Comparative Studies on molecular mechanisms utilized by HTLV-1 and HTLV-2 in viral replication and induction of T-cell transformation
Year: 2005
Type: article
Abstract: Human T-cell leukemia virus (HTLV)-1 and HTLV-2 are closely related human retroviruses that have similar genetic organization and biological properties.However, they display distinct pathogenicity.HTLV-1 has been identified as a causal agent for two human diseases, adult T-cell leukemia (ATL) and HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP), whereas HTLV-2 appears much less pathogenic without conclusive disease association.Studies in this thesis focus on the specific strategies utilized by HTLV-1 and/or HTLV-2 to replicate and induce cellular transformation.HTLV cellular transformation and disease induction are dependent on expression of the viral Tax oncoprotein.HTLV-1 Tax (Tax-1), but not HTLV-2 Tax (Tax-2), contains a PDZ domain binding motif (PBM) that promotes Tax-1 interaction with several cellular PDZ proteins.PDZ is a modular protein interaction domain involved in cell polarity, synapse, and cellular proliferation.In Chapter 2 of this thesis, we investigate the contribution of the Tax-1 PBM in HTLV-induced proliferation and immortalization of primary T-cells in vitro and viral survival in an infectious rabbit animal model.We generated several HTLV-1 and HTLV-2 Tax viral mutants including HTLV-1∆PBM, HTLV-2+C22(+PBM), and HTLV-2+C18(∆PBM).Tax mutations did not alter the ability to activate the CREB/ATF or NFκB signaling pathways.Coculture microtiter iii proliferation assays revealed that the Tax-1 PBM significantly increases both HTLV-1 and HTLV-2-induced primary T-cell proliferation.In addition, Tax-1 PBM was responsible for the micronuclei induction activity of Tax-1 relative to that of Tax-2.Viral infection and persistence were severely attenuated in rabbits inoculated with HTLV-1∆PBM.Our results provide the first direct evidence that PBM-mediated associations between Tax-1 and cellular proteins play a key role in HTLV-induced cell proliferation and genetic instability in vitro and facilitate viral spread and persistence in vivo.Previous studies have indicated that in vivo HTLV-1 has a preferential tropism for CD4+ T cells, whereas HTLV-2 in vivo tropism is less clear but appears to favor CD8+ T cells.This tropism difference has been recapitulated in in vitro transformation assays.Taking advantage of the high homology between HTLV-1 and HTLV-2, we chose to generate chimeric HTLV proviral clones in order to identify the viral determinant(s) responsible for their distinct in vitro transformation tropisms.Our previous studies have shown that Tax and its overlapping Rex sequence do not confer the distinct transformation tropisms between HTLV-1 and HTLV-2.In Chapter 3, two pairs of recombinant HTLV were generated, in which the viral LTR or env gene was exchanged between HTLV-1 and HTLV-2.All the recombinants are functional based on the p19 Gag expression from proviral clones transfected into 293T cells.Stable transfectants expressing recombinant viruses were established, irradiated, and cocultured with human peripheral blood monocytes (PBMCs).All recombinants were competent to transform T-lymphocytes in in vitro coculture transformation assays.Flow cytometry analysis of transformed PBMCs indicated that exchange of the viral LTR does not alter HTLV iv transformation tropisms.However, when the env gene was exchanged, HTLV transformation tropism changed correspondingly, i.e.HTLV-2/Env1 preferentially transforms CD4+ T lymphocytes, and HTLV-1/Env2 has similar cellular tropism pattern with wild type HTLV-2 (CD8+ T cells).The results indicate that the env gene is a major viral determinant for HTLV T-cell transformation tropism and provide strong evidence implicating its contribution to the different pathobiologies of HTLV-1 versus HTLV-2.Rex phosphoprotein functions to increase the level of viral structural and enzymatic gene products, which are expressed from the unspliced and incompletely-spliced viral mRNAs.Recently, it has been demonstrated that the phosphorylation of HTLV-2 Rex (Rex-2), particularly at serine residues 151 and 153 within the carboxy-terminal phosphorylation domain (CTPD), is critical for its function.Replacement of both serine residues with phosphomimetic aspartic acid (S151D/S153D) has been shown to lock Rex-2 in an active conformation.This mutant phenotype provides us with a unique reagent to evaluate the role of Rex-2 functional regulation in viral replication and cellular transformation in vitro and persistence in vivo.Since the mutation in RexS151D/S153D also affects viral oncoprotein Tax amino acid sequences and its transactivation activities, in Chapter 4 several additional Rex mutants in this region were generated and characterized to facilitate these studies, two aspartic acid substitution mutants, RexP152D and RexA157D and two carboxy-terminus deletion mutants, RexS151Term and RexS158Term.All mutants localized predominantly to the nucleus/nucleolus and display a locked active functional conformation similar to RexS151D/S153D.In short-term microtiter proliferation assays, RexA157D enhances HTLV-2-mediated cellular proliferation of human peripheral blood mononuclear cells (hPBMCs), whereas hPBMCs in coculture v with 729HTLV-2/RexS151Term or 729HTLV-2/RexS158Term display substantially decreased proliferation.In long-term immortalization assays, HTLV-2/RexP152D and HTLV-2/RexA157D have the capacity to immortalize primary T cells as efficiently as wtHTLV-2, whereas both HTLV-2/RexS151Term and HTLV-2/RexS158Term were deficient in immortalization of hPBMCs.Our studies indicate that constitutive active Rex-2, particularly the phosphomimetic mutants, can increase viral protein expression, contributing to the enhanced primary T-cell proliferation mediated by HTLV-2 infection.However, constitutive active Rex-2 mutants without CTPD impair HTLV-2-induced cellular proliferation and transformation, implying that the CTPD is required for proper functional regulation of Rex-2 in the context of the virus.Interestingly, HTLV-2/RexP152D was impaired in inoculated rabbits as compared to wtHTLV-2 supporting the conclusion that this domain plays an important regulatory role in vivo.This is the first report of the critical role of HTLV Rex regulatory control in viral mediated cellular proliferation and immortalization, and provides insight into viral-host interaction in HTLV-2-infected cells.Overall, the work described in this thesis employ methods that manipulate viral elements in the context of full-length proviral clones and analyze their function and mechanism of action in a system closely mimicking in vivo HTLV infection.In this way, our studies provide important insight into the molecular pathogenesis of HTLV-1 and HTLV-2.
Author: Li Xie
Institution: The Ohio State University
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2280966463