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

Abstract

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...

HTLV-1 Tax Functions as a Ubiquitin E3 Ligase for Direct IKK Activation via Synthesis of Mixed-Linkage Polyubiquitin Chains

“…Tax can transform rodent fibroblasts and human primary T cells in the presence of IL-2 [ 5 ]. In addition, Tax-transformed lymphoid cells and fibroblasts form tumors when inoculated into immunodeficient nude mice [ 6 , 7 ], and Tax-based transgene expression induces an ATL-like syndrome in the T cell compartment in mice [ 8 ] and plasmatocyte proliferation in Drosophila [ 9 ]. Moreover, HTLV-1 genome without Tax loses its transformation ability [ 10 ].…”

The HTLV-1 oncoprotein Tax is modified by the ubiquitin related modifier 1 (Urm1)

“…The RNAi line for Urm1 P{GD15862}v48364 was from Vienna Drosophila Resource Center, Vienna, Austria [ 33 ]. UAS:Urm1 WT [ 32 ] and UAS:Tax WT [ 34 ] have been described previously.…”

HTLV-1, the Other Pathogenic Yet Neglected Human Retrovirus: From Transmission to Therapeutic Treatment

“…Thus, Tax favors both the proliferation of infected cells and the accumulation of genetic alterations. Finally, Tax has been shown to induce transformation of rodent fibroblasts [ 94 , 95 ], but also cell transformation in transgenic Drosophila melanogaster and mice expressing Tax [ 96 , 97 ]. Even though the expression of Tax alone seems sufficient for immortalization, Tax poorly transforms human T-cells [ 98 ].…”