Human T-cell lymphotropic virus type 3: complete nucleotide sequence and characterization of the human tax3 protein

Abstract

We and others have recently uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3), the third member of the HTLV family. We have now sequenced the full-length HTLV-3Pyl43 provirus. As expected, HTLV-3Pyl43 contains open reading frames corresponding to the gag, pol, env, tax, and rex genes. Interestingly, its long terminal repeat (LTR) includes only two Tax-responsive elements, as is the case for type 3 simian T-cell lymphotropic viruses (STLV-3). Phylogenetic analyses reveal that HTLV-3Pyl43 is closely related to central African STLV-3. Unexpectedly, the proximal pX region of HTLV-3Pyl43 lacks 366 bp compared to its STLV-3 counterpart. Because of this deletion, the previously described RorfII sequence is lacking. At the amino acid level, Tax3Pyl43 displays strong similarities with HTLV-1 Tax, including the sequence of a PDZ class I binding motif. In transient-transfection assays, Tax3Pyl43 activates the transcriptions from HTLV-3, HTLV-1, and HTLV-2 LTRs. Mutational analysis indicates that two functional domains (M22 and M47) important for transactivation through the CREB/ATF or NF-kappaB pathway are similar but not identical in Tax1 and Tax3Pyl43. We also show that Tax3Pyl43 transactivates the human interleukin-8 and Bcl-XL promoters through the induction of the NF-kappaB pathway. On the other hand, Tax3Pyl43 represses the transcriptional activity of the p53 tumor suppressor protein as well as the c-Myb promoter. Altogether, these results demonstrate that although HTLV-3 and HTLV-1 have only 60% identity, Tax3Pyl43 is functionally closely related to the transforming protein Tax1 and suggest that HTLV-3, like HTLV-1, might be pathogenic in vivo.

Figures

FIG. 1.

HTLV-3Pyl43 genome. (A) PCR strategy for amplifying the complete HTLV-3Pyl43 provirus. The 19 PCR fragments are shown. (B) Schematic representation of the HTLV-3Pyl43 genome. (C) The HTLV-3 proximal pX sequence is shorter than that in the prototypical STLV-3 strain. PCR amplification using high-molecular-weight DNA extracted either from HTLV-3Pyl43 (lanes 1 and 4) or from STLV3PH969 (lanes 3 and 6). The primers used for both nested-PCR experiments are described in Table S1 in the supplemental material. DNA was not added to the PCR mixture in lanes 2 and 5. Lanes 3 and 6, expected sizes of 1,027 bp and 1,097 bp for STLV-3PH969; lanes 1 and 6, observed sizes of 660 bp and 730 bp for HTLV-3Pyl43. (D) Organization of the proximal pX region. The deleted region, which encompasses OrfII, is shown. nt, nucleotides.

FIG. 2.

HTLV-3Pyl43 belongs to the central African STLV-3 group. The unrooted phylogenetic tree was generated with the neighbor-joining method, performed with the PAUP program (v4.0b10), using 6,812 bp of the concatenated gag-pol-env-tax genes of the Pyl43 strain and the complete sequences of PTLVs available in GenBank. Bootstrap values (1,000 replicates) are noted on the branches of the tree. Branch lengths are drawn to scale, and the bar indicates a 0.1-nucleotide substitution per site.

FIG. 3.

Schematic display of the different putative HTLV-3Pyl43 Tax functional regions. Representative HTLV-1, HTLV-2, and STLV-3 sequences as well as all published HTLV-3 sequences are shown. HTLV-2MO is used as a prototypical sequence. The HTLV-1 Tax NLS, nuclear export signal (NES), CBP/p300 binding sequence, M22 domain, M47 domain, and CR2 binding domain as well as the PDZ binding motif are shown as reported in references , , , , , and .

FIG.4.

Tax3Pyl43's intracellular localization is similar to that of Tax1. (A) HeLa cells were transiently transfected with GFP (a), GFP-Tax1 (b), GFP-Tax2 (c), and various GFP-Tax3Pyl43 (d, e, and f) or GFP-Tax3602 (g) plasmids. Images of cells that are representative of the entire population are shown. (B) Western blot analysis of GFP, GFP-Tax1, GFP-Tax2, GFP-Tax3Pyl43, GFP-Tax3Pyl43 M22, GFP-Tax3Pyl43 M47, and GFP-Tax3602. Cell lysates (70 μg) were subjected to electrophoresis and probed with a GFP or a β-tubulin antibody. ns, nonspecific.

FIG.4.

Tax3Pyl43's intracellular localization is similar to that of Tax1. (A) HeLa cells were transiently transfected with GFP (a), GFP-Tax1 (b), GFP-Tax2 (c), and various GFP-Tax3Pyl43 (d, e, and f) or GFP-Tax3602 (g) plasmids. Images of cells that are representative of the entire population are shown. (B) Western blot analysis of GFP, GFP-Tax1, GFP-Tax2, GFP-Tax3Pyl43, GFP-Tax3Pyl43 M22, GFP-Tax3Pyl43 M47, and GFP-Tax3602. Cell lysates (70 μg) were subjected to electrophoresis and probed with a GFP or a β-tubulin antibody. ns, nonspecific.

FIG. 5.

Tax3Pyl43 contains a PDZ binding domain. (A) Alignments of the different Tax1, Tax2, and Tax3 C-terminal sequences. (B) Coimmunoprecipitations of GFP-Tax with pSGF-cl.15-FLAG, a PDZ domain-containing protein. 293T cells were transiently transfected with the different GFP constructs together with pSGF-cl.15 DNA. (C) GFP-Tax input. Lanes 1 to 7, direct Western blot (WB) using 1:10 dilutions of the extracts used for the immunoprecipitations (IP). NS, nonspecific.

FIG. 6.

Tax3Pyl43 is a viral transactivator of the HTLV-1, HTLV-2, and HTLV-3 promoters. Jurkat cells were transiently transfected with 250 ng of HTLV-3 LTR-luc (A), HTLV-1 LTR-luc (B), or HTLV-2 LTR-luc (C) or 200 ng of NF-κB-luc together with 2 μg of the pSG5M empty vector or with the different pSG5M-Tax constructs (D). (A to D) Results presented are average values from at least three independent experiments. (E) Detection of the different tax3 mRNAs by RT-PCR in the RNA extracted from mock-transfected (lanes 1 and 2) or Tax3 transiently transfected (lanes 3 to 8) Jurkat cells. Lanes 1, 3, 5, and 7, RT was not added to the PCR mix.

FIG. 7.

The ability of Tax3Pyl43 to transactivate or to transrepress cellular promoters is similar to that of Tax1. (A, top) Tax3Pyl43 and Tax3Pyl43 M22 but not Tax3Pyl43 M47 repress human p53 functions. HeLa cells were transiently transfected with 100 ng of PG13py-luc, 200 ng of the different Tax3Pyl43 constructs, or 200 ng of the empty pSG5M vector and 100 ng of a p53-expressing plasmid, as previously reported (26). (A, bottom) Sixty micrograms of protein extracts from the lysates obtained after transfection was run and probed for p53 with the DO-1 antibody or with anti-β-tubulin antibody. Lane 1, empty vector; lane 2, p53 and no Tax; lane 3, p53 plus Tax3; lane 4, p53 plus Tax3M22; lane 5, p53 plus Tax3M47. (B) Tax3Pyl43 and Tax3Pyl43 M47 but not Tax3Pyl43 M22 repress the c-Myb promoter through the activation of the NF-κB pathway. Jurkat cells were transiently transfected with 1 μg of MRE-luc and 2 μg of the different Tax3 constructs or with the empty pSG5M plasmid. (C) Tax3 Pyl43 transactivates the Bcl-XL promoter through the activation of the NF-κB pathway. Jurkat cells were transfected with 500 ng of the Pr-Bcl-XL construct together with 2 μg of the different Tax3 constructs or the pSG5M empty plasmid. When needed, 1 μg of the pCMV4-HA-IκBS32/36Α dominant negative plasmid was added. (D) Tax3Pyl43 transactivates the IL-8 promoter through NF-κB induction. Jurkat cells were transfected with 100 ng of the IL-8536 plasmid (30) together with 2 μg of the different Tax3 and Tax1 constructs. When needed, 1 μg of the IκBαS32/36A dominant negative plasmid was added. (A to D) Results presented are average values from at least two independent experiments.