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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.
Nonacute murine leukemia viruses (MuLVs) do not encode oncogenes yet induce hematopoietic malignancies in susceptible mice after numerous rounds of infection in vivo. Prior to tumor formation, multiple proviral DNAs are inserted into the host genome. These proviruses contribute to tumor formation either by functionally disrupting cellular genes (i.e., insertional mutagenesis) or by inducing overexpression of nearby proto-oncogenes (2, 47). Proto-oncogene overexpression is a consequence of the strong enhancer activity of nearby proviral sequences. These enhancer sequences are generally contained in two or more copies of directly repeated (DR) sequences present in the unique 3′ (U3) region of the proviral long terminal repeats (LTRs). Each DR sequence contains multiple DNA elements predicted to bind cellular proteins that regulate transcription and thus influence the magnitude of virus expression in vivo and the expression levels of nearby cellular proto-oncogenes. In addition, the DR sequences influence the type of neoplastic disease induced by a particular virus as well as the time to disease onset (4, 7–10, 13–15, 17, 28–31, 36, 45, 50).
MCF 247 induces T-cell lymphomas 2 to 4 months after inoculation into susceptible mice. The two DR sequences strongly influence the pathogenic potential of this virus (29, 30). The 5′ portion of each DR sequence contains four highly conserved motifs (binding sites for LVb, AML-1, NF-1, and GRE) thought to provide a framework for enhancer function (26) (Fig. (Fig.1A).1A). Other putative binding sites for known transcriptional regulators are dispersed in the DR sequences and in sequences proximal to the promoter (43). However, the proteins that bind these sequences in MCF 247 and that modulate enhancer activity have not been determined experimentally.
We recently demonstrated that the insertion of 14 bp into the sequence at the junction between the two DR sequences significantly reduced the pathogenic potential of a mink cell focus-inducing (MCF) virus otherwise identical to MCF 247 (17). We have since demonstrated that this altered U3 region is less active than the wild-type sequences in transient gene expression assays (N. L. DiFronzo, C. K. Leung, M. K. Mammel, and Q. N. Pham, unpublished data). These findings indicate that either the sequence formed at the junction of the two DR sequences or the distance or spatial orientation between the DR sequences or both modulate the biological activities of this retrovirus. To examine the former possibility, we analyzed the U3 region of MCF 247 for putative binding sites for known transcriptional regulators (43). The analysis revealed that the junction sequence contains a putative binding site for Ikaros, a transcriptional regulator required for early lymphoid-cell differentiation (23, 22, 39, 46). Interestingly, this DNA element (Fig. (Fig.1A,1A, site 2) was disrupted in the weakly pathogenic MCF virus containing the 14-bp insertion between the two DR sequences (17). This suggested to us the possibility that Ikaros contributes to the enhancer activity of MCF 247 U3 sequences.
Ikaros, the founding member of a family of Krüppel-like zinc finger transcription factors related to Drosophila melanogaster protein Hunchback (24, 27, 32, 37, 41), was identified by its ability to bind to the enhancer of a gene encoding early-T-cell-differentiation antigen CD3-δ (24, 27, 32, 37, 41). In vitro, Ikaros stimulates expression of reporter genes under the control of multimerized Ikaros-binding sites (39), where the core consensus binding site is 5′-GGGA-3′ (32, 35, 41). In vivo, Ikaros is predicted to regulate the expression of genes whose products determine lymphoid-cell fate and function (3, 23, 25, 48). Moreover, this protein is proposed to provide a checkpoint that regulates T-cell proliferation (49).
By means of alternative splicing, the Ikaros gene encodes at least eight isoforms of Ikaros (22, 27, 34, 39, 46). All isoforms contain C-terminal zinc fingers required for protein dimerization and protein-protein interactions, and six of these (Ikaros-1, Ikaros-2, Ikaros-2A, Ikaros-3, Ikaros-4, and Ikaros-4A) contain N-terminal zinc fingers required for sequence-specific DNA-binding activity (39). Ikaros-1 and Ikaros-2, containing four and three N-terminal zinc fingers, respectively, are abundantly expressed in the nuclei of developing (34, 39) and mature lymphocytes (39) and strongly stimulate the activity of reporter genes under the control of Ikaros-binding sites (39).
In this report, we provide the first evidence that Ikaros up-regulates the level of transcription from the U3 region of a retrovirus. We show that Ikaros binds putative Ikaros-binding sites in the U3 sequences of MCF 247 and enhances LTR-mediated reporter activity in T cells in vitro. Mutational analyses of the U3 region reveal that multiple Ikaros-binding sites cooperate in regulating reporter activity. We also demonstrate that Ikaros expressed in murine splenocytes binds Ikaros-binding sites in the U3 region of MCF 247 and that Ikaros is readily detectable in MCF-induced lymphomas. Finally, we show that, compared to the wild-type virus, a replication-competent MCF virus containing a point mutation in one Ikaros-binding site is attenuated in its abilities to replicate in vivo and to induce thymic lymphomas.
Murine fibroblast cell line NIH 3T3 and human epithelial cell line 293T were grown in Dulbecco’s modified Eagle’s medium containing a high concentration of glucose supplemented with 10% fetal bovine serum (FBS). Human T-lymphoblastoid-cell line Jurkat and the murine T-lymphoblastoid-cell line SL3H were grown in RPMI 1640 supplemented with 10% FBS. Cells were maintained at 37°C in 95% humidity and 5% CO2.
The reporter plasmid MCF 2dr contains the PstI-to-SmaI LTR fragment from molecular clone p247-W inserted upstream of the chloramphenicol acetyltransferase (CAT) gene in vector pCAT-basic. The LTR fragment contains most of U3, including two DR sequences and the viral promoter (Fig. (Fig.1).1). Reporter plasmids containing mutations in Ikaros-binding sites were generated from MCF 2dr using oligonucleotide-directed mutagenesis (QuikChange site-directed mutagenesis; Stratagene). All mutations were verified by sequencing the U3 regions in both directions (CEQ 2000 dye terminator cycle sequencing; Beckman). Plasmids were purified by banding twice in cesium chloride gradients. Two clones of each reporter were evaluated by transient gene expression assays.
Complementary oligonucleotides spanning different portions of the U3 region from MCF viruses were synthesized and used as probes for electrophoretic mobility gel shift assays (EMSA) and competition gel shift assays. Probes were prepared by end labeling oligonucleotides with [α2-P]ATP, annealing the labeled oligonucleotide to its complement, and purifying the double-stranded oligonucleotides on 8% nondenaturing polyacrylamide gels.
Nuclear extracts were prepared from primary murine splenocytes and from 293T human kidney epithelial cells transfected with a control vector (CDM8) or a vector expressing full-length Ikaros (CDM8-IK1) tagged with an epitope of the influenza hemagglutinin (HA) using published methods (18, 46).
Protein-DNA interactions were examined by EMSA and competition gel shift assays (6). Protein preparations, including Ikaros purified from bacteria, nuclear extracts from 293T cells transfected with vectors expressing Ikaros tagged with HA, and nuclear extracts from murine splenocytes, were incubated for 15 min at room temperature with double-stranded 32P-labeled oligonucleotide probes, and the resulting complexes were examined by EMSA using antibodies against the HA epitope or Ikaros (see below). Species-specific immunoglobulins (Ig) were added to parallel reaction mixtures as negative controls. Control oligonucleotide IK-BS1 (5′-TCAGCTTTTGGGAATACCCTGTCA-3′), containing a known high-affinity Ikaros-binding site (underlined) (39), was included to verify Ikaros binding. In competition reactions, proteins were incubated with a molar excess of unlabeled oligonucleotide prior to the addition of the probe. The protein-DNA complexes were separated by electrophoresis at 4°C in a 5% polyacrylamide gel containing low-ionic-strength TAE (6.7 mM Tris, 3.3 mM sodium acetate, 1.0 mM sodium EDTA), pH 7.6, with buffer recirculation. Gels were dried and autoradiographed.
Mouse (clone MOPC-12; Sigma) and rabbit (Pierce) nonspecific IgGs, mouse (clone TEPC 183; Sigma) nonspecific IgM, a mouse monoclonal antibody reactive with an HA epitope (α-HA; clone 12CA5; Boehringer Mannheim), a rabbit polyclonal antiserum against Ikaros (α-IK), and a monoclonal antibody directed against the MCF envelope glycoprotein (Hy7; gift from L. Evans, National Cancer Institute, Rocky Mountain Laboratories, Hamilton, Montana) were used in these studies.
NIH 3T3 cells were cotransfected (Lipofectamine; Gibco BRL) with MCF 2dr reporter plasmid (0.125 μg) plus control vector (CDM8) or combinations (1.375 μg total) of an Ikaros expression vector (CDM8-IK1) plus control vector. Forty-eight hours posttransfection, the cells were harvested and processed for protein and CAT assays as described below.
To transfect Jurkat cells, plasmids (7 μg) were diluted in OptiMEM (1.2 ml; Gibco BRL) containing DMRIE-C (10 μl; Gibco BRL) and added to individual wells of a six-well tissue culture dish. After a 30-min incubation, 7 × 105 cells were added to each well and the contents of the wells were incubated for 4.5 h at 37°C and supplemented with 2.4 ml of RPMI 1640 containing 15% FBS. Twenty-four hours posttransfection, the cells were treated with phorbol myristate acetate (PMA; 25 ng/ml) and ionomycin (1.4 ng/ml). Twenty-four hours after the addition of PMA and ionomycin, the cells were harvested and processed for protein determinations and CAT assays. SL3H cells were transfected as described for Jurkat cells but were not treated with PMA and ionomycin.
Cellular proteins were extracted in Promega reporter lysis buffer and stored at −80°C. Protein concentrations were determined using the bicinchoninic acid protein assay (BCA kit; Pierce). Nonchromatographic CAT assays (44) were performed using 20 μl (SL3H or NIH 3T3) or 30 μl (Jurkat) of cytoplasmic extract in a final reaction volume of 175 μl containing 0.15 μCi of [14C]chloramphenicol, 1.7 mM n-butyryl coenzyme A (Pharmacia Biotech), 1.9% glycerol, and 186 mM Tris-Cl, pH 7.5. Reaction mixtures were incubated for 2 h at 37°C, and reactions were terminated by extraction with 500 μl of mixed xylenes (Aldrich). After a thorough mixing, the samples were centrifuged (5 min, 14,000 × g) and the organic phase was collected and back-extracted twice with 0.25 mM Tris, pH 7.5. Conversion of [14C]chloramphenicol into butyrylated species was quantitated in 100 μl of the organic-phase mixture. The activity of each sample was converted to total CAT activity per unit of protein (disintegrations per minute per microgram). The transcriptional activity of each reporter plasmid was evaluated in triplicate in two or more separate experiments. For each experiment, the transcriptional activities of all other reporters were normalized relative to that of MCF 2dr, the reporter containing the wild-type U3 sequences. Analyses of variance were performed on the normalized data pooled from at least two separate transfections, and the significance of the differences between the mean activities of individual reporters were determined using Tukey multiple-comparison tests (51).
Mutation of the third Ikaros-binding site in the U3 region of an infectious molecular clone of MCF 247 was achieved using the QuikChange site-directed mutagenesis kit and oligonucleotide OP103 and its complement (Fig. (Fig.1B).1B). The U3 region of the resulting plasmid, the vMCF (3 IK) virus plasmid, was sequenced in both directions to confirm the presence of the point mutation. The DNA fragment containing the genomic sequences of vMCF (3 IK) was gel purified, ligated to form concatemers with identical LTRs, and transfected into Mus dunni cells to generate virus (16). Supernatants containing variant virus vMCF (3 IK) were collected approximately 3 weeks later (37) and titered using freshly infected M. dunni cells and the Hy7 antibody (22). Stocks of the variant MCF and wild-type viruses had similar titers. Newborn (24- to 48-h-old) AKR/J mice were injected intraperitoneally with approximately 5 × 104 infectious units of the variant or the wild-type virus.
Single-cell suspensions were prepared from the thymuses of individual mice. Thymocytes (106) were incubated in parallel reactions with Hy7 and an isotype-matched mouse control Ig. After being washed, the cells were incubated with phycoerythrin (PE)-conjugated goat anti-mouse Ig (Southern Biotechnology). In some experiments, biotin-conjugated goat anti-mouse Ig (PharMingen) and streptavidin-conjugated PE (PharMingen) were used to amplify the primary antibodies. The cells were subsequently incubated with a cocktail of monoclonal antibodies containing Cy-Chrome-conjugated rat anti-mouse CD4 (PharMingen; clone H129.19), fluorescein isothiocyanate-conjugated rat anti-mouse CD8 (PharMingen; clone 53-6.7), and allophycocyanin-conjugated hamster anti-mouse CD3- (PharMingen; clone 145-2C11), washed, fixed with 0.5% paraformaldehyde, and analyzed on a FACSCalibur cytometer using Cell Quest software (Becton Dickinson). In total, thymocytes from 17 naive mice, 21 mice inoculated with vMCF (3 IK), and 18 mice inoculated with the wild-type virus were analyzed. Because of the amplification step in the second protocol, the results obtained with the two immunofluorescence assay (IFA) protocols could not be pooled. Nevertheless, the patterns of MCF gp70 expression by both IFA protocols were similar, and the conclusions drawn from the results were similar.
Analysis of the MCF 247 U3 sequences revealed that the enhancer region, namely, the DR and promoter-proximal sequences, contains five predicted Ikaros-binding sites (Fig. (Fig.1A)1A) (43). The possibility that Ikaros regulates the MCF promoter was suggested to us by our finding that an MCF virus with 14 bp inserted into one of these Ikaros-binding sites was less lymphomagenic than the wild-type virus (17). We therefore undertook a series of studies to determine whether Ikaros binds predicted Ikaros-binding sites in the enhancer region of MCF 247. For these studies, we used oligonucleotides containing the U3 sequences of MCF 247 (henceforth called U3 probes) in EMSAs and competition gel shift assays (Fig. (Fig.22 and and33 and data not shown).
The EMSA shown in Fig. Fig.2A2A (and data not shown) illustrates that Ikaros expressed in mammalian cells and in the presence of other nuclear proteins binds wild-type Ikaros-binding sites in the enhancer region of MCF 247. Nuclear proteins from 293T cells overexpressing Ikaros tagged with an HA epitope bound a substantial amount of a U3 probe containing the fifth Ikaros-binding site (WT 5; lanes 1 to 3) and considerably less of the homologous U3 probe lacking this DNA element (MUT 5-1, lanes 4 to 6). An antibody specific for the HA tag (α-HA) supershifted two complexes formed with the U3 probe containing the fifth Ikaros-binding site (lane 2), demonstrating that Ikaros was present in these protein-probe complexes. Isotype-matched, nonspecific Ig did not supershift these complexes (lane 3). Conversely, α-HA did not supershift proteins bound to the MUT 5-1 probe (lane 5), indicating that Ikaros is not present in the complex formed with MUT 5-1. A similarly sized, nonspecific complex formed when nuclear extracts from cells transfected with control vector were incubated with the U3 probe containing the fifth Ikaros-binding site (WT 5, lanes 7 to 9). Furthermore, antibodies specific for tagged Ikaros (α-HA) did not react with this complex (lane 8), demonstrating that this antibody does not cross-react with other 293T cell nuclear proteins present in this complex. It should be noted that, although Ikaros does not bind the terminal Ikaros-binding site (site 4) present in the WT 5 and MUT 5-1 probes, Ikaros binds specifically to the fourth Ikaros-binding site when it is flanked on both sides by U3 sequences (data not shown).
We used this same approach, as well as competition gel shift assays, to demonstrate that HA-tagged Ikaros binds U3 probes containing each of the predicted Ikaros-binding sites in the enhancer region of MCF 247. In addition, we demonstrated that Ikaros does not bind these sites when mutated by single base pair changes (Fig. (Fig.2B2B and data not shown). The U3 probes containing mutant Ikaros-binding sites are aligned beneath wild-type U3 sequences in Fig. Fig.1B.1B. The EMSA in Fig. Fig.2B2B illustrates that multiple complexes form on the U3 probe containing a point mutation in the third Ikaros-binding site (MUT 3, lanes 1 to 3) and on the wild-type probes containing an unmutated third Ikaros-binding site (WT 3, lanes 4 to 6) or unmutated first and second Ikaros-binding sites (WT 1-2, lanes 7 to 9). Ikaros bound to probes containing wild-type Ikaros-binding sites (WT 3 and WT 1-2), as judged by supershift of a complex with α-HA (lanes 5 and 8). This complex did not form on the U3 probe containing a point mutation predicted to eliminate the third Ikaros-binding site (MUT 3, lanes 1 to 3). Isotype-matched, nonspecific Ig did not supershift the protein-probe complexes (lanes 3, 6, and 9). Thus, Ikaros binds a U3 probe containing the third Ikaros-binding site, but not when a point mutation is introduced into this DNA element. These and other results (not shown) demonstrate that Ikaros expressed in mammalian cells and in the presence of other nuclear proteins binds specifically on the predicted Ikaros-binding sites in the enhancer region of MCF 247 (Fig. (Fig.1A,1A, 1 to 5).
Ikaros is expressed abundantly in lymphocytes present in the thymuses and spleens of developing and mature mice (22, 41). We next tested the in vitro DNA-binding activity of Ikaros expressed in unsorted murine splenocytes, which contain 50 to 60% T cells, the natural cellular target for most MCF viruses (Fig. (Fig.3A).3A). The U3 probe MUT 5-2 contains sequences from a nonpathogenic MCF virus (MCF 30-2) identical to those of probe WT 5, except that MUT 5-2 contains a point mutation in the fifth Ikaros-binding site; this point mutation is predicted to eliminate binding of Ikaros to the probe. Splenocyte nuclear proteins bound the MUT 5-2 probe minimally (lanes 2 to 4). In contrast, multiple complexes were detected on the probe containing a wild-type Ikaros-binding site (WT 5, lanes 6 to 8). At least two of the faster-migrating complexes contained Ikaros (lanes 6 and 8), as judged by their supershift by antiserum specific for Ikaros (lane 7). Since the intensity of the supershifted complex (lane 7) is greater than the combined intensities of the two faster-migrating complexes abolished by α-IK, it is possible that a minor fraction of a more slowly migrating complex was also supershifted or that α-IK stabilized these protein-DNA complexes. These data demonstrate that Ikaros present in the nuclei of murine lymphoid cells binds an Ikaros-binding site in U3 sequences from the wild-type virus.
Virus-infected and transformed T cells are the predominant cells in the thymuses, lymph nodes, and spleens from mice with advanced leukemia (16). Western analysis (Fig. (Fig.3B)3B) illustrates that Ikaros is readily detectable in lymphoid organs from mice with advanced leukemia. Taken together, the data in this section and in the previous section support the hypothesis that Ikaros, together with other nuclear proteins, has the potential to influence enhancer function.
As Ikaros binds specifically to DNA elements in the MCF LTR, we tested the functional significance of this binding by transactivation assays using MCF 2dr, a CAT reporter plasmid containing the majority of the wild-type U3 sequences (Fig. (Fig.1A,1A, PstI-SmaI fragment). MCF 2dr was cotransfected into NIH 3T3 cells with increasing amounts of an expression vector containing Ikaros-1 cDNA (Fig. (Fig.4).4). In each transfection, the amount of DNA was held constant by the addition of a control vector without insert. NIH 3T3 murine fibroblasts were chosen for these experiments because they support the in vitro replication of MuLVs yet do not express Ikaros (40). In the absence of Ikaros, the MCF 2dr reporter exhibited strong transcriptional activity (Fig. (Fig.44 and data not shown), indicating that NIH 3T3 fibroblasts express other cellular proteins that regulate transcription from the MCF virus promoter. When increasing amounts of the Ikaros-expression vector were cotransfected with the reporter plasmid, a dose response was observed. At the highest ratio of Ikaros expression vector to reporter plasmid (8:1), CAT expression was increased sevenfold relative to that of the reporter alone (0:1). These data demonstrate that Ikaros increases transcription from the MCF promoter.
To test whether Ikaros-binding sites potentiate enhancer activity from the LTR, we generated reporter plasmids with point mutations in one or more Ikaros-binding sites and tested their activities in T cells by transient gene expression assays. The oligonucleotides used to generate the mutant Ikaros-binding sites are shown in Fig. Fig.1B;1B; EMSA and competition gel shift assays demonstrated that Ikaros does not bind the mutated sequences (Fig. (Fig.2B2B and data not shown). The activities of each reporter, relative to that of the reporter containing the wild-type U3 sequences, are shown in Fig. Fig.5.5. The reporter plasmids are named to indicate the Ikaros-binding site(s) mutated in the genetic context of MCF 2dr. For example, reporter plasmid MCF (1 IK) contains the U3 region of MCF 2dr with a point mutation in the first Ikaros-binding site, while reporter plasmid MCF (1-2 IK) contains point mutations in both the first and second Ikaros-binding sites. Except for the reporter containing a mutation in the fourth Ikaros-binding site, MCF (4 IK), individual reporters exhibited similar activities in human T-cell line Jurkat (Fig. (Fig.5A)5A) and in murine T-cell line SL3H (Fig. (Fig.5B5B).
Of the five reporters containing a single mutant Ikaros-binding site, the CAT activity of the reporter containing a mutation in the third Ikaros-binding site, MCF (3 IK), was reduced the most dramatically. The activity of MCF (3 IK) was 36% ± 5% of wild type (P < 0.05) in Jurkat cells and 51% ± 12% of wild type (P < 0.05) in SL3H cells. CAT activities from cells transfected with reporters containing mutations in either the first, second, or fifth Ikaros-binding site were unaltered relative to that of the wild-type reporter in both cell lines, whereas cells transfected with a reporter containing a mutation of the fourth Ikaros-binding site had significantly reduced CAT activity in one of the T-cell lines (Jurkat; Fig. Fig.5A).5A). In contrast to the reporters containing mutations in either the first, second, fourth, or fifth Ikaros-binding sites, reporters with two adjacent mutant Ikaros-binding sites, namely, MCF (1-2 IK) and MCF (4-5 IK), displayed significantly reduced CAT activity (P < 0.05) in both the human and murine T-cell lines. Reporter activities were further reduced when the U3 sequences contained three or more mutant Ikaros-binding sites, namely, MCF (1-3 IK), MCF (3-5 IK), and MCF (1-2, 4-5 IK) (P < 0.05). These data suggest that the MCF virus enhancer region is more efficient in T cells when multiple Ikaros-binding sites are functional and that the third site is the single most important Ikaros-binding site for MCF 247 enhancer activity in vitro. We conclude that Ikaros-binding sites, together with other DNA elements in MCF U3, functionally regulate the viral promoter.
MCF viruses are T cell tropic. We demonstrated that a single base pair change in the third Ikaros-binding site in the U3 region of MCF 247 markedly reduced MCF-driven transcription of the CAT gene in two T-cell lines (Jurkat and SL3H; Fig. Fig.5)5) and abolished Ikaros binding to this DNA element (Fig. (Fig.2C).2C). To determine whether this mutation was relevant to the biological properties of MCF virus in vivo, we introduced this single base pair change into the U3 sequence of the wild-type virus to generate a variant MCF virus, called vMCF (3 IK) (Fig. (Fig.1B,1B, OP103). Since virion proteins are synthesized from viral mRNAs transcribed from proviruses integrated in cellular DNA, we used the expression of a virus-specific protein (MCF gp70) on thymocytes from mice injected with the variant MCF virus as an indicator of the levels of transcription from the integrated provirus and virus replication in vivo. Thymocytes from age-matched naive (control) mice and mice injected with the wild-type virus were analyzed in parallel.
Figure Figure6A6A shows the levels of MCF gp70 expression on thymocytes from control mice and mice inoculated at birth with either variant or wild-type virus, as judged by staining with an amplified IFA protocol and analysis by flow cytometry. At each of the three time points examined, MCF gp70 expression was generally lowest on thymocytes from control mice and highest on thymocytes from mice inoculated with the wild-type virus. Thymocytes from mice inoculated as newborns with the variant virus expressed lower levels of MCF gp70 than thymocytes from mice inoculated with the wild-type virus. Nonetheless, by 77 days of age, two of the three mice inoculated with variant MCF virus expressed levels of MCF gp70 comparable to that from mice inoculated with the wild-type virus. These results suggest that replication of an MCF virus is dependent, in part, on the presence of Ikaros-binding sites in the U3 region.
To determine whether the injected viruses replicate equivalently in thymocyte subpopulations, we analyzed MCF gp70 expression on CD4− CD8−, CD4+ CD8+, CD4− CD8+, and CD4+ CD8− thymocytes (Fig. (Fig.6B).6B). MCF gp70 was readily detected on CD4− CD8−, CD4+ CD8+, CD4− CD8+, and CD4+ CD8− thymocytes from most mice inoculated with the wild-type virus at each time point analyzed. Conversely, MCF gp70 was expressed differentially on thymocyte subpopulations in mice infected with the variant MCF virus. In younger (35- and 49-day-old) mice, MCF gp70 was evident on the surface of CD4+ CD8+ thymocytes but barely detectable on the most-immature thymocytes (CD4− CD8−) and on the most-mature thymocyte subpopulations (CD4+ CD8− and CD4− CD8+). In older (77-day-old) mice, the magnitude of MCF gp70 expression was highest on both immature thymocyte subpopulations (CD4− CD8− and CD4+ CD8+). These results are consistent with the conclusion that a mutation in the third Ikaros-binding site, in the context of a replication-competent MCF virus, reduces transcription of viral mRNA in biologically relevant cells and hinders virus replication in vivo.
The LTR sequences of integrated proviruses regulate the expression of cellular loci important for tumor induction by MuLVs (2, 47). To determine whether Ikaros binding to proviral U3 sequences affects MCF virus-induced lymphomagenesis, we compared levels of lymphoma induction in mice inoculated with variant and wild-type MCF viruses (Fig. (Fig.7).7). By 180 days postinoculation (p.i.), the variant virus induced lymphomas in fewer inoculated mice (10 of 12) than the wild-type virus (12 of 12). In addition, the onset of lymphoma induction in mice inoculated with the variant virus was delayed 34 days compared to the onset of lymphoma induction in mice inoculated with wild-type virus. These data indicate that mutation of the third Ikaros-binding site impairs replication of the variant MCF virus in vivo as well as the ability of this MCF virus to induce lymphomas. These data are consistent with the hypothesis that the Ikaros-binding sites in the U3 region of MCF 247 are biologically relevant to integrated proviruses in vivo.
The U3 region of MCF 247 contains multiple DNA elements predicted to bind Ikaros, a lymphoid-cell-restricted transcriptional regulator. Here, we provide evidence that Ikaros binds to Ikaros-binding sites present in the DR sequences and in sequences proximal to the promoter (Fig. (Fig.1A,1A, sites 1 to 5). Moreover, we provide several pieces of evidence that these sites are functional. First, ectopic expression of Ikaros in murine cells that do not normally express this protein elevated the activity of a reporter containing wild-type U3 sequences. Consistent with this, U3 sequences with site-directed mutations in multiple Ikaros-binding sites are transcribed less efficiently than wild-type sequences in T cells that express endogenous Ikaros. In addition, one Ikaros-binding site (site 3; Fig. Fig.1A),1A), when mutated alone, reduced transcription from the MCF promoter significantly. These studies indicate that Ikaros-binding sites, along with other DNA elements in U3, functionally regulate the virus promoter. Additional evidence demonstrating that the Ikaros-binding sites in U3 are functional is provided by studies with a variant MCF virus containing a point mutation in the third Ikaros-binding site. This variant virus replicated less efficiently than the wild-type virus in thymocytes, suggesting that the mutation in the third Ikaros-binding site reduces transcription from integrated MCF proviruses in vivo. Last, mice infected with this variant MCF virus developed frank leukemia with a delayed onset relative to mice infected with the wild-type virus. We conclude that the Ikaros-binding sites in the U3 sequences of integrated proviruses are functional and contribute to the expression of cellular loci important for T-cell lymphoma induction.
To our knowledge, this is the first demonstration that Ikaros binds predicted Ikaros-binding sites in the U3 sequences of a retrovirus. This was documented by EMSA and competition assays using U3 probes with wild-type and mutant Ikaros-binding sites, as well as specific antibodies. We note that the U3 regions from several human retroviruses and lentiviruses (human T-cell leukemia virus type 1 and human immunodeficiency virus type 1, respectively), other MCF viruses (Moloney, Friend, Rauscher, and MCF 1233 viruses), as well as Cas-Br-E, SL3H, Moloney, and Friend MuLVs, contain predicted Ikaros-binding sites in their U3 regions (43). In fact, several probes used in this study are identical with the U3 sequences from other MuLVs, including those of an endogenous xenotropic virus (Bxv-1) and other polytropic viruses isolated from lymphomas in AKR/J mice (MCF13, MCFr35, MCF 30-2, and Tikaut) (1). It is therefore likely that Ikaros binds predicted Ikaros-binding sites in the regulatory sequences of other retroviruses, in addition to the binding observed in MCF 247.
The functional significance of the Ikaros-binding sites in the U3 region of MCF 247 was first tested by transient-gene expression assays. Here, ectopic expression of Ikaros in murine cells that do not normally express this protein increased the activity of a reporter gene under the control of native MCF U3 sequences. Similar studies with T cells (that express endogenous Ikaros) demonstrated that the five Ikaros-binding sites were not equal and that the third site is a key Ikaros-binding site in the U3 region of MCF 247.
Studies with a variant MCF virus containing a point mutation in the third Ikaros-binding site verified that this DNA element plays a role in enhancer function in vivo. Since virion proteins are synthesized from viral mRNAs transcribed from integrated proviral DNAs, the expression of a virus-specific protein (MCF gp70) on thymocytes was used as an indicator of transcription from the integrated provirus and virus replication in vivo. At 5 and 7 weeks p.i., MCF gp70 was less abundant on thymocytes from mice injected with the variant MCF virus than on those from mice injected with wild-type virus; by 10 weeks p.i., the difference in MCF gp70 abundance on thymocytes from these two groups of mice was less apparent. We recognize that viral recombination events, or rearrangements, might have generated viral genomes with an MCF env sequence and U3 sequences different than those in the injected virus. In fact, we previously documented recombination between an injected MuLV and the endogenous viral sequences carrying the MCF env (16). However, this recombinant virus was first detected at 14 weeks p.i. Thus, although formally possible, it is unlikely that a recombinant virus having U3 sequences different from that of the injected virus contributes to the expression of MCF gp70 at early times (5 to 7 weeks) after inoculation of the variant MCF virus. The low levels of MCF gp70 on thymocytes from mice inoculated with the variant virus are consistent with the conclusion that the mutation in the third Ikaros-binding site present hindered virus replication in vivo. This same mutation delayed (by 34 days) the induction of lymphoma by the variant MCF virus. These data provide the first evidence that a mutation in an Ikaros-binding site diminishes the pathogenic potential of an MuLV. Since Ikaros-binding sites are predicted in the U3 sequences of other retroviruses, we anticipate that Ikaros or other Ikaros family members bind these DNA elements and contribute to the enhancer activities of these retroviruses.
Reduced levels of virus replication and the delayed onset and slightly delayed time course of lymphoma induction by the variant MCF virus may reflect the time needed to select an MCF virus that replicates in thymocytes more efficiently than the variant virus. This virus, if generated, may contain a reversion of the point mutation in the third Ikaros-binding site, rearrangements, or other compensatory mutations in the U3 sequences. Our preliminary studies of the U3 sequences expressed in tumors induced by the variant MCF virus indicate that second-site mutations occur more frequently than the reversion of the mutation in site 3 (N. L. DiFronzo, J. O. Benson, and Q. N. Pham, unpublished data). Reversions and second-site mutations in viruses evolving from MuLVs engineered with point mutations in sequences that bind AML-1 (also known as core binding factor) and nuclear factor 1 have been documented; these reversions and second-site mutations frequently increase transcriptional activity of the LTR and increase virus-induced lymphomagenesis (19–21, 38, 42). We are currently investigating whether the U3 sequences of viruses expressed in tumors induced by our variant MCF virus evolved in vivo and whether they increase transcription from MCF U3 sequences.
Ikaros-binding sites are present in the regulatory sequences of several lymphoid-cell-restricted genes, including loci encoding CD2, CD4, CD8-α, the interleukin-2 (IL-2) enhancer, IL-2 receptor α, beta interferon, major histocompatibility complex class II, as well as subunits of the T-cell receptor (α, β, and δ) and CD3 (δ, γδ, and ) (39); however, the functional relevance of these Ikaros-binding sites has not been determined experimentally. In this report, we demonstrate that Ikaros-binding sites in the U3 region of an MCF MuLV are functional in T cells. Mutation of one of these sites, in the U3 region of a replication-competent MCF virus, reduced virus replication in vivo and delayed the onset of lymphoma induction. We conclude that this Ikaros-binding site, in the genetic context of its native enhancer and promoter sequences, regulates expression of viral sequences and cellular loci important for transformation.
Ikaros is a hematopoietic-cell-restricted transcriptional regulator required for lymphoid lineage commitment (23, 22, 48). The molecular mechanism by which Ikaros dictates this process is largely unclear but is most likely related to the ability of Ikaros to either activate or repress transcription of lymphoid-cell-specific cellular loci (12). These two activities depend on the genetic context of the Ikaros-binding sites, transcription factor-binding sites adjacent to the Ikaros-binding sites, and the cellular proteins physically associated with Ikaros (5, 33, 35, 39). It is possible that these two factors contribute to the differential effects of mutant Ikaros-binding sites on transcriptional activity from the viral promoter. At the present time, the array of protein binding sites in the MCF 247 U3 sequences that regulate virus replication in thymocytes and lymphoma induction have not been determined. Further studies are necessary to identify these protein-binding sites and to identify proteins that bind U3 sequences and interact with Ikaros to more fully elucidate molecular mechanisms regulating the LTR of integrated proviruses.
We thank A. Colberg-Poley and J. Koipally for valuable discussions and critical reading of the manuscript.
An Avery Scholar Award (N.L.D.) and Discovery Funds from Children’s Research Institute, the Board of Lady Visitors, and the Milheim Cancer Foundation supported N.L.D. and her laboratory.
†This work is dedicated to the memory of Ralph L. DiFronzo.