Activity of synthetic peptides from the Tat protein of human

To determine which of the 86 amino acids in the Tat protein of human inmunodeficiency virus type 1 (HIV-1) are important for transactivation, peptides from Tat were synthesized and their activity was measured in cells containing a chloramphenicol acetyltransferase reporter gene under control of the HIV long terminal repeat promoter. Although the Tat sequence contains arginineand cysteine-rich stretches that are difficult to synthesize, it was possible to prepare pure peptides in good yield by using fluoren9-ylmethoxycarbonyl (Fmoc) chemistry. A peptide containing residues 1-58 had 5-10% the activity of full-length Tat. Deleting 4 amino acids from the N terminus of this peptide further reduced activity, while peptides with more extensive N-terminal deletions and peptides missing the basic region at the C terminus had no detectable activity. A peptide previously reported to transactivate, Tat-(37-62), was completely inactive in our assays. Inactive peptides were also tested as possible inhibitors of transactivation. Tat-(21-38), which contains the cysteine-rich region and can form heterodimers with intact Tat in vitro, showed inhibition at high peptide concentrations. However, this effect was not specific for Tat or for the HIV promoter, since the peptide also inhibited expression from the simian virus 40 early promoter. The Tat protein from human immunodeficiency virus (HIV) is a potent viral transactivator (1) that is essential for viral replication (2, 3). Although the precise mechanism of transactivation remains unclear, Tat may cause accumulation of mRNA by increasing the rate of transcription from the long terminal repeat (LTR) promoter (4, 5), by acting as a transcriptional antiterminator (6), or by stabilizing viral mRNAs (7-10). Tat may also increase translational efficiency (7-12). A region of about 40 base pairs just 3' to the start of transcription [the transactivation response (TAR) site] is necessary for transactivation by Tat (13-15). The Tat protein is 86 amino acids long and contains a highly basic region (with 2 lysines and 6 arginines in 9 residues) and a cysteine-rich region (with 7 cysteines in 16 residues) (16, 17). The basic region is important for nuclear localization (18, 19). The cysteine-rich region mediates the formation of metal-linked dimers in vitro (20), although it is not known whether the metal-linked dimer is important for transactivation. An 18-amino acid peptide containing just the cysteinerich region of Tat can also form metal-linked dimers and can form heterodimers with intact Tat in vitro (21). It was suggested that, ifdimerization is essential for transactivation, this peptide might inhibit Tat activity by forming inactive heterodimers (20, 21). It is now possible to directly measure the activity of Tat peptides and to test whether peptides can inhibit transactivation. Assays have recently been developed that use a procedure known as "scrape-loading" to introduce Tat into tissue culture cells that contain the HIV-1 promoter linked to a reporter gene (22, 23). It is believed that this procedure transiently damages cell membranes, allowing molecules in the medium to enter the cytoplasm. During development of the scrape-loading assay, it was also found that Tat could be taken up by cells simply growing in tissue culture and could enter the nucleus and transactivate the HIV-1 promoter (22). Uptake and transactivation could be stimulated as much as 1000-fold with lysosomotrophic agents such as chloroquine, which apparently prevent proteolysis of Tat (22). We use these assays to measure the activity of Tat peptides synthesized by fluoren-9-ylmethoxycarbonyl (Fmoc) chemistry and show that the N-terminal region, the cysteine-rich region, and the basic region are all necessary for transactivation. We also show that peptides that are capable of forming heterodimers with intact Tat in vitro do not specifically inhibit Tat transactivation in vivo. MATERIALS AND METHODS Synthesis of Tat Peptides. Syntheses were performed using Fmoc chemistry on a MilliGen/Biosearch model 9600 peptide synthesizer with a peptide amide linker-norleucine-4-methylbenzhydrylamine (PAL-Nle-MBHA) polystyrene resin (MilliGen/Biosearch; 0.5 g). The benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate/1-hydroxybenzotriazole (BOP/HOBt) coupling method (24) was used with coupling times of 1-4 hr and with double coupling of His-33. Protecting groups were t-butyl ester (for Glu and Asp), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Arg), t-butyloxycarbonyl (Lys), trityl (His and Cys), t-butyl (Ser, Thr, and Tyr), and trimethoxybenzyl (Asn and Gln). All peptides were synthesized as their C-terminal amides. After synthesis was completed, protecting groups were removed and the peptide chains were cleaved from the resin with trifluoroacetic acid/ ethanedithiol/thioanisole/anisole (90:3:5:2, vol/vol). The mixture was filtered and the products were obtained by addition of cold anhydrous diethyl ether to the filtrate. The precipitate was collected by filtration, thoroughly washed with ether, and dried. Purification and Analysis ofTat and Peptides. Peptides were treated with 0.5 M dithiothreitol at 370C for 30 min to ensure complete reduction ofthe cysteines and were purified on a C4 HPLC column (Vydac) using an acetonitrile gradient in 0.1% trifluoroacetic acid. Amino acid composition was determined by hydrolysis in 6M HCl containing 0.5% phenol at 110TC and analysis on an LKB Alpha Plus analyzer. Peptide purity (>90%6) was determined by HPLC using an acetonitrile gradient of <0.5% per min. Tat-(37-62)-peptide (15 nmol) was sequenced (27 cycles on a model 470A Applied BiosysAbbreviations: CAT, chloramphenicol acetyltransferase; FAB, fastatom bombardment; Fmoc, fluoren-9-ylmethoxycarbonyl; HIV, human immunodeficiency virus; LTR, long terminal repeat. tTo whom reprint requests should be addressed. 7397 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 86 (1989)