HTLV-I antisense transcripts initiating in the 3'LTR are alternatively spliced and polyadenylated
© Cavanagh et al; licensee BioMed Central Ltd. 2006
Received: 23 December 2005
Accepted: 02 March 2006
Published: 02 March 2006
Antisense transcription in retroviruses has been suggested for both HIV-1 and HTLV-I, although the existence and coding potential of these transcripts remain controversial. Thorough characterization is required to demonstrate the existence of these transcripts and gain insight into their role in retrovirus biology.
This report provides the first complete characterization of an antisense retroviral transcript that encodes the previously described HTLV-I HBZ protein. In this study, we show that HBZ-encoding transcripts initiate in the 3' long terminal repeat (LTR) at several positions and consist of two alternatively spliced variants (SP1 and SP2). Expression of the most abundant HBZ spliced variant (SP1) could be detected in different HTLV-I-infected cell lines and importantly in cellular clones isolated from HTLV-I-infected patients. Polyadenylation of HBZ RNA occurred at a distance of 1450 nucleotides downstream of the HBZ stop codon in close proximity of a typical polyA signal. We have also determined that translation mostly initiates from the first exon located in the 3' LTR and that the HBZ isoform produced from the SP1 spliced variant demonstrated inhibition of Tax and c-Jun-dependent transcriptional activation.
These results conclusively demonstrate the existence of antisense transcription in retroviruses, which likely plays a role in HTLV-I-associated pathogenesis through HBZ protein synthesis.
Natural antisense transcription has been described in several eukaryotic organisms and has been ascribed several functions [1–3]. Retroviruses have long been thought to lack antisense transcription and to rely on a single sense transcript for viral gene expression. Unspliced and spliced sense transcripts are thought to produce all viral proteins required for replication and survival in the infected host. Although a few studies have suggested that retroviruses might produce antisense transcripts with coding potential [4–10], the existence of such atypical RNAs has not been conclusively demonstrated. Recent identification of the HBZ (HTLV-I bZIP) protein, surprisingly encoded on the antisense strand of human T-cell leukemia virus type I (HTLV-I), revived the likely existence of antisense transcription among retroviruses .
HTLV-I is the etiological agent of adult T cell leukemia/lymphoma (ATLL) and HTLV-I-associated myelopathy (also termed tropical spastic paraparesis) (HAM/TSP) [12–17]. In the sense strand, the HTLV-I genome encodes typical retroviral proteins as well as other more HTLV-I-specific proteins, such as Tax. The viral Tax protein has been suggested to play an important role in the diseases occurring in HTLV-I-infected patients. Tax is an important transactivator and acts upon the HTLV-I gene expression by promoting protein complexes involving CREB and the CREB binding Protein (CBP) on the TRE1 regions present in the HTLV-I long terminal repeat (LTR) promoter region.
In this report, we have focussed on the characterization of the HBZ-encoding antisense transcript produced from the HTLV-I genome. Our results show that HBZ-encoding transcripts initiate in the 3' LTR, are polyadenylated and are alternatively spliced. Furthermore, the HBZ isoform produced from the most abundant spliced form possesses similar functional properties to the one previously attributed to the former HBZ isoform. These results will strongly impact the field of retrovirology, being the first clear demonstration of the existence of antisense transcription in retroviruses.
Results and discussion
Detection of the antisense transcript in transfected 293T cells and HTLV-I-infected cell lines
The identification of the HBZ gene has raised several important issues regarding the various mechanisms governing retroviral gene expression. Its atypical positioning in the HTLV-I genome (Fig. 1A) warranted further investigation and a more thorough characterization of the HBZ-encoding RNA was thus conducted.
These results clearly demonstrated the existence of an antisense transcript in HTLV-I, which included the HBZ sequence. The use of HTLV-I proviral DNA clones and of infected cell lines demonstrated that a wide range of HTLV-I clones is capable of producing this transcript. Furthermore, data from the transfected 293T cells with the 5'LTR-deleted proviral DNA construct also argued that sense transcription could impede antisense transcription, which might be expected.
HBZ transcripts initiate in the 3' LTR at different position
These results hence demonstrated that the HBZ transcript initiated in the 3' LTR at multiple positions. This multiplicity of initiation sites might be a consequence of the absence of TATA boxes at close distance. Our results parallel the data presented on the localisation of the transcription initiation sites specific for HIV-1 antisense transcripts, which were near or in the 3' LTR region [6, 7]. Similar to HIV-1, based on the positioning of the transcription initiation sites, it is expected that the promoter region for HTLV-I antisense transcription would be present in the 3'LTR region as initially suggested by Larocca et al. . Further investigations are required to determine the mechanism of regulation of this promoter region and to evaluate the possible involvement of adjacent cellular DNA in these regulatory mechanisms.
HBZ transcripts are alternatively spliced
Another important feature of the SP1 RNA was the presence of the splice acceptor downstream of the AUG initiation codon initially suggested by Gaudray et al. . However, further analysis of the SP1 RNA sequence originating in the 3' LTR revealed a new in frame AUG initiation codon that permits proper initiation of HBZ translation (Fig. 4B). In contrast, no in frame AUG was identified within the HBZ SP2 RNA sequence flanking the splice junction and downstream of the first stop codon. It could however be possible that a non-AUG initiation codon (for example, GUG or CUG) could allow proper initiation of translation from this RNA. In fact, non-AUG initiation codons have been proposed for other HTLV-I proteins . Amino acid sequence changes introduced limited variation in overall amino acid composition between these two potentially new HBZ isoforms and the previously published HBZ amino acid sequence . For example, seven amino acids from the amino terminus of the original HBZ isoform would be substituted by four amino acids in the SP1-encoded isoform.
To demonstrate that both HBZ splice variants existed in HTLV-I-infected and transfected cells, RT-PCR analysis was performed on isolated RNA with the forward primer 20-19 derived from the transcribed spliced 3' LTR and the reverse primer 21-5 located downstream of the identified splice acceptor (see Fig. 4A). This RT-PCR strategy was expected to generate a 684 bp signal for the HBZ SP1 RNA and a 544 bp signal for the HBZ SP2 RNA. Indeed for both tested HTLV-I-infected cell lines, i.e. C8166-45 and MJ, an amplified signal of the expected size for SP1 was present (Fig. 4C). However, the SP2 variant was only weakly detected in these infected cell lines. Similar analyses conducted in 293T cells transfected with K30, K30-3'/5681 and a different proviral DNA clone, i.e. ACH amplified the spliced HBZ SP1 and SP2 templates (very faint for SP2). Because of nucleotide sequence variation of the LTR region complementary to primer 20-19, the forward primer 20–27 (similar to the 20-19 primer, but with nucleotide sequence specificity for ACH) was used for RT-PCR analyses of ACH-transfected cells. To further demonstrate the existence of these spliced transcripts, the detection of HBZ spliced variants was evaluated in cell clones derived from HTLV-I-infected individuals (Fig. 4D). Taking in consideration the variability occurring in between HTLV-I isolates in the LTR region, primers from the HBZ-coding sequence that encompass the highly conserved splice junctions of SP1 and SP2 were used to detect antisense transcripts. Analysis of amplified products indeed demonstrated expression of the HBZ SP1 RNA variant in certain cell clones while other clones appeared negative. As a control, HTLV-I-infected MT4 cells were similarly analyzed and demonstrated amplification of the expected band. However, no signals were observed with primers overlapping the splice SP2 junction (data not shown).
These data thereby provide evidence for the existence of splicing events occurring in the HTLV-I antisense transcripts. A recent study has also confirmed the spliced nature of the HBZ RNA, having demonstrated the existence of the SP1 HBZ transcript . In our study, we further suggest that, although the SP1 RNA variant represents the most abundant transcript, other spliced variants could exist (such as SP2). We have also importantly demonstrated that SP1 RNA variant is present in patient-derived cell clones, and unlike Satou et al. , not all tested cell clones were found to be positive for HBZ expression. Although more data is needed to understand the significance of these findings, these data might be indicative of a possible relationship between lack of HBZ expression and disease outcome. Furthermore, it is possible that the various identified HBZ RNA variants might contribute differently to HBZ protein synthesis. However, our PCR analysis has not permitted us to detect unspliced HBZ RNA in HTLV-I-infected cells or transfected 293T cells. Obviously, the designed PCR protocol used above favours shorther size PCR fragments derived from spliced HBZ RNA. Nonetheless, the formerly described HBZ isoform  could be produced from unspliced HBZ RNA although possible mechanisms might be needed for proper translation to occur from the resulting long 5' untranslated region of such a transcript. It should also not be excluded that other splice variants could also exist and contribute to post-transcriptional regulation of HBZ expression. Further experiments are presently underway to clearly establish if these other transcripts are indeed produced in infected cells.
Positioning of the polyA addition site
These results hence have permitted to identify the 3'end of the spliced HBZ transcript. Taking into account the results of Fig. 4, we predict the size of the more abundant HBZ SP1 transcript to be 2.4 kb. This characterization of the HTLV-I antisense transcript hence agrees with previous findings of Larocca et al., who detected a 2.5 kb antisense transcript . Our results also confirm the Northern blot data of this former study as to the possible existence of an intron at a similar position in the antisense transcript of HTLV-I. Furthermore, presence of the 3' untranslated region might suggest a potential role for this region in post-transcriptional regulation of HBZ expression. Further experiments will be needed to assess this possibility.
Synthesis of the various HBZ isoforms
To confirm these data and extend our analyses to other splice consensus sequences and to the two different possible AUG initiation codon, mutations of the K30-3'/4089 construct specifically targeting SD/SA consensus sequences, as well as both putative AUG translation initiation codons, were specifically generated (Fig. 8C). Following transfection of wild-type and mutated K30-3'/4089 constructs into 293T cells, the HBZ protein was detected by Western blot (Fig. 8D). Significantly less HBZ protein was detected when the proviral DNA was mutated in the SA or SP1 SD sequence, or the SP1-specific AUG, suggesting that SP1 mRNA is important for HBZ protein synthesis. On the other hand, mutation of the intronic AUG or the SP2 SD sequence had little impact on HBZ protein levels. Interestingly, transfection of 293T cells with a vector expressing the original HBZ isoform produced HBZ protein of a higher molecular weight than K30 HBZ protein, which may depend on presence of the Myc tag and differences in amino terminus.
These data indeed suggested the possible existence of different HBZ isoforms. In agreement with our RT-PCR analysis, our results suggest that the SP1 RNA-translated HBZ isoform contributes importantly to overall HBZ protein synthesis. It should be noted that, in our Western blot analyses, a constant shift in migration of the SP1-derived isoforms is observed when compared to the other HBZ isoforms. Although these results are unexpected given the small differences in amino acid composition between the various HBZ isoforms, we could speculate that the SP1 isoform is differently modified at a post-translational level, which would then account for these suggested variations. Further experiments are needed to address this issue.
Functional properties of the SP1 RNA-derived HBZ isoform
In this study, we have thoroughly characterized the antisense transcripts produced from the HTLV-I retrovirus and responsible for the synthesis of the previously described HBZ protein. Using different RT-PCR approaches, our results first demonstrated that antisense transcripts could be detected in HTLV-I-infected cell lines and 293T cells transfected with proviral DNA and initiated in the R and U5 segments of the LTR. Transcripts were alternatively spliced at a varying frequency and produced two new isoforms with translation initiating in exon 1, at least for the most abundant variant. PolyA site was positioned at a distance of 1450 nt form the HBZ stop codon and occurred next to known polyA signals. Mutation experiments also showed the importance of the SP1 mRNA for HBZ protein synthesis. Transfection experiments also indicated that the isoform produced from HBZ SP1 mRNA demonstrated suppression of AP-1- and Tax-dependent transcriptional activation.
Our results strongly argue that the major spliced antisense transcript is responsible for producing the HBZ protein. However, the minor spliced form and the unspliced HBZ transcript may be important sources of HBZ expression in other cellular contexts or states. More data are needed to indeed confirm that the SP2 transcript is indeed produced in several other HTLV-I-infected cells and that both SP2- and unspliced derived HBZ isoforms can be detected at the protein level in infected cells. In light of the possible existence of multiple HBZ RNA variants, it could then be postulated that transcriptional and post-transcriptional mechanisms might regulate HBZ mRNA and protein levels and drive the type of transcript (and isoform) being produced. These mechanisms might involve other HTLV-I viral proteins. Regulation of HBZ protein levels and functions will likely modulate HTLV-I latency and pathogenesis. Detection of varying levels of the major spliced form of HBZ RNA in several cellular clones isolated from infected patients (even in the same patient) is highly relevant in this regard. Future investigations will need to address the different mechanisms regulating HBZ protein synthesis.
Our study has an important impact on the field of retrovirology, in general. These data provide the strongest evidence for the existence of retroviral antisense transcripts, which have previously been seen as potential artefacts. It is likely that antisense transcripts are also produced in other retroviruses (human and non-human) and could encode for proteins as previously proposed for HIV-1 and FIV [5, 8, 22, 27]. Based on our data, further studies on antisense transcription are warranted, specifically in complex retroviruses. The presence of one or more potentially new genes in these transcripts would provide important new insights into retroviral regulation and function, resulting in a more complete understanding of these viruses. It will be of great interest to determine whether regulatory processes linked to antisense transcription are active in HTLV-I, such as the antisense effect previously suggested for these transcripts in HIV-1 [28, 29].
Cell lines and antibodies
All T-cell lines were maintained in RPMI-1640 culture medium supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 2 mM glutamine, 100 U/ml penicillin G, and 100 μg/ml streptomycin. 293T cells were grown in supplemented DMEM. Peripheral blood mononuclear cells (PBMCs) from HTLV-I-infected individuals were cloned by limiting dilution (0.1 cell per well) in the presence of feeder cells (γ-irradiated allogeneic PBMCs (5 × 105 cells/ml)) and in complete RPMI 1640 containing 10% filtered human serum AB, recombinant IL-2 (100 U/ml), PHA (1 μg/ml). Positive cultures were transferred into 96 U-bottom plates and stimulated every 14 days with PHA and fresh feeder cells (1 × 106 cells/ml). Derived cellular clones were identified as YB034 to YB356 (patient 1), J1+ (patient 2), 1P8 (patient 3) and Jas081 (patient 4). The anti-HBZ antiserum has been described previously . Mouse anti-Myc antibody 9E10 was purchased from Sigma. Goat anti-mouse and anti-rabbit IgG antibodies coupled to the horse radish peroxidase were obtained from Amersham Bioscience.
Vectors and site-directed mutagenesis
HTLV-I proviral DNA constructs used in this study were ACH  and K30 . The K30-3'/5681 and K30-3'/4089 constructs were derived from K30 DNA by subcloning 3' segments (positions 5681 to 9043 and 4089 to 9043, respectively) in pBlueScript KS. The K30-LUC proviral DNA construct contains the luciferase reporter gene cloned in frame to the ATG initiation codon of the envelope gene has been previously reported . The K30-3'-asLUC construct was generated from the K30-3'/5681 vector by introducing a NcoI site at position 1791 (antisense strand) located in the HBZ-coding region and downstream of the splice acceptor with the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) and the primer 5'-GCTTGCCTGTGACCATGGCCGGAGGACCTGC-3' and the complementary primer. A luciferase reporter gene/SV40 polyA cassette isolated from pGL3-Basic (Promega) was cloned in the NcoI/SalI sites concomitantly deleting the sequence positioned downstream of the mutated region of the HBZ ORF. Mutagenesis of the splice acceptor region at position 1766 (antisense strand) was similarly conducted using the primer 5'-CTTTGTATCTGTCTGGCTGTTTCGATGCTTGCCTG-3' (with the mutated sequence indicated in bold) generating K30-3'-asLUC mSA. Other mutagenesis strategies were undertaken in the K30-3'/4089 construct in order to mutate the splice acceptor (as indicated above), splice donor 1 (position 368; 5'-GGCGGCCTCAGCAAGGGCGGCGGG-3'), splice donor 2 (position 228; 5'-GCCGGCTGAGTCTAGCAAGGCTCCAAGGG-3'), the intronic ATG (position 1746; 5'-GTGGGCTGATAATAAGCCTAGTTAACTTTGTATCTG-3') and the exon 1 ATG (position 356; 5'-CAACCGGCGTGGAACGCGGCCTCAGGTAGGG-3'). The pActin-β-gal vector contains the β-galactosidase gene under the control of the β-actin promoter. SP1 and SP2 HBZ cDNAs (including the 5' untranslated region (UTR)) were amplified and cloned in the pcDNA3.1-Myc-His A expression vector generating pcDNA3.1-Myc-His HBZ SP1 and pcDNA3.1-Myc-His HBZ SP2, respectively. An equivalent construct bearing the HBZ SP1 cDNA without the 5' UTR was also produced (pcDNA3.1-Myc-His HBZ SP1 Δ 5'UTR). The construct expressing a Myc-tagged version of the former HBZ isoform (pcDNA3.1-Myc-His HBZ), the collagenase promoter-luciferase and pcDNA3.1-c-Jun vectors have been previously described [18, 20].
Transfection and gene reporter assays
293T cells were transfected with 5–10 μg of DNA through the calcium phosphate protocol as previously described . CEM cells were transfected according to a previously described protocol . In transfection experiments with K30-LUC or collagenase promoter-luciferase vectors, the pcDNA3.1-Myc-His A empty vector was used to standardize DNA quantity in between transfection samples. Transfected cells were lysed 48 hours post-transfection in a lysis buffer (25 mM Tris phosphate, pH 7.8, 2 mM DTT, 1% Triton X-100, 10% glycerol) and luciferase activity read out was performed with the MLX microplate luminometer (Dynex Technologies) with a single injection of a luciferase buffer (20 mM tricine, 1.07 mM (MgCO3)4·Mg(OH)2·5H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 220 μM Coenzyme A, 4.7 μM D-Luciferin potassium salt, 530 μM ATP, 33.3 mM DTT). Each sample was co-transfected with a β-gal-expressing vector for normalisation. The β-galactosidase activity was measured using the Galacto-Light™ kit (Applied Biosystems, Bedford, MS) according to manufacturer's suggestions. Luciferase activity are presented in Relative Light Units (RLU) and represent the calculated mean ± SD of three transfected samples normalised by the measured β-galactosidase activity.
RT-PCR and 5'/3' RACE analyses
Total RNA was extracted by the Trizol reagent (Invitrogen) from HTLV-I-infected cell lines or transfected 293T cells. PolyA+ RNA was purified from lysed cell samples using the Poly(A)Purist™ Kit (Ambion) and according to manufacturer's instructions. RT-PCR analyses were conducted using RT primer 21-5 (5'-AACTGTCTAGTATAGCCATCA-3'). Prior to RT, RNAs were treated with DNAseI and incubated at 70°C for 5 min. RNA (5 ug) was then added to 1.5 μM RT primer, 1 mM dNTPs, 15 U AMV reverse transcriptase (USB), 1× AMV Reaction buffer and 10 U SUPERase·In RNAse inhibitor (Ambion) and RT reactions were incubated for 2 hours at 42°C. Aliquots from the RT reactions were then PCR amplified in the presence of 1.25 U Taq DNA polymerase (New England BioLabs Inc.), 1× ThermoPol buffer, 20 μM dNTP, 1.5 uM of each primer and 4% DMSO using a Tgradient thermocycler (Biometra). Primers added to the PCR reactions were the reverse 21-5 primer and the forward primer 21-4 (5'-TGCTGGTGGAGGAATTGGTGG-3'), 23-3 (5'-CAAGGAGGAGGAGGAAGCTGTGC-3'), 20-19 (in the 3' LTR: 5'-CGCAGAGTTGAACAAGCAGG-3') and 20–27 (in the 3' LTR specific for ACH: 5'-CGCAGAGGTGAGCAAACAGG-3'). PCR conditions were as follow: a first step of denaturation at 94°C for 5 min followed by 35 to 40 cycles of denaturation (94°C for 1 min.), annealing (60°C for 1 min.) and extension (72°C for 1 min.) and a final extension at 72°C for 5 min. In RT-PCR analyses of the cellular clones isolated from infected patients, a modified RT-PCR approach designed to reduce autopriming was carried out using the RT primer 42-5 consisting of a HBZ-specific 3' end (in bold) and a non-hybridizing 5' end (5'-AGTAGAGTATCGACGATACACAACTGTCTAGTATAGCCATCA-3) followed by PCR amplification with reverse primer 24-21 (specific to the 5' end of the RT primer: 5'-AGTAGAGTATCGACGATACACAAC-3') and forward primer 18-4 (5'-ATGGCGGCCTCAGGGCT-3') (overlapping the SP1 splice junction). For amplification of the SP2 spliced variant, in these experiments, primer 18-4 was substituted by forward primer 19-19 (5'-CGGCTGAGTCTAGGGCTGTTT-3') during PCR amplification. A similar strategy was used for RT-PCR analysis of transfection experiment with the different K30-3'-asLUC constructs, in which the RT primer was 49-1 (luciferase-specific 3' end in bold) (5'-CCATCATCACATTGGAATATCGCCTTTCTTTATGTTTTTGGCGTCTTCC-3) and forward and reverse primers for PCR were 20-19 and 25–30 (specific to the 5'end of the RT primer: 5'-AGTAGAGTATCGACGATACACAAC-3') respectively. RT-PCR amplifications were controlled for DNA contamination (RNA samples with no RT step) and autopriming (cDNA synthesis reaction in the presence of RT with no added specific primer). Extremities of the HBZ RNA (5' and 3') were analyzed from isolated total RNA with the FirstChoice RLM-RACE kit from Ambion according to the manufacturer's instructions. For the 5'RACE protocol, cDNAs were synthesized with random decamers and the subsequent two PCR rounds were conducted with the supplied 5'RACE outer and inner primers and HBZ-specific primers 21-9 (5'-TCCTCTTTCTCCCGCTCTTTT-3') and 20-18 (5'-CCGCGGCTTTCCTCTTCTAA-3') successively. For the 3'RACE protocol, cDNA synthesis was performed in the presence of the supplied 3'RACE adapter; PCR amplification was achieved through 3'RACE inner and outer primers and primers 24-20 (5'-CGAGGATGTGGTCTAGGTTAGA-3') and 22-15 (5'-GGCTGGGTTCGGTATTAAGGAA-3') derived from the sequence downstream of the HBZ stop codon. Amplified products were then directly sequenced or first cloned in pBlue Script KS+ (Stratagene) before sequencing.
Western blot analysis
Transfected 293T cells were lysed and total protein or nuclear extracts were prepared as previously described [26, 35]. Equal quantities of extracts were run on a SDS-12% PAGE and transferred to PVDF membranes (Millipore). The blot was next blocked in PBS 1X/5% milk and incubated with a mouse anti-Myc 9E10 antibody (dilution 1/1000) or anti-HBZ antiserum (dilution 1/1000). After several washes, signals were revealed by the addition of peroxydase-conjugated goat anti-mouse IgG (dilution 1/2000) or goat anti-rabbit IgG (dilution 1/10000) antibodies and subsequent incubation with the ECL reagent (Amersham Pharmacia Biotech). Membranes were exposed on hyperfilms ECL (Amersham Pharmacia Biotech).
List of abbreviations
human immunodeficiency virus type 1
human T-cell leukemia virus type I
long terminal repeat
This work was supported by the Canadian Institutes of Health Research (grant n° HOP-67257) and The Cancer Research Society (B.B.) and by grants from the Centre National de la Recherche Scientifique (CNRS)-Université Montpellier I (J.M.M.), from the Association pour la Recherche sur le Cancer (ARC n° 3606) (J.M.M.) and from the Ligue Nationale contre le Cancer (É.W.). B.B. was supported by a FRSQ scholarship (Junior 2) and presently holds a Canada Research Chair (Tier 2). We would like to thank J.N. Brady and C. Power for carefully reading the manuscript and for their helpful comments. We are also thankful to Éric Legault for excellent technical assistance.
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