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Transformation studies of human t-cell leukemia virus with emplhasis on the role of tax and rex

Ye, Jianxin
http://rave.ohiolink.edu/etdc/view?acc_num=osu1060451751

Abstract Details

2003, Doctor of Philosophy, Ohio State University, Molecular, Cellular, and Developmental Biology.
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 and differences have been identified between Tax-1 and Tax-2, we hypothesized that the viral determinant of transformation tropism is encoded by tax. Using molecular clones of HTLV-1 (Ach) and HTLV-2 (pH6neo) we constructed recombinants in which tax and overlapping rex genes of the two viruses were exchanged. p19 Gag expression from proviral clones transfected into 293T cells indicated that both recombinants contained functional Tax and Rex, but with significantly altered activity as compared to the wild-type clones. Stable transfectants expressing recombinant viruses were established, irradiated, and cocultured with peripheral blood mononuclear cells (PBMC). Both recombinants were competent to transform T-lymphocytes with efficiency similar to the parental viruses. Flow cytometry analysis indicated that HTLV-1 and HTLV-1/TR2 had a preferential tropism for CD4+ T cells and HTLV-2 and HTLV-2/TR1 had a preferential tropism for CD8+ T cells. Our results indicate that tax/rex in different genetic backgrounds display altered functional activity, but ultimately do not contribute to the different in vitro transformation tropism. Secondly, we investigated the contribution of Rex in HTLV-1-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 primary T-lymphocytes. These cells carried the HTLVRex- genome and expressed tax/rex mRNA, but produced no detectable Rex or p19 Gag. Rabbits inoculated with irradiated 729HTLVRex- cells and 729HTLVRex- cells transiently transfected with a Rex cDNA expression plasmid failed to become persistently infected or mount a detectable antibody response to the viral gene products. Together, our results provide the first direct evidence that Rex and its function to modulate viral gene expression and virion production is not required for in vitro immortalization by HTLV-1. However, Rex is critical for efficient infection of cells and persistence in vivo. Efficient HTLV replication requires Rex/RxRE regulation of incompletely spliced viral mRNAs that encode the viral enzymatic and structural proteins. Other viruses including simple retroviruses contain elements in their genome that interact with cellular proteins to regulate this process. To ultimately generate an HTLV-1 that replicates independent of Rex/RxRE function, Spleen necrosis virus (SNV), Rous sarcoma virus (RSV), or hepatitis B virus (HBV) posttranscriptional regulatory elements were cloned in the appropriate orientation in a HTLV-1 proviral clone. We found that SNV post-transcriptional control element (SNV-PCE) at the 5’ LTR could partially substitute for Rex/RxRE function, however, it impaired HTLV-1 viral gene expression when it was inserted into the 3’ LTR. HBV post-transcriptional regulatory element (HBV-PRE) inserted at the end of tax also had the capacity to partially substitute for Rex/RxRE function, as measured by 5-fold increase in p19 Gag expression in the absence of Rex. The RSV direct repeat (DR) failed to substitute for Rex/RxRE function. Furthermore, coculture transformation assay revealed that HTLVPRERex- maintained the capacity to transform PBMCs indicting that HBV-PRE did not affect HTLV-1 mediated cellular transformation. However, p19 Gag expression in the HTLVPRERex- transformed PBMCs was below the detection limit suggesting that HBV-PRE did not efficiently function in PBMCs. 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. Together this work provides important information on the role of Tax and Rex on HTLV replication and cellular transformation and further insight into the biological differences between HTLV-1 and HTLV-2.
Patrick Green (Advisor)
146 p.
Biology, Molecular
HTLV; Transformation; Tax; Rex; tropism

Recommended Citations

Citations

  • Ye, J. (2003). Transformation studies of human t-cell leukemia virus with emplhasis on the role of tax and rex [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1060451751

    APA Style (7th edition)

  • Ye, Jianxin. Transformation studies of human t-cell leukemia virus with emplhasis on the role of tax and rex. 2003. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1060451751.

    MLA Style (8th edition)

  • Ye, Jianxin. "Transformation studies of human t-cell leukemia virus with emplhasis on the role of tax and rex." Doctoral dissertation, Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=osu1060451751

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.