A 10-Amino Acid Domain within Human T-cell Leukemia Virus Type 1 and Type 2 Tax Protein Sequences Is Responsible for Their Divergent Subcellular Distribution

Abstract

Human T-cell leukemia virus type 1 and type 2 (HTLV-1/2) are related retroviruses that infect Tlymphocytes. Whereas HTLV-1 infection can cause leukemia, HTLV-2 has not been demonstrated to be the agent of a hematological malignant disease. Nevertheless, the virally encoded Tax-1 and Tax-2 transactivators display a high percentage of similarity. Tax-1 is a shuttling protein that contains a noncanonical nuclear localization signal as well as a nuclear export signal. The presence of the nuclear localization signal and the nuclear export signal domains in the Tax-2 sequence has not been determined. The distribution of Tax-2 in infected cells is not known but has been assumed to be similar to that of Tax-1. By using a Tax-2-specific antibody, we report here that Tax-2 is located predominantly in the cytoplasm of the HTLV-2 immortalized or transformed infected T-cells. These results were confirmed after transient transfection of untagged Tax-1 and Tax-2 constructs, histidine tag Tax1/Tax2, GFPTax, and Tax-GFP fusion constructs in several cell lines. We show that this unanticipated localization is not due to a default in the Tax-2 nuclear localization signal functions nor to major differences in Tax-2 versus Tax-1 binding to the IKK␥/NEMO protein. In addition, we demonstrate that inhibiting the proteasome results in a relocalization of Tax-1 in the cytoplasm, similar to that of Tax-2. By using a series of Tax-1/Tax-2 chimeras, we determined that the minimal domain that is necessary for Tax-2 peculiar distribution encompasses amino acids 90 -100. Finally, we show a high correlation between intracellular localization of Tax and their NF-B or CREB transactivating ability.

The tax protein from the primate T-cell lymphotropic virus type 3 is expressed in vivo and is functionally related to HTLV-1 Tax rather than HTLV-2 Tax

“…We then undertook an extensive molecular analysis of Tax3 properties: since Tax localization in the nucleus is critical for its ability to transactivate the viral promoter , we initially took advantage of the previously described GFP-Tax-based system to determine the intracellular localization of the protein (Meertens et al, 2004a). In transient transfection assays, the GFP-Tax3 S fusion protein has a strong nuclear localization that is similar to that of Tax1 and different from that of Tax2, suggesting that it contains a functional NLS domain (Figure 3a).…”

“…Finally, tax3 cDNA was cloned into a pSG5M vector. The nucleotide sequence of the constructs was determined using the DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences, Piscataway, NJ, USA) on an Applied Biosystems 373A DNA sequencer as previously described (Meertens et al, 2004a). The pSG5M-Tax3 M22 mutant was constructed by replacing the M 131 amino-acid residue by S 131 using the Quickchange site-directed mutagenesis (Stratagene, La Jolla, CA, USA).…”

“…For microscopic analyses, HeLa cells were seeded on an eightwell chamber glass slide at a concentration of 3.5 Â 10 4 cells/ well and transfected the next day with 0.3 mg of the different GFP plasmids using Effectene reagent (QIAGEN) as described (Meertens et al, 2004a). At 24 h after transfection, the cells were washed with PBS, fixed with 4% paraformaldehyde (Sigma) and washed with PBS.…”

“…Luciferase assays CEM cells (10 Â 10 6 ) were electroporated using a Biorad apparatus (250 V, 950 mF) in 0.4 cm cuvettes with HTLV-1-LTR-luc, HTLV-2-LTR-luc, NF-kB-luc plasmids together with pSG5M-Tax plasmids as previously described (Meertens et al, 2004a). Transient transfection experiments were also performed with Jurkat and 293T cells using the Superfect and the Polyfect procedures, respectively (QIAGEN), as previously described (Meertens et al, 2004b).…”

“…All the transfections were carried out in the presence of a phRG-TK vector in order to normalize the results for the transfection efficiencies. Reporter activities were assayed 24 h post-transfection using the Dual-Luciferase reporter assay system (Promega, Charbonnieres, France) with a Berthold LB9500C luminometer as described (Meertens et al, 2004a). Because the complete HTLV-3 LTR sequence is yet not available, a 445 bp region encompassing the U3 (i.e.…”