HTLV-I antisense transcripts initiating in the 3'LTR are alternatively spliced and polyadenylated

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Detection of the HTLV-I antisense transcript in HTLV-I-infected cell lines. (A) Positioning of the HBZ antisense ORF in the HTLV-I proviral DNA. Primers used for RT-PCR experiments and the expected size of the amplified signal are indicated above the enlarged HBZ ORF. (B) RT-PCR analyses were performed on RNA samples from HTLV-I-infected cell lines using the 21-5 primer for RT and primer combinations presented in A for PCR analysis. Samples were tested for DNA contamination in RNA samples (lanes 1–2; no RT and no RT primer) and autopriming (lanes 3–4; in the presence of RT with no added RT primer). CTL represents PCR analysis with no added cDNA or RNA. M = 100 bp marker (the asterisk indicates the 600 bp band). Lanes 5 and 6 show the results of PCR using primers 23-3/21-5 and 21-4/21-5 to generate products of 400 bp and 450 bp, respectively.

Figure 2

Detection of the HTLV-1 antisense transcript in HTLV-I-producing 293T cells. (A) K30 and K30-3'/5681 proviral DNA constructs are depicted. The deleted region for the latter construct is shown. (B-C) 293T cells were transfected with 5 μg K30 (B) or K30-3'/5681 (C). RT-PCR analyses was then conducted on RNA isotated from transfected 293T cells. RT-PCR conditions and controls were performed as in fig. 1. M = lambda DNA (EcoRI/HindIII) marker.

Figure 3

HTLV-I antisense transcription initiates in the 3' LTR. (A) 5'RACE analysis was conducted using RNA samples from 293T cells transfected with the K30-3'/5681 proviral DNA construct. The resulting amplified products were run on an agarose gel. M = 100 bp marker (the asterisk indicates the 600 bp band). (B) Position of the identified CAP sites for antisense transcripts are depicted in the 3' LTR. Nucleotide numbering corresponds to the sense strand.

Figure 4

HBZ transcripts are alternatively spliced. (A) The position of splice junctions within the two HBZ SP1 and SP2 RNA are positioned relative to the 3'LTR and the HBZ ORF. Nucleotide numbering corresponds to the antisense strand. (B) Predicted amino acid sequences for all potential HBZ isoforms are shown above each cDNA sequence. Sequences from exons 1 and 2 are separated and identified accordingly. The AUG initiation codon in unspliced and SP1 HBZ RNAs are highlighted in bold. (C) RNA isolated from HTLV-I-infected cell lines and 293T cells transfected with 5 μg K30, K30-3'/5681 or ACH was analyzed by RT-PCR using RT primer 21-5 and PCR primers 21-5 and 20-19 (or 20–27 for ACH) (see panel A for positioning). (D) RNAs from cellular clones isolated from four different infected patients and from MT4 cells were analyzed by a modified RT-PCR protocol using a PCR primer overlapping the SP1 splice junction. M = 100 bp marker (asterisk indicates the 600 bp band).

Figure 5

Sequence comparison of the HBZ splice acceptor, splice donors SD1 and SD2 and encoding regions between various HTLV-I and STLV-I isolates. STLV-I and HTLV-I sequences taken from GenBank were compared with different segments of the antisense strand of the K30 proviral DNA (accession number L03561): position 1756–1779 (splice acceptor) (A), position 350–379 (splice donor 1) (B) and position 182–239 (splice donor 2) (C). Comparisons were also made with the splice acceptor and splice donor consensus sequences (shown below compared stretches) and the corresponding K30 sequence is underlined. Coding regions are presented in bold and amino acid sequences are also indicated above the compared nucleotide sequence. GenBank accession numbers are provided for each compared STLV-I and HTLV-I proviral DNA clones.

Figure 6

Identification of the polyA addition site of the HBZ transcript. (A), PolyA+ RNA and total RNA from 293T cells transfected with 5 μg K30-3'/4089 or ACH were analyzed by RT-PCR with the primers 21-5 and 20-19 (20–27 for ACH-transfected cells). Controls were performed for DNA contamination (lane 2) and autopriming (lane 3). CTL represents PCR amplification conducted in the absence of cDNA or RNA samples. M = 100 bp marker (the asterisk indicates the 600 bp band). (B) RNA samples from 293T cells transfected with 5 μg K30 or HTLV-I-infected MJ cells were analysed by 3' RACE. Amplified products were run next to a 100 bp marker (M). (C) Position of the polyA addition site (indicated with arrow) next to a consensus polyA signal and a GU-rich consensus sequence. The structure of the HBZ mRNA with the most representative HBZ spliced variant (SP1) and the 3' polyA tail is shown below. Dark boxes represent the coding portion of the transcript. The complete proviral DNA and the former HBZ ORF are also shown below. (D) HTLV-I sequences taken from GenBank were compared with polyA signals (position 3821–3880) located on the antisense strand of the K30 proviral DNA (accession number L03561). Comparisons were focussed on the AATAAA polyA signal, the cleavage site deduced from our 3'RACE results and the GT-rich sequence (underlined in the K30 proviral DNA sequence). GenBank accession numbers are provided for each compared HTLV-I proviral DNA clones.

Figure 7

Synthesis of the various HBZ isoforms. Cell extracts were prepared from 293T cells transfected with 4 μg pcDNA3.1-Myc-His HBZ, pcDNA3.1-Myc-His HBZ SP1, pcDNA3.1-Myc-His HBZ SP2 or the empty vector (-). HBZ isoforms were detected by Western blot using anti-Myc antibodies. The position of the SP1- and SP2-derived HBZ isoforms is indicated by arrows.

Figure 8

Importance of the SD/SA sequences and of the SP1-specific ATG for HBZ protein synthesis (A) 293T cells were co-transfected with 5 μg K30-3'-asLUC or K30-3'-asLUC mSA and 2 μg pActin-β-gal. Luciferase activities represent the mean value of three measured samples ± S.D and are expressed as normalised RLU for 5 × 10⁶ cells. (B). 293T cells were co-transfected with 5 μg K30-3'-asLUC or K30-3'-asLUC mSA and 2 μg pActin-βgal. RNA samples from transfected cells were analysed by a modified RT-PCR protocol (see Materials and Methods). Controls for DNA contamination (lanes 2 and 5) and autopriming (lanes 3 and 6) were included. M = 100 bp marker (the asterisk indicates the 600 bp band). (C) The K30-3'/4089 construct was mutated at the splice acceptor (mSA), the splice donor of SP1 (mSD1), the splice donor of SP2 (mSD2), the presumed ATG initiation codon of SP1 (mATG/e1) or the initially identified ATG initiation codon (mATG/int). Comparison of sequences between wild-type and mutated versions of K30-3'/4089 are depicted. (D) 293T cells were transfected with 2 μg pActin-β-gal and 5 μg pcDNA3.1-Myc-His HBZ, wild-type K30-3'/4089 or versions mutated for SA, SD1, SD2, ATG/e1 or ATG/int and nuclear extract from samples transfected with equal efficiency (based on β-gal read-outs) were analysed by Western blot using anti-HBZ antiserum. The position of the SP1-specific HBZ isoform is indicated by an arrow.

Figure 9

Functional properties of the SP1-derived HBZ isoform. (A) 293T cells were co-transfected with 2 μg of K30-LUC and increasing concentrations of pcDNA3.1-Myc-His HBZ SP1 Δ 5'UTR, along with the β-gal reporter vector. (B) CEM cells were co-transfected with the collagenase promoter-driven luciferase reporter construct (2 μg), pcDNA-c-Jun (1 μg), pcDNA3.1-Myc-His HBZ SP1 Δ 5'UTR (2 and 5 μg), and the β-gal reporter vector (5 μg). Luciferase activities represent the mean value of three measured samples ± S.D and are expressed as normalised RLU for 5 × 10⁶ cells. Fold inductions in panel B were calculated with respect to CEM cells transfected in the absence of pcDNA-c-Jun (set at a value of 1).