Phosphorylation of Two Serine Residues Regulates Human T-Cell Leukemia Virus Type 2 Rex Function

Abstract

The function of the human T-cell leukemia virus (HTLV) Rex phosphoprotein is to increase the level of the viral structural and enzymatic gene products expressed from the incompletely spliced viral RNAs containing the Rex-responsive element. The phosphorylation of HTLV type 2 Rex (Rex-2), predominantly on serine residues, correlates with an altered conformation, as detected by a gel mobility shift, and is required for specific binding to its viral RNA target sequence. Thus, the phosphorylation state of Rex in the infected cell may be a switch that determines whether the virus exists in a latent or a productive state. A mutational analysis of Rex-2 that focused on serine and threonine residues was performed to identify regions or domains within Rex-2 important for function, with a specific emphasis on identifying Rex-2 phosphorylation mutants. We identified mutations near the carboxy terminus that disrupted a novel region or domain and abrogated Rex-2 function. Mutant M17 (with S151A and S153A mutations) displayed reduced phosphorylation that correlated with reduced function. Replacement of both serine residues 151 and 153 with phosphomimetic aspartic acid restored Rex-2 function and locked Rex-2 in a phosphorylated active conformation. A mutant containing threonine residues at positions 151 and 153 displayed a phenotype indistinguishable from that of wild-type Rex. Furthermore, this same mutant showed increased threonine phosphorylation and decreased serine phosphorylation, providing conclusive evidence that one or both of these residues are phosphorylated in vivo. Our results provide the first direct evidence that the phosphorylation of Rex-2 is important for function. Further understanding of HTLV Rex phosphorylation will provide insight into the regulatory control of HTLV replication and ultimately the pathobiology of HTLV.Human T-cell leukemia virus (HTLV) types 1 (HTLV-1) and 2 (HTLV-2) are complex retroviruses that have been causally associated with leukemia and neurological disorders in humans (21). In addition to structural and enzymatic genes gag, pol, and env, HTLV encodes two trans-acting regulatory gene products, Tax and Rex, both of which are essential for viral replication (15,17,24). Tax acts to increase the rate of transcription from the viral long terminal repeat (LTR). In addition, Tax modulates the transcription of various cellular genes involved in growth and differentiation and disrupts cell cycle control and DNA repair processes (3,4,38,45,47). These pleiotropic effects of Tax on cellular processes are likely important in the ability of HTLV to mediate oncogenesis. Consistent with this hypothesis, Tax was recently shown to be essential for cellular transformation of primary human T lymphocytes in cultures (39, 41).Rex, the other trans-acting regulatory protein, specifically binds unspliced and singly spliced viral RNAs, resulting in RNA stabilization and their selective trafficking to the cytoplasm (6, 30). These RNAs encode viral structural and enzymatic gene products. Rex function is media...

The Human T-cell leukemia virus Rex protein

“…Replacement of these serines with alanine relocalizes Rex-2 to the cytoplasm and renders it functionally inactive, whereas aspartic acid substitutions lock the protein in a constitutively active form that localizes mainly to the nucleolus (48). Several pieces of evidence suggest that Rex-2 is also phosphorylated on other residues, leading to the hypothesis that additional phosphorylation sites are required to stabilize the active form of Rex-2.…”

“…Both Rex-1 and Rex-2 have homologous nuclear localization signals (NLS), RNA binding domains (RBD), multimerization domains, and an activation domain which encompasses the nuclear export signal (NES) (48,(62)(63)(64)(65)(66). In addition, a unique C-terminal domain has been described for Rex-2 that is a target for serine phosphorylation and may also contribute to efficient nucleocytoplasmic shuttling (54).…”

“…When analyzed by SDS-PAGE, Rex-1 has an apparent size of 27 kDa, and Rex-2 is detected as two major bands of 24 and 26 kDa 1 . The two isoforms of Rex-2, p24 rex and p26 rex , have the same amino acid backbone and differ by a post-translational modification, specifically a conformational change induced by serine phosphorylation (47,48). HTLV-1 and HTLV-2 also produce truncated forms of Rex from alternatively spliced mRNAs.…”

Repression of Human T-Cell Leukemia Virus Type 1 and Type 2 Replication by a Viral mRNA-Encoded Posttranscriptional Regulator

“…In addition, Tax is highly immunogenic in vivo (16,23). Rex acts posttranscriptionally by preferentially binding, stabilizing, and selectively exporting the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm, thus controlling the expression of the structural and enzymatic proteins (1,28,31).…”

Human T-Cell Leukemia Virus Type 1 Expressing Nonoverlapping Tax and Rex Genes Replicates and Immortalizes Primary Human T Lymphocytes but Fails To Replicate and Persist In Vivo

“…All experiments were performed independently three times in triplicate, and results were normalized for transfection efficiency using ␤-galactosidase. The Rex functional assay was performed as described elsewhere (38). Briefly, 0.4 g of control plasmid, HTLV-1, or H1IT proviral clone was cotransfected with 0.1 g CMV-Luc, 0.1 g pctat, and 0.3 g of pCgagRxRE-I reporter.…”