Transformation studies of human T-cell leukemia virus with emphasis on the role of Tax and Rex

Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are related but distinct oncogenic retroviruses. Both of them transform human primary T cells, however the precise transformation mechanism remains to be elucidated. In this thesis, we studied two HTLV regulatory proteins, Tax and Rex, and their role of in HTLV-mediated cellular transformation. It has been shown that HTLV-1 has a preferential transformation tropism of CD4+ T cells, whereas HTLV-2 transforms primarily CD8+ T cells. Since Tax has been shown to be critical for cellular transformation, we hypothesized that the viral determinant of transformation tropism is encoded by tax. Recombinant HTLVs were constructed in which tax and overlapping rex genes were exchanged between HTLV-1 and HTLV-2. p19 Gag expression from recombinants transfected 293T cells was significantly altered. Stable transfectants expressing recombinant viruses were established, irradiated, and cocultured with peripheral blood mononuclear cells (PBMC). Both recombinants were competent to transform T-lymphocytes. Flow cytometry analysis indicated that tax/rex in different genetic backgrounds did not contribute to the distinct HTLV transformation tropism.;Secondly, we investigated the contribution of Rex in HTLV-I-mediated immortalization of primary T-cells in vitro and viral survival in a rabbit animal model. Rex functions to regulate the expression of the viral structural and enzymatic genes and it is essential for efficient viral replication. A Rex deficient HTLV-1 (HTLVRex−) was constructed and 293T cells transiently transfected with the HTLVRex− proviral clone produced low detectable levels of p19 Gag. 729HTLVRex− stable transfectants were established, irradiated and cocultured with PBMCs and resulted in sustained IL-2 dependent growth of PBMCs. These cells carried the HTLVRex− genome and expressed tax/rex mRNA. Rabbits inoculated with irradiated 729HTLVRex− cells failed to become persistently infected. Together, our results indicated that Rex is not required for in vitro immortalization by HTLV-1 but critical for efficient infection and persistence in vivo. In order to generate a HTLV-1 that replicates independent of Rex, post-transcriptional regulatory elements were cloned into HTLV-1 to replace Rex/RxRE function. We found that SNV-PCE at the 5′ LTR could partially substitute for Rex/RxRE function but not in the 3 ′ LTR. HBV-PRE had the capacity to partially substitute for Rex/RxRE function. RSV-CTE failed to substitute for Rex/RxRE function. Overall, our results indicate that post-transcriptional control elements identified in other viruses have a partial capacity to substitute for HTLV Rex/RxRE function. However, the low activities of these elements are not sufficient to maintain viral replication and virus spread in culture.