Human T-cell leukemia virus types 1-4 are related complex retroviruses that are members of the genus Deltaretrovirus
]. HTLV-1 and HTLV-2 are the most prevalent worldwide, whereas HTLV-3 and HTLV-4 were discovered recently in a limited number of individuals in Africa [2
]. Of the HTLV isolates, only HTLV-1 infection has been clearly linked to the development of adult T-cell leukemia/lymphoma (ATL), an aggressive CD4+ T-lymphocyte malignancy, and various lymphocyte-mediated inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) [5
]. However, a few cases of atypical hairy cell leukemia or neurologic diseases have been associated with HTLV-2 infection [8
]. Although the difference in pathology between HTLV-1 and HTLV-2 has yet to be elucidated, it likely results from differential activities of the regulatory and accessory proteins.
In addition to the typical structural and enzymatic retroviral genes gag
, and env
, HTLV encodes two trans-regulatory genes, tax
, which are essential for efficient viral replication/transformation, as well as several accessory genes important for viral infection and persistence in vivo
]. The viral oncoprotein Tax increases the rate of transcription from the viral promoter located in the 5' long terminal repeat (LTR) [13
] and modulates the transcription and activity of numerous cellular genes involved in cell growth, cell cycle control, DNA repair, and cell differentiation [16
]. The pleiotropic effects of Tax make it essential for efficient viral replication as well as cellular transformation and oncogenesis [21
HTLV-1 Rex (Rex-1) is a nuclear-localizing and shuttling phosphoprotein that acts post-transcriptionally by preferentially binding, stabilizing, and selectively exporting the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm, thus controlling expression of the structural and enzymatic proteins that are essential for production of viral progeny [24
]. Therefore, it has been proposed that Rex-1 regulates the switch from the early latent phase to the late productive phase of HTLV infection. Rex-1 binds viral RNAs via a cis
-acting RNA sequence termed the Rex-response element (RxRE), which is located in the R region of the viral LTR [27
]. Mutational analysis of Rex-1 has identified several critical domains including an arginine-rich N-terminal sequence that functions as an RNA binding domain (RBD) that overlaps with a nuclear localization signal (NLS), a leucine-rich central core activation domain that contains a nuclear export signal (NES), two flanking Rex-Rex multimerization domains, and a C-terminal stability domain [28
Phosphorylation is a well known reversible regulatory event that controls the activity/function of proteins in eukaryotic cells [38
]. It has been demonstrated that both Rex-1 and Rex-2 are phosphoproteins, and that this modification is critical for their function [26
]. One study investigating the possible relationship of Rex-1 function and phosphorylation showed that treatment of HTLV-1 infected cells with the protein kinase C inhibitor H-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] specifically blocked cytoplasmic accumulation of Rex-dependent gag-pol
]. The same group reported that Rex-1 is phosphorylated on Ser-70, Ser-177, and Thr-174, with Ser-70 phosphorylation being 12-O
-tetradecanoyl-phorbol-13-acetate (TPA)-dependent [39
]. However, a complete phosphorylation map and the identification of the key residues required for function have yet to be elucidated.
In this study, we combined liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis [43
] of affinity-purified Rex-1 protein in combination with substitution mutational analysis to identify and functionally characterize key phosphorylation sites. The LC-MS/MS analysis achieved 100% coverage of the Rex-1 sequence and revealed five novel phosphorylation sites. We also identified two specific amino acid phosphorylation events found to be critical for Rex-1 function (Ser-97 and Thr-174). Overall, this work highlights the importance of phosphorylation and how it regulates the biological properties of Rex-1, ultimately controlling the distribution of viral gene expression and productive viral replication.