviral vectors in human primitive hematopoietic cells. Cord blood cells were cultured on top of the mixed cell layers of the stromal cell line, HESS-M28, and retroviral vector-producing cells. The FMEV-type vector SF/Lyt contained the spleen focus-forming virus U3 and the MESV primer-binding site (PBS), while MO3/Lyt contained the U3 region and PBS from Mo-MuLV. After transduction by the FMEV-type and Mo-MuLV-based vectors, expression of the marker gene murine CD8 (mCD8) was examined in CD34-, CD34+, and CD34+CD38- cells. In CD34+ and CD34+CD38- cells, expression of mCD8 was higher with the FMEV-type vector, SF/Lyt, compared with the cells transduced by the Mo-MuLV-based vector MO3/Lyt, although the expression was comparable in CD34- cells. Expression of marker genes was also confirmed in long-term culture-initiating cells (LTC-ICs) and SCID-repopulating cells (SRCs).

The nucleotide sequence of the high-leukemogenic murine retrovirus SL3-3 reveals a patch of mink cell focus forming-like sequences upstream of the ecotropic envelope gene. Brief report.

We report the complete nucleotide sequence of the potent T-lymphomagenic murine retrovirus SL3-3. The non-LTR regions of the virus show 98% sequence identity to the endogenous ecotropic Akv murine leukemia virus. While the region encoding the surface envelope protein is completely identical to that of Akv, a approximately 200 nucleotide stretch in the integrase encoding region upstream of env is similar to the sequence of mink cell focus forming (MCF) viruses and shows a complete match with the mouse retrovirus 10A1. The history of SL3-3 may therefore include recombination involving an Akv-like virus and a virus with MCF-like sequences.

Retroviral insertions in Evi12, a novel common virus integration site upstream of Tra1/Grp94, frequently coincide with insertions in the gene encoding the peripheral cannabinoid receptor Cnr2.

The common virus integration site (VIS) Evi11 was recently identified within the gene encoding the hematopoietic G-protein-coupled peripheral cannabinoid receptor Cnr2 (also referred to as Cb2). Here we show that Cnr2 is a frequent target (12%) for insertion of Cas-Br-M murine leukemia virus (MuLV) in primary tumors in NIH/Swiss mice. Multiple provirus insertions in Evi11 were cloned and shown to be located within the 3' untranslated region of the candidate proto-oncogene Cnr2. These results suggest that proviral insertion in the Cnr2 gene is an important step in Cas-Br-M MuLV-induced leukemogenesis in NIH/Swiss mice. To isolate Evi11/Cnr2 collaborating proto-oncogenes, we searched for novel common VISs in the Cas-Br-M MuLV-induced primary tumors and identified a novel frequent common VIS, Evi12 (14%). Interestingly, 54% of the Evi11/Cnr2-rearranged primary tumors contained insertions in Evi12 as well, which suggests cooperative action of the target genes in these two common VISs in leukemogenesis. By interspecific backcross analysis it was shown that Evi12 resides on mouse chromosome 10 in a region that shares homology with human chromosomes 12q and 19p. Sequence analysis demonstrated that Evi12 is located upstream of the gene encoding the molecular chaperone Tra1/Grp94, which was previously mapped to mouse chromosome 10 and human chromosome 12q22-24. Thus, Tra1/Grp94 is a candidate target gene for retroviral activation or inactivation in Evi12. However, Northern and Western blot analyses did not provide evidence that proviral insertion had altered the expression of Tra1/Grp94. Additional studies are required to determine whether Tra1/Grp94 or another candidate proto-oncogene in Evi12 is involved in leukemogenesis.

Receptors for polytropic and xenotropic mouse leukaemia viruses encoded by a single gene at Rmc1.

The onset of leukaemia caused by type C retroviruses (MLV) in mice is accelerated by the emergence of recombinant polytropic or mink cell focus-forming (MCF) viruses. Susceptibility to infection by polytropic/MCF and also by closely related xenotropic MLV has been mapped to Rmc1 on mouse chromosome 1 (refs 5-7). To identify this gene, we introduced an expression cDNA library prepared from mouse NIH3T3 fibroblasts into nonpermissive hamster cells and screened these cells for acquired susceptibility to MCF viruses encoding beta-galactosidase and G418 resistance. From hamster cell clones identified in the screen, we recovered a mouse cDNA that maps to Rmc1 and confers MCF MLV infection when expressed in nonpermissive cell lines. It encodes a membrane protein related to Syg1p (suppressor of yeast G alpha deletion; ref. 8). The receptor-binding domain of the MCF MLV envelope protein binds specifically to Xenopus laevis oocytes that express mouse Syg1, suggesting it functions as a receptor that mediates virus entry. We also obtained the cDNA encoding human SYG1. When expressed in hamster cells, it establishes infectivity by MCF MLV as well as xenotropic MLV, which do not infect laboratory mice.

Sequence-specific and/or stereospecific constraints of the U3 enhancer elements of MCF 247-W are important for pathogenicity.

The oncogenic potential of many nonacute retroviruses is dependent on the duplication of the enhancer sequences present in the unique 3' (U3) region of the long terminal repeat (LTR). In a molecular clone (MCF 247-W) of the murine leukemia virus MCF 247, a leukemogenic mink cell focus-inducing (MCF) virus, the U3 enhancer sequences are tandemly repeated in the LTR. We mutated the enhancer region of MCF 247-W to test the hypothesis that the duplicated enhancer sequences of this virus have a sequence-specific and/or a stereospecific role in enhancer function required for transformation. In one virus, we inserted 14 nucleotide bp into the novel sequence generated at the junction of the two enhancers to generate an MCF virus with an interrupted enhancer region. In the second virus, only one copy of the enhancer sequences was present. This second virus also lacked the junction sequence present between the two enhancers of MCF 247-W. Both viruses were less leukemogenic and had a longer mean latency period than MCF 247-W. These data indicate that the sequence generated at the junction of the two enhancers and/or the stereospecific arrangement of the two enhancer elements are required for the full oncogenic potential of MCF 247-W. We analyzed proviral LTRs within the c-myc locus in tumor DNAs from mice injected with the MCF virus with the interrupted enhancer region. Some of the proviral LTRs integrated upstream of c-myc contain enhancer regions that are larger than those of the injected virus. These results are consistent with the suggestion that the virus with an interrupted enhancer changes in vivo to perform its role in the transformation of T cells.

Genetic and immunological parameters governing in vivo susceptibility/resistance to retrovirally induced murine malignant histiocytosis.

Malignant histiocytosis sarcoma virus (MHSV) arose as a recombinant of c-Harvey-ras murine sarcoma virus (Ha-MuSV) and Friend mink cell focus-forming virus (F-MCFV). It is a defective acute transforming retrovirus that, along with Friend murine leukemia helper virus (F-MuLV), induces malignant histiocytosis (MH) in susceptible adult mice. We have assessed the in vivo susceptibility to MHSV in inbred homozygous, F1 hybrid, congenic, and recombinant inbred (RI) mice. We have shown that: (1) in vivo resistance to MHSV is multigenic, regulated by MHC and non-MHC genes in a different fashion than with F-MCFV, F-MuLV, or Ha-MuSV; (2) using BXD RI mice, the resistance phenotype is linked with 95.8% probability to two linked loci, Pmv-9 and Iapls3-14, on chromosome 13 (homologous to the area of human chromosome 5 for which a chromosomal break point at position 5q35 is associated with human MH); and (3) CD4+ T cells are critical for MHSV resistance.

Spacing between the enhancer and promoter of the long terminal repeat of a murine leukaemia retrovirus is required for transcriptional activation in T cells.

In T cells, transcriptional activation by the long terminal repeat (LTR) of the mink cell focus-forming murine leukaemia virus requires some spacing between the enhancer and promoter. A large size-range of intervening sequences (11-93 bp) is able to activate transcription efficiently. Neither a specific nucleotide sequence nor stereospecific alignment of the spacer is important.

FMEV vectors: both retroviral long terminal repeat and leader are important for high expression in transduced hematopoietic cells.

FMEV retroviral vectors combine the long terminal repeat of Friend mink cell focus-forming viruses with the 5' untranslated leader region of the murine embryonic stem cells virus. These modules were connected to achieve high transgene expression in hematopoietic progenitor and stem cells. Here, we report the cloning of safety-improved and versatile FMEV vectors allowing module-wise exchange of crucial elements for comparative studies. By transfer and expression of four different marker genes (neomycin phosphotransferase, lacZ, enhanced green fluorescent protein and truncated low affinity nerve growth factor receptor), we formally demonstrate that both the long terminal repeat and the leader contribute to the high expression of FMEV in transduced hematopoietic cells. Most prominent are the data recorded in the absence of selection in myelo-erythroid progenitor cells. Here, FMEV vectors mediate up to two orders of magnitude increased transgene expression levels when compared with vectors based on the Moloney murine leukemia virus.

Low-frequency loss of heterozygosity in Moloney murine leukemia virus-induced tumors in BRAKF1/J mice.

To identify potential involvement of tumor suppressor gene inactivation during leukemogenesis by Moloney murine leukemia virus (M-MuLV), a genome-wide scan for loss of heterozygosity (LOH) in tumor DNAs was made. To assess LOH, it is best to study mice that are heterozygous at many loci across the genome. Accordingly, we generated a collection of 52 M-MULV-induced tumor DNAs from C57BR/cdJ x AKR/J F1 (BRAKF1) hybrid mice. By using direct hybridization with oligonucleotides specific for three different classes of endogenous MuLV-related proviruses, 48 markers on 16 of 19 autosomes were simultaneously examined for allelic loss. No allelic losses were detected, with the exception of a common loss of markers on chromosome 4 in two tumors. The three autosomes that lacked informative endogenous proviral markers were also analyzed for LOH by PCR with simple-sequence length polymorphisms (SSLPs); one additional tumor showed LOH on chromosome 15. Further screening with chromosome 4 SSLPs identified one additional tumor with LOH on chromosome 4. Therefore, in total, the average fractional allelic loss was quite low (0.002), but the LOH frequency of 6% on chromosome 4 was highly statistically significant (P < 0.0005). Detailed SSLP mapping of the three tumors with LOH on chromosome 4 localized the region of common LOH to the distal 45 centimorgans, a region syntenic with human chromosomes 1 and 9. Candidate tumor suppressor genes, Mts1 (p16INK4a) and Mts2 (p15INK4b), have been mapped to this region, but by Southern blot analysis, no homozygous deletions were detected in either gene. One of three tumors with LOH on chromosome 4 also showed a proviral insertion near the c-myc proto-oncogene. These results suggested that tumor suppressor inactivation is generally infrequent in M-MuLV-induced tumors but that a subset of these tumors may have lost a tumor suppressor gene on chromosome 4.

Identification of nucleotide sequences that regulate transcription of the MCF13 murine leukemia virus long terminal repeat in activated T cells.

The region downstream of the enhancer (DEN) of the long terminal repeat of the mink cell focus-forming murine leukemia virus is important for viral pathogenicity. Another important activity of DEN is its control of transcription in activated T cells, and we have determined that an NF-kappaB site is critical for this activity.

Leukemogenesis by Moloney murine leukemia virus: a multistep process.

Moloney murine leukemia virus is a prototypical simple retrovirus that has been an extremely useful model for leukemogenesis. Important steps in leukemogenesis include proviral activation of cellular proto-oncogenes, generation of mink cell focus-inducing recombinants, and early (preleukemic) virus-induced changes in hematopoiesis.

Improved retroviral vectors for hematopoietic stem cell protection and in vivo selection.

Therapeutic gene transfer into hematopoietic cells is critically dependent on the evolution of methods that allow ex vivo expansion, high-frequency transduction, and selection of gene-modified long-term repopulating cells. Progress in this area needs elaboration of defined culture and transduction conditions for long-term repopulating cells and improvement of gene transfer systems. We have optimized retroviral vector constructions based on murine leukemia viruses (MuLV) to overcome the transcriptional repression encountered with the use of conventional Moloney MuLV (MoMuLV) vectors in early hematopoietic progenitor cells (HPC). Novel retroviral vectors, termed FMEV (for Friend-MCF/MESV hybrid vectors), were cloned that mediate greatly improved gene expression in the myeloerythroid compartment. Transfer of the selectable marker multidrug resistance 1 (mdr1), FMEV, in contrast to conventional MoMuLV-related vectors currently in use for clinical protocols, mediated background-free selectability of transduced human HPC in the presence of myeloablative doses of the cytostatic agent paclitaxel in vitro. Furthermore, FMEV also greatly improved chemo-protection of hematopoietic progenitor cells in a murine model system in vivo. Finally, when a second gene was transferred along with mdr1 in an FMEV-backbone, close to 100% coexpression was observed in multidrug-resistant colonies. These observations have significant consequences for a number of ongoing and planned gene therapy trials, for example, stem cell protection to reduce the myelotoxic side effects of anticancer chemotherapy, correction of inherited disorders involving hematopoietic cells, and antagonism of HIV infection.

Genetic basis for resistance to polytropic murine leukemia viruses in the wild mouse species Mus castaneus.

Cultured cells derived from the wild mouse species Mus castaneus were found to be uniquely resistant to exogenous infection by polytropic mink cell focus-forming (MCF) murine leukemia viruses (MuLVs). This MCF MuLV resistance is inherited as a genetically recessive trait in the progeny of F1 crosses between M. castaneus and MCF MuLV-susceptible laboratory mice. Examination of the progeny of backcrosses demonstrated that susceptibility is inherited as a single gene which maps to chromosome 1. The map location of this gene places it at or near the locus Rmc1, the gene encoding the receptor for MCF/xenotropic MuLVs, suggesting that resistance is mediated by the M. castaneus allele of this receptor.

Activity of Friend mink cell focus-forming retrovirus during myelo-erythroid hematopoiesis.

Friend mink cell focus-forming (FMCF) viruses are recombinants between the Friend murine leukemia virus (F-MuLV) and endogenous polytropic retroviruses involved in a number of retrovirus-induced malignancies of the myelo-erythroid compartment. To analyze the contribution of the viral cis regulatory elements to the host range determinants within the hematopoietic system, we performed a series of marker gene experiments using both transient transfection and retroviral-mediated stable transduction of indicator cell lines representing distinct developmental stages. According to our data, the U3 region in the long terminal repeat (LTR) of FMCF viruses possesses an enhancer assembly that allows efficient transcription in both early and late myelo-erythroid stem and progenitor cells. Retroviral gene expression, however, is subjected to stage-dependent transcriptional controls during blood cell maturation. We obtained evidence that a repressor element overlapping with the primer binding site in the viral leader region compromises U3-mediated gene expression in a stage-dependent manner, with the strongest restriction observed in the most primitive cells analyzed, FDCP-mix. In addition, our data indicate a second hurdle for retroviral gene expression in early hematopoietic cells that is independent of the primer binding site and most likely related to inefficient utilization of U3-located enhancers. These data shed light on the mechanisms of host range restriction within the hematopoietic system and define a basis for the design of retroviral vectors aimed to overcome transcriptional inefficiency in early hematopoietic cells. Thus, we developed novel retroviral vectors combining FMCF-type U3 regions with a permissive leader from the murine embryonic stem cell virus. These vectors are highly efficient for gene transfer and expression in both early and late myelo-erythroid cells, indicating that they will be of great use for a variety of experimental and therapeutic applications.