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Mutations in the Bovine Leukemia Virus Tax Protein Can Abrogate the Long Terminal Repeat-Directed Transactivating Activity Witho

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Mutations in the Bovine Leukemia Virus Tax Protein Can Abrogate the Long Terminal Repeat-Directed Transactivating Activity Witho Proc. Nail. Acad. Sci. USA Vol. 89, pp. 3957-3961, May 1992 Medical Sciences Mutations in the bovine leukemia virus Tax protein can abrogate the long terminal repeat-directed transactivating activity without concomitant loss of transforming potential (leukemogenesis/transactivation/zinc finger/retrovirus) Luc WILLEMS*, CATHERINE GRIMONPONT*, HUBERTINE HEREMANSt, NICOLE REBEYROTTOE, GAO CHEN*, DANIEL PORTETELLE*, ARStNE BURNY*§, AND RICHARD KETTMANN* *Department of Molecular Biology, Faculty of Agronomy, B5030 Gembloux, Belgium; tREGA Institute, B3000 Leuven, Belgium; tInstitut National de la Sante et de la Recherche Mddicale, F33076 Bordeaux, France; and §Department of Molecular Biology, University of Brussels, B1640 Rhode-St-Genese, Belgium Communicated by Marc Van Montagu, January 27, 1992 ABSTRACT The bovine leukemia virus Tax protein trans- In the human T-cell lymphotropic virus (HTLV) system, the activates gene expression directed by the viral long terminal Tax-I protein activates transcription of the genes encoding repeat (LTR) and contributes to immortalization of primary interleukin 2 and its receptor, allowing preferential prolifer- cells. Theoretical analysis of the protein sequence revealed the ation of the HTLV-infected T cells by an autocrine stimula- presence of a putative zinc ringer structure at its amino end. tion process (6). (ii) The Tax protein forms a complex with a Selected mutations in that region completely abolished trans- putative tumor-suppressor gene. Such interactions are in- activation, demonstrating its importance for LTR-directed volved in tumor induction by DNA tumor viruses (adenovi- gene regulation. However, these mutations did not interfere rus, papillomavirus). A role in regulating the expression of with the ability of tax to bind zinc or to contribute to immor- genes involved in cell proliferation, in differentiation, or in talization of primary cells. Thus, transactivation of bovine controlling the initiation ofDNA synthesis may be envisaged. leukemia virus LTR and target cell transformation are inde- According to the first hypothesis, transactivation and pendent functions of Tax and involve different functional transformation by Tax seem to be tightly correlated. In domains of the protein. contrast, the interaction of Tax with a putative tumor- suppressor gene could well involve sequences of the Tax The bovine leukemia virus (BLV) tax gene product (the Tax protein different from those implicated in transactivation. In protein, p34tax) is involved in viral replication and probably this case, it should be possible to isolate transactivation- in leukemogenesis. Tax is a nuclear phosphoprotein trans- deficient tax mutants that are still transformation-competent. lated from the 3' long open reading frame ofthe BLV provirus In this report, we demonstrate that Tax mutated in its and is able to increase long terminal repeat (LTR)-directed putative zinc finger structure may completely lose its trans- gene expression without direct binding to the LTR sequences activating activity while maintaining its transforming capac- (1, 2). As such, Tax acts as a positive regulator of viral ity. transcription. Another property of Tax is its ability to im- mortalize rat embryo fibroblast (REF) cells in culture (3). Furthermore, the Tax protein in cooperation with the Ha-ras MATERIALS AND METHODS oncogene induces foci in vitro that develop into tumors in Plasmids. Plasmids pSV2neo (7), pSV2neoEJ (8), and vivo. BLV tax can thus be classified in the immortalizing pLTRCAT (3) have been described. oncogene subgroup including the adenovirus ElA, simian with the virus 40 T-antigen, and myc genes. These data emphasize the Site-directed mutagenesis was performed pSGtax causal role of tax in BLV-induced tumorigenesis. plasmid (3). Single-stranded DNA was produced by using Previous direct mutagenesis of the tax gene failed to M13K07 helper phage and was extracted as described by identify the regions implicated in its functional activity (4). Promega. Mutants 1, 3, 4, and 5 (pSGtaxMl, -3, 4, and -5) All modifications (amino acid deletions or insertions) abro- were obtained by the Eckstein procedure using the Amer- gated transactivation, suggesting that the present BLV p341 sham kit and the oligonucleotides 5'-ATGCCTGGGGC- structure results from heavy evolutionary constraints. How- CCCCTCT-3', 5'-GCGGGCCCGCTGAGCGAC-3', 5'- ever, by taking advantage of hybrids between well-defined CGCTCACCGGCGAGACCC-3', and 5'-GCGAGACCGC- regions of Tax and the yeast GAL4 transactivator protein, a TCGTATCA-3', respectively. Mutant 2 (pSGtaxM2) was specific domain (amino acids 157-197) was identified as an constructed by the polymerase chain reaction (PCR) using activating region (5). This segment is located approximately two overlapping oligonucleotides containing the desired mu- in the middle of p34tax and is globally neutral (net charge of tation (5'-GCCCCCTCGGCGGGCCCC-3' and 5'-GGGGC- zero). This Tax domain contains 24% of the leucine residues CCGCCGAGGGGGC-3'). All the mutants were sequenced possibly involved in heterologous protein interactions. in order to verify the presence ofthe desired mutation and the The molecular mechanisms by which Tax induces cell absence of other modifications. transformation are not known. However, two main hypoth- The pTITtax+ vector is a prokaryotic expression plasmid eses (that are not mutually exclusive) can be put forward. (i) containing the tax gene cloned downstream of the T7 RNA The Tax protein transactivates transcription ofa cellular gene polymerase promoter (N.R. and R. Mamoun, unpublished involved in cell division or differentiation. The identification work). The Cla I-Nde I fragment of the pSGtaxMl-5 plas- of these genes and their mode of action remains conjectural. mids (these unique sites flank the sequence encoding the zinc The publication costs of this article were defrayed in part by page charge Abbreviations: BLV, bovine leukemia virus; CAT, chloramphenicol payment. This article must therefore be hereby marked "advertisement" acetyltransferase; HTLV, human T-cell lymphotropic virus; LTR, in accordance with 18 U.S.C. §1734 solely to indicate this fact. long terminal repeat; REF, rat embryo fibroblast. 3957 Downloaded by guest on September 24, 2021 3958 Medical Sciences: Willems et al. Proc. Natl. Acad. Sci. USA 89 (1992) finger) was introduced into the same sites of the pTITtax+ vector, giving rise to pTITtaxM1-5. Expression in Bacteria, Zinc Column Chromatography, and Western Blotting. HMS174 bacteria were transformed with the pTITtax+ and pTITtaxM1-5 vectors, cultivated in M9 medium containing ampicillin, and infected with bacterio- phage CE6 as described by Studier et al. (9). After 2 hr of M3 incubation at 370C, bacteria were pelleted and resuspended in ;ALA36 -.4-HIS36 /,, phosphate-buffered saline. M4 For the zinc column purification, the bacterial pellets were M2 GLY 33 -- CYS33 CYS s _GLY50 resuspended in 20 mM Hepes, pH 7.9/20% (vol/vol) glycer- -Zn ol/50 mM KCl/1 mM dithiothreitol/0.5 mM phenylmethyl- ml 30-.-Q-CYS30 HIS53 ----ALA53 sulfonyl fluoride/6 M guanidinium chloride and incubated for IGLY 1.5 hr at room temperature. The sample was then progres- M5 sively diluted with 20 mM sodium phosphate, pH 7.5/0.5% MET 1 PHE309 Tween 20/0.5 M NaCl to decrease the guanidinium chloride FIG. 1. Schematic organization of the Tax protein [from the concentration to 1 M. The samples were loaded on a 2-ml initiator methionine (MET 1) to the last amino acid (PHE 309)]. The fast-flow Sepharose column (Pharmacia) saturated with theoretical tetrahedral structure ofthe finger is coordinated by a zinc ZnCl2. After extensive washing (five column volumes) with ion (Zn) interacting with three cysteines (positions 30, 33, and 50) and 20 mM sodium phosphate, pH 7.5/0.5% Tween 20/0.5 M one histidine (position 53). Amino acid modifications in the mutants NaCl, bound proteins were eluted with the same buffer (Ml to M5) are indicated. containing 250 mM EDTA. Bacterial lysate proteins were separated in an SDS/10% and histidine residues were modified to either alanine or polyacrylamide gel. Proteins were transferred onto nylon glycine codons (Fig. 1). After mutagenesis, the nucleotide membranes and incubated with a mixture of anti-p34t1 sequences of the mutants were checked for the presence of monoclonal antibodies (dilution 1:200) (4). Immune com- the expected mutations and for the lack of nondirected plexes were revealed by the phosphatase colorimetric pro- modification. cedure (Promega). Interactions Between Zn2+ and Wild-Type or Mutant Tax. Chloramphenicol Acetyltransferase (CAT) Assays. D17 cells The tax gene was cloned into the pTIT prokaryotic expres- (from canine osteosarcoma) or primary REFs were trans- sion vector downstream ofthe 17 RNA polymerase promoter fected by the calcium phosphate coprecipitation method (10). (N.R. and R. Mamoun, unpublished work). After transfor- In short, 3 ,ug of pLTRCAT (which contains the BLV LTR mation of Escherichia coli HMS174 cells and infection with cloned upstream of the CAT gene) and 2 ,g of effector bacteriophage CE6 (which codes for the T7 RNA polymer- plasmid (pSGtax or pSGtaxMl-5) were used to transfect 3 x ase), p341 was correctly expressed as demonstrated by 105 cells. Cytoplasmic extracts were prepared 48 hr after Western blot analysis using a mixture of monoclonal anti- transfection and CAT activity was determined (11). bodies directed against the Tax protein (Fig. 2A, lane a). Transformation and Tumorigenicity Assays. REFs were In order to gain insight into the interaction between p34tax obtained by mincing 14-day Fischer rat embryos (Iffa Credo, and the Zn2+ ion, the bacterial lysate containing the Tax Brussels) in trypsin solution and were cultured in Opti-MEM protein was denatured with 6 M guanidinium chloride and (GIBCO) supplemented with 10% heat-inactivated fetal bo- chromatographed on a fast-flow Sepharose column primed vine serum. One million cells were transfected with 10 ,ug of with Zn2+. After washing, p34tax was absent from the eluate plasmid DNA by calcium phosphate coprecipitation (10). [Fig. 2A, lane b(+)]. p34tax was then eluted in the presence Ability of immortal cells to grow in the presence of 1% or of EDTA [Fig. 2A, lane c(+)].
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