HTLV Gene Regulation: Because Size Matters, Transcription is Not Enough

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j Human T-cell lymphotropic virus type 1 (HTLV-1)-induced adult T-cell leukemia/lymphoma is an aggressive malignancy. HTLV-2 is genetically related to HTLV-1 but does not cause any malignant disease. HTLV-1 Tax transactivator (Tax-1) contributes to leukemogenesis via NF-B. We describe transgenic Drosophila models expressing Tax in the compound eye and plasmatocytes. We demonstrate that Tax-1 but not Tax-2 induces ommatidial perturbation and increased plasmatocyte proliferation and that the eye phenotype is dependent on Kenny (IKK␥/NEMO), thus validating this new in vivo model. Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy secondary to HTLV-1 (human T-cell lymphotropic virus type 1) infection (1). Although HTLV-1 and HTLV-2 are similar in genetic organization, they display major differences in pathogenesis and disease manifestation. HTLV-1 is capable of transforming T lymphocytes in infected individuals and subsequently leads to ATL, whereas HTLV-2 has not been clearly associated with malignant diseases but only with lymphocytosis (2, 3).Transgenic mouse models overexpressing Tax-1 demonstrate its oncogenic properties (4-6). However, cellular pathways and in vivo Tax-1 partners that mediate Tax-1-induced cellular transformation are still unexplored. Drosophila melanogaster is a valuable model because of the availability of facile genetic screens, a nearly complete collection of mutants, RNA interference (RNAi) lines, and advanced genetic technologies, in addition to highly conserved pathways such as NF-B. Acquisition of a "rough-eye" phenotype is an accepted surrogate for cell transformation in the Drosophila model. Using Drosophila transgenic models expressing Tax proteins (Tax-1 and Tax-2), we present evidence demonstrating the ability of Tax-1 but not Tax-2 to induce transformation in Drosophila and show that this transformation is dependent primarily on Kenny, the Drosophila orthologue of IKK␥/NEMO, upstream of Relish (NF-B) activation.Briefly, we overexpressed Tax-1 specifically in developing imaginal eyes by using the glass multimer reporter promoter GMRGal4 (number 9146; Bloomington Drosophila Stock Center, NIH P40OD018537). Tax-1 was amplified from pSG5M-Tax (7) and cloned into the Drosophila expression vector pUAST (GenScript) with an N-terminal Myc tag. Plasmid pUAST-Tax-1 was used for the generation of transgenic fly strains after injection into wildtype white-eyed (white Ϫ ) embryos (BestGene). Successful transgenesis was monitored through the appearance of the red-eye phenotype (white ϩ ). Analysis of the ommatidial structure by scanning electron microscopy was performed with a Tescan Mira3 LMU field emission gun scanning electron microscope. A grading system based on the severity of the eye phenotype (number of ommatidial fusions and extent of bristle organization) was developed, and statistical tests were done by one-way analysis of variance. While control flies displayed normal eyes (Fig. 1A, left panel), the eyes of adult flies expressing a single copy of Tax-1 under the contr...

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confidence: 99%

A Transgenic Drosophila melanogaster Model To Study Human T-Lymphotropic Virus Oncoprotein Tax-1-Driven Transformation In Vivo

Shirinian

Kambris

Hamadeh

et al. 2015

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“…In the canonical pathway, activation of the IKK complex leads to the ubiquitination and degradation of IB␣ and to the translocation of RelA/p65-p50 heterodimers into the nucleus (42). Although Tax1 directly interacts with IKK-␥/NEMO (43), the exact mechanism that leads to canonical NF-B activation is still a matter of investigation (36,(44)(45)(46)(47) (for a review, see reference 48). Tax1 undergoes posttranslational modifications, including phosphorylation, acetylation, ubiquitination, and SUMOylation.…”

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confidence: 99%

Human T Cell Leukemia Virus Type 2 Tax-Mediated NF-κB Activation Involves a Mechanism Independent of Tax Conjugation to Ubiquitin and SUMO

Journo

Bonnet

Favre-Bonvin

et al. 2013

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f Permanent activation of the NF-B pathway by the human T cell leukemia virus type 1 (HTLV-1) Tax (Tax1) viral transactivator is a key event in the process of HTLV-1-induced T lymphocyte immortalization and leukemogenesis. Although encoding a Tax transactivator (Tax2) that activates the canonical NF-B pathway, HTLV-2 does not cause leukemia. These distinct pathological outcomes might be related, at least in part, to distinct NF-B activation mechanisms. Tax1 has been shown to be both ubiquitinated and SUMOylated, and these two modifications were originally proposed to be required for Tax1-mediated NF-B activation. Tax1 ubiquitination allows recruitment of the IKK-␥/NEMO regulatory subunit of the IKK complex together with Tax1 into centrosome/Golgi-associated cytoplasmic structures, followed by activation of the IKK complex and RelA/p65 nuclear translocation. Herein, we compared the ubiquitination, SUMOylation, and acetylation patterns of Tax2 and Tax1. We show that, in contrast to Tax1, Tax2 conjugation to endogenous ubiquitin and SUMO is barely detectable while both proteins are acetylated. Importantly, Tax2 is neither polyubiquitinated on lysine residues nor ubiquitinated on its N-terminal residue. Consistent with these observations, Tax2 conjugation to ubiquitin and Tax2-mediated NF-B activation is not affected by overexpression of the E2 conjugating enzyme Ubc13. We further demonstrate that a nonubiquitinable, non-SUMOylable, and nonacetylable Tax2 mutant retains a significant ability to activate transcription from a NF-B-dependent promoter after partial activation of the IKK complex and induction of RelA/p65 nuclear translocation. Finally, we also show that Tax2 does not interact with TRAF6, a protein that was shown to positively regulate Tax1-mediated activation of the NF-B pathway.

“…More recently, using a high-throughput transcriptomic approach, we demonstrated that the Tax-3 protein was phenotypically related to Tax-1, thus suggesting that HTLV-3 might indeed be pathogenic (39). Others have also shown that HTLV-3 and -4 encode antisense transcripts (APH-3 and APH-4, respectively) that repress viral expression (40), as is the case for the HTLV-1 and HTLV-2 HBZ and APH-2 proteins, respectively (41)(42)(43). The ability of APH-3 and -4 to drive cellular proliferation and/or transformation has not yet been investigated.…”

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confidence: 67%

Discovery and Characterization of Auxiliary Proteins Encoded by Type 3 Simian T-Cell Lymphotropic Viruses

Turpin

Journo

et al. 2015

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Human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of auxiliary protein-encoding open reading frames (ORFs) in HTLV-3, the latest HTLV to be discovered, is unknown. Simian T-cell lymphotropic virus type 3 (STLV-3) is almost identical to HTLV-3. Given the lack of HTLV-3-infected cell lines, we took advantage of STLV-3-infected cells and of an STLV-3 molecular clone to search for the presence of auxiliary transcripts. Using reverse transcriptase PCR (RT-PCR), we first uncovered the presence of three unknown viral mRNAs encoding putative proteins of 5, 8, and 9 kDa and confirmed the presence of the previously reported RorfII transcript. The existence of these viral mRNAs was confirmed by using splice site-specific RT-PCR with ex vivo samples. We showed that p5 is distributed throughout the cell and does not colocalize with a specific organelle. The p9 localization is similar to that of HTLV-1 p12 and induced a strong decrease in the calreticulin signal, similarly to HTLV-1 p12. Although p8, RorfII, and Rex-3 share an N-terminal sequence that is predicted to contain a nucleolar localization signal (NoLS), only p8 is found in the nucleolus. The p8 location in the nucleolus is linked to a bipartite NoLS. p8 and, to a lesser extent, p9 repressed viral expression but did not alter Rex-3-dependent mRNA export. Using a transformation assay, we finally showed that none of the STLV-3 auxiliary proteins had the ability to induce colony formation, while both Tax-3 and antisense protein of HTLV-3 (APH-3) promoted cellular transformation. Altogether, these results complete the characterization of the newly described primate T-lymphotropic virus type 3 (PTLV-3). IMPORTANCETogether with their simian counterparts, HTLVs form the primate T-lymphotropic viruses. HTLVs arose from interspecies transmission between nonhuman primates and humans. HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of ORFs encoding auxiliary proteins in HTLV-3 or STLV-3 genomes was unknown. Using in silico analyses, ex vivo samples, or in vitro experiments, we have uncovered the presence of 3 previously unknown viral mRNAs encoding putative proteins and confirmed the presence of a previously reported viral transcript. We characterized the intracellular localization of the four proteins. We showed that two of these proteins repress viral expression but that none of them have the ability to induce colony formation. However, both Tax and the antisense protein APH-3 promote cell transformation. Our results allowed us to characterize 4 new retroviral proteins for the first time.