Multiple functional domains of Tat, thetrans-activator of HIV-1, defined by mutational analysis

Abstract

The tat gene of HIV-1 is a potent trans-activator of gene expression from the HIV long terminal repeat (LTR). To define the functionally important regions of the product of the tat gene (Tat) of HIV-1, deletion, linker insertion and single amino acid substitution mutants within the Tat coding region of strain SF2 were constructed. The effect of these mutations on trans-activation was assessed by measuring the expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene linked to the HIV-LTR. These studies have revealed that four different domains of the protein that map within the N-terminal 56 amino acid region are essential for Tat function. In addition to the essential domains, an auxiliary domain that enhances the activity of the essential region has also been mapped between amino acid residues 58 and 66. One of the essential domains maps in the N-terminal 20 amino acid region. The other three essential domains are highly conserved among the various strains of HIV-1 and HIV-2 as well as simian immunodeficiency virus (SIV). Of the conserved domains, one contains seven Cys residues and single amino acid substitutions for several Cys residues indicate that they are essential for Tat function. The second conserved domain contains a Lys X Leu Gly Ile X Tyr motif in which the Lys residue is essential for trans-activation and the other residues are partially essential. The third conserved domain is strongly basic and appears to play a dual role. Mutants lacking this domain are deficient in trans-activation and in efficient targeting of Tat to the nucleus and nucleolus. The combination of the four essential domains and the auxiliary domain contribute to the near full activity observed with the 101 amino acid Tat protein.

The Human Immunodeficiency Virus Transactivator Tat Interacts with the RNA Polymerase II Holoenzyme

“…To address further the specificity of the Tat-affinity chromatography, crude nuclear extracts were incubated with mutant Tat-streptavidin beads. These mutations in the Tat activation domain (C22G, C30G, and K41A) were previously demonstrated to abolish Tat transactivation in vivo (31) and in vitro (data not presented). Western blotting (Fig.…”

“…Cysteine at position 30 in the activation domain of Tat is essential for Tat transactivation (31). Cell lysates were immunoprecipitated with the same ␣RAP74 antibody that we used previously to immunoprecipitate the holoenzyme from crude nuclear extracts (36).…”

“…The mutant construct was sequenced by dideoxy sequencing (U.S. Biochemicals, Cleveland, Ohio) to confirm the integrity of the Tat sequence. The C30G mutation abolished Tat transactivation from the HIV-1 promoter but did not affect the levels of protein expression (31). The hybrid CD8-Nef protein has been described elsewhere (4).…”

The Glutamine-rich Region of the HIV-1 Tat Protein Is Involved in T-cell Apoptosis

“…Tat is a short HIV-encoded trans-activating regulatory protein (86 -106 amino acids) whose primary role is in regulating productive and processive transcription from the HIV-1 long terminal repeat (11)(12)(13)(14)(15). It is encoded by two exons and has six different regions, each having specific biochemical and functional characteristics (16). Region II (residues 22-37) has seven conserved cysteines, which are required for the trans-activational activity of Tat.…”

TOE1 is an inhibitor of HIV-1 replication with cell-penetrating capability

“…Interestingly, and similarly to Tat, TOE1 harbors a long lysine/arginine-rich NLS (amino acids 335-347) composed of a largely uninterrupted sequence of 12 basic residues (Table 1). For Tat, this basic region (amino acids 49-57) is indispensable for the recognition and binding to the TAR sequence in the viral LTR, as well as for efficient elongation and capping of the viral transcript, and it also serves as the protein's cell penetrating peptide (20)(21)(22)(23). Therefore, we tested the hypothesis that the TOE1 NLS might also function as a cell-penetrating peptide.…”