Human T-Cell Leukemia Virus Type 2 Rex Carboxy Terminus Is an Inhibitory/Stability Domain That Regulates Rex Functional Activity and Viral Replication

Abstract

Human T-cell leukemia virus (HTLV) regulatory protein, Rex, functions to increase the expression of the viral structural and enzymatic gene products. The phosphorylation of two serine residues (S151 and S153) at the C terminus is important for the function of HTLV-2 Rex (Rex-2). The Rex-2 phosphomimetic double mutant (S151D, S153D) is locked in a functionally active conformation. Since rex and tax genes overlap, Rex S151D and S153D mutants were found to alter the Tax oncoprotein coding sequence and transactivation activities. Therefore, additional Rex-2 mutants including P152D, A157D, S151Term, and S158Term were generated and characterized ("Term" indicates termination codon). All Rex-2 mutants and wild-type (wt) Rex-2 localized predominantly to the nucleus/nucleolus, but in contrast to the detection of phosphorylated and unphosphorylated forms of wt Rex-2 (p26 and p24), mutant proteins were detected as a single phosphoprotein species. We found that Rex P152D, A157D, and S158Term mutants are more functionally active than wt Rex-2 and that the Rex-2 C terminus and its specific phosphorylation state are required for stability and optimal expression. In the context of the provirus, the more active Rex mutants (A157D or S158Term) promoted increased viral protein production, increased viral infectious spread, and enhanced HTLV-2-mediated cellular proliferation. Moreover, these Rex mutant viruses replicated and persisted in inoculated rabbits despite higher antiviral antibody responses. Thus, we identified in Rex-2 a novel C-terminal inhibitory domain that regulates functional activity and is positively regulated through phosphorylation. The ability of this domain to modulate viral replication likely plays a key role in the infectious spread of the virus and in virus-induced cellular proliferation.

Site-Specific Phosphorylation Regulates Human T-Cell Leukemia Virus Type 2 Rex Function In Vivo

“…Moreover, we recently reported that the carboxy terminus of Rex-2 contains a stability/inhibitory domain that is positively regulated through phosphorylation (54). However, a complete map of site-specific phosphorylation of Rex-2 is lacking, and the precise role it plays in protein function remains unclear.…”

“…1). In addition to the nuclear localization signal/RBD, central core activation domain/nuclear export signal, and multimerization domains that were also shared with the functionally homologous HTLV-1 Rex (32), we also reported that Rex-2 contained a unique C-terminal inhibitory/stability domain that was found to be important for proper nucleocytoplasmic shuttling, subcellular localization, and function (32,54). This domain also encompasses at least one phosphorylated serine residue (S151), identified by substitutional mutational and peptide phosphoamino acid analysis, that is critical for Rex-2 function in vivo (31,32).…”

“…Although the carboxy termini of the two related proteins at the amino acid level are quite divergent with no apparent Ser-151, Ser-153, and Thr-164 counterpart in Rex-1 (31, 32), we have previously shown that, like that of Rex-2, the carboxy terminus of Rex-1 does contain a stability domain (54). Rex-1 is known to be phosphorylated (1,2), but further studies are needed to determine whether Rex-1 sitespecific phosphorylation has a significant functional role.…”

“…Rex mutational analysis has defined several domains that are critical for its functions, including an arginine-rich N-terminal sequence that functions as an RNA binding domain (RBD), which also overlaps with a nuclear localization signal, a leucine-rich central core activation domain that contains a nuclear export signal, and two flanking Rex-Rex multimerization domains (4,5,18,21,32,34,37,45,51). More recently, it has been reported that Rex-2 contains a novel C-terminal inhibitory/stability functional domain that is positively regulated through phosphorylation (31,32,54).…”

Human T-lymphotropic virus proteins and post-translational modification pathways

“…The authors mapped also HTLV-2 Rex and found that phosphorylations on Thr-164, Ser-151 and Ser-153 are functional [136] . In particular, Ser-151 phosphorylation correlates with Rex nuclear/nucleolar localization [136] and with an active Rex protein [137] . For unknown reasons, Rex expression promotes the expression of the Src kinase FynB, usually absent in T cells, thus altering the TCR pathway [41] .…”