Department of Medicine, University of Cambridge, Level 5, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QQ, UK1
Author for correspondence: Nicola Rose. Fax +44 1223 336846. e-mail njr1004{at}mole.bio.cam.ac.uk
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Abstract |
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Transcription of an HTLV-I provirus requires the viral trans-activating protein, Tax. Tax regulates transcription through indirect binding, via secondary proteins, to various motifs within the viral 5' long terminal repeat (LTR), most notably the binding of the cAMP-responsive element binding protein (CREB) to the 21 bp imperfect repeats in the U3 region (reviewed in Flint & Shenk, 1997 ). In addition Tax is increasingly recognized as affecting the regulation of a number of cellular gene promoters (reviewed in Mesnard & Devaux, 1999
).
A striking feature of the T-cell clones is the selective inhibition of the autonomous proliferation by rapamycin (Höllsberg et al., 1992 ). This immunosuppressant (reviewed in Abraham & Wiederrecht, 1996
) has recently been shown to selectively suppress the mitogen-induced translation of a subset of mRNAs. The translation of most mRNAs remains unaffected (Terada et al., 1994
; Nielsen et al., 1995
; Mendez et al., 1996
; Jeffries et al., 1997
). A feature of the affected mRNAs is the presence of a 5'-terminal oligopyrimidine tract (5' TOP) downstream of their N7-methylguanosine (m7G) cap structures. This motif is composed of between 5 and 15 nucleotides the first of which is invariably cytosine. It has been proposed that rapamycin binds a member of the FK506-binding protein family, FKBP-12, creating a complex which binds with the FKBP-12rapamycin-associated protein (FRAP) kinase (reviewed in Brown & Schreiber, 1996
). Kinase activity is abrogated preventing phosphorylation, and hence activation, of eukaryotic initiation factor 4E (eIF4E)-binding protein, 4E-BP1 (Pause et al., 1994
). eIF4E possesses helicase activity: unphosphorylated 4E-BP1 sequesters free eIF4E forming an inactive complex thus preventing association of the elongation factor with mRNA cap structures. This coupling is essential for efficient translation as it is speculated that further initiation factors are subsequently attracted whose function is to assist unwinding of secondary structure in the 5'untranslated regions of mRNAs. This pathway is distinct from previously described JakStat and RasMAP kinase pathways (Brown & Schreiber, 1996
).
Downstream of the U3 region lies the R region of the HTLV-I 5' LTR; this region harbours six motifs conforming to 5' TOP structural constraints. The first has a thymine as the initial base but is in the most conserved position for a rapamycin effect; the other five all begin with a cytosine. We investigated whether these motifs act as control elements for rapamycin-induced translational regulation through the use of LTR- and mutant LTR-chloramphenicol acetyltransferase (CAT) reporter plasmids. The first polypyrimidine motif of the wild-type, CR-CAT construct [the CAT gene driven by the HTLV-I 5' LTR (pU3R-I; Sodroski et al., 1984 ); Fig. 1
] was altered to a polypurine motif by site-directed mutagenesis (Kunkel et al., 1987
) via a pBluescript KSII(+) (Stratagene) intermediate which harboured a 716 bp XhoIHindIII LTR-containing fragment from CR-CAT. The sequence of the antisense mutagenic oligonucleotide used was 5' GCCGGGCGCGTCCTTCCTCCCTGCGAGCCCCCTC 3'. The CR-CAT R region deletion mutant [CR(
R)-CAT; Fig. 1
] was generated by PCR mutagenesis using Pfu. DNA polymerase (Stratagene). Sequence flanking the region to be deleted from CR-CAT was amplified using the opposing oligonucleotides
Ra (5' CTCGCATCTCTCCTTCAggcctctgta 3') and
Rs (5' gccccgataTCTGTTCTGCGCCGTTAC 3') where sequences in uppercase are complementary to LTR sequence and those in italics introduce a StuI (
Ra) or an EcoRV (
Rs) restriction site. 10 µg CR-CAT DNA was amplified in a 100 µl reaction volume comprising 1xnative reaction buffer (Stratagene), 4·5 mM MgCl2, 0·25 mM each dNTP, 0·12 µM each oligonucleotide and 0·02 units Pfu. DNA polymerase. A single incubation at 96 °C for 45 s was followed by 25 cycles comprising 96 °C for 45 s, 37 °C for 45 s and 72 °C for 10 min and an additional single extension step at 72 °C for 10 min. Gel-purified amplicon was restricted with the appropriate enzymes and circularized using a Rapid Ligase kit (Boehringer Mannheim). All plasmids were confirmed by sequencing.
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Transfection efficiency was assessed by transfection of plasmid CMV-GFP [green fluorescent protein (GFP) under control of the CMV promoter] into COS-1 cells in the presence and absence of rapamycin. The percentage of cells expressing GFP across the rapamycin concentration range was assessed following image capture on an Olympus IX70 fluorescent microscope using Image Pro Plus software. The ratio of GFP-expressing to non-expressing cells was calculated in three independent transfections and did not reflect the decrease of CAT activity seen across the same rapamycin concentration gradient (data not shown).
We investigated the HTLV-I-specific protein levels derived from cotransfected cells to confirm that Tax protein levels remained constant across the rapamycin concentration. Total cellular protein from 1x106 CR-CAT-transfected cells was harvested and 1/20 vol. blotted onto a nitrocellulose membrane using standard techniques. The primary detection antibody was a rabbit polyclonal antibody raised against the C terminus of Tax applied at a 1:400 dilution. The secondary, donkey anti-rabbit horseradish peroxidase-linked antibody (Amersham), was applied at a 1:1000 dilution. Proteins were detected using ECL reagents (Amersham), according to the manufacturers instructions, followed by autoradiography. There was no significant decrease in Tax levels with increasing concentrations of rapamycin (data not shown). This indicates that the observed decrease in acetylated CAT products is not due to reduced levels of Tax protein available to activate the 5' LTR in the co-transfection experiments.
A further observation made with the T-cell clones was that, despite being sensitive to rapamycin, the autonomous proliferation was not affected by cyclosporin A or FK506 (tacrolimus). These immunosuppressants are usually able to prevent T-cell proliferation by interfering with IL-2 transcription (Sehgal, 1998 ). We have shown that FK506 (Fujisawa GmbH, Munich, Germany) restores rapamycin-mediated abrogation of acetylation levels. Using plasmids CR-CAT and pcDNA3Tax/Rex in our reporter assay, transfected cells were grown in media containing (i) neither drug, (ii) 20 nM rapamycin, (iii) 40 µM FK506 or (iv) 20 nM rapamycin+40 µM FK506. Reporter gene activity was restored to near-wild-type levels when FK506 was present with rapamycin in the culture medium. FK506 alone had no suppressive effect on promoter activity as expected (Fig. 3
). This mirrors the effects witnessed in the T-cell clones (Höllsberg et al., 1992
). FK506 and rapamycin show considerable structural similarity (Abraham & Wiederrecht, 1996
). It is likely that FK506 binds the same receptor as rapamycin competitively and is able to restore gene expression by displacing it. As these experiments utilized a large molar excess of FK506 over that of rapamycin it is likely that FK506 saturates the binding site thus preventing binding of rapamycin. Higher concentrations of rapamycin are required to inhibit FRAP activity in vitro than in vivo (Brown et al., 1995
; Brunn et al., 1996
; Chen et al., 1997
; Scott et al., 1998
). It is therefore not unexpected that the required molar excess of FK506 over rapamycin in vitro might be larger than for the T-cell clones (Höllsberg et al., 1992
). This observation is mirrored by the concentrations used in our study. The crystal structure of the ternary complex formed by rapamycin, FKBP-12 and the FRAP-binding domain (FRB) revealed that rapamycin binds to the hydrophobic regions of the two molecules drawing them together (Choi et al., 1996
). It is possible that the rapamycinFKBP-12 complex has a greater affinity for FRAP than does FK506; thus abolition of the above association requires a high molar excess of FK506.
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Acknowledgments |
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The authors wish to acknowledge the scientific support of the HTLV European Research Network. This work was supported by a grant from the Wellcome Trust.
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References |
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Received 11 September 2000;
accepted 25 October 2000.