ABSTRACTtat, the trans-activator protein for human immunodeficiency virus 1 (HIV-1), has been expressed in Escherichia coli from synthetic genes. Purified tat binds specifically to HIV-1 trans-activation-responsive region (TAR) RNA in gel-retardation, filter-binding, and immunopripitation assays. tat does not bind detectably to anfisense TAR RNA sequences, cellular mRNA sequences, variant TAR RNA sequences with altered stem-oop structures, or TAR DNA.

The HIV‐1 trans‐activator protein, tat, is an RNA binding protein with a high affinity for a U‐rich bulge near the tip of the stem in the RNA stem‐loop structure encoded by the trans‐activation responsive region (TAR). A Scatchard analysis of tat binding has shown that the purified protein forms a one‐to‐one complex with HIV‐1 TAR RNA with a dissociation constant of Kd = 12 nM. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6‐ to 8‐fold lower affinity than wild‐type TAR. Introduction of a point mutation expected to destabilize base pairing in nearby residues of the TAR stem‐loop structure reduced tat binding 10‐fold. In contrast, mutations that alter the sequence of the six nucleotide long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson‐Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs carrying TAR sequences encoding any of the mutations known to produce transcripts which bind tat weakly, are not stimulated efficiently by tat in vivo.

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The human immunodeficiency virus type 1 rev protein binds with high affinity (Kd < 1-3 nM) to a purine-rich "bubble" containing bulged GG and GUA residues on either side of a double-helical RNA stem-loop located toward the 5' end of rev-response element RNA. High-anity rev binding is maintained when the bubble is placed in heterologous stem-loop structures, but rev binding is reduced when either the bulged residues or flanking base pairs in the stem are altered. Rev binding to the purine-rich bubble nucleates assembly of long ifiamentous ribonucleoprotein structures containing polymers of rev bound to anking RNA sequences. It is proposed that rev regulates human immunodeficiency virus RNA expression by selectively packaging viral transcripts carrying the rev-response element sequence into rod-like nucleoprotein complexes that block splicing of the packaged mRNAs.Transcription of human immunodeficiency virus type 1 (HIV-1) begins with the production of short fully spliced mRNAs encoding the regulatory proteins tat and rev. As the infection progresses, the combined activity of the rev gene product and a cis-acting RNA sequence called the revresponsive element (RRE) allows the expression of the unspliced virion RNA and partially spliced mRNAs, including the mRNA for the env gene (1-3). It is still unknown whether rev blocks splicing directly (4) or whether it increases the rate of transport of unspliced mRNAs from the nucleus (3).Recent experiments have shown that the interaction between rev and RRE RNA is due to direct binding (5-7). Here we demonstrate that rev binds with high affinity to a purinerich "bubble" containing GG and GUA residues on either side of a double-stranded stem-loop structure present in RRE RNA. Additional rev molecules can then polymerize and form long filamentous ribonucleoprotein structures in association with the RNA sequences that flank the high-affinity binding site. These observations suggest that rev regulates HIV mRNA expression by packaging unspliced mRNA precursors carrying RRE sequences into protein coats, thus preventing access to the splicing machinery. MATERIALS AND METHODSExpression and Purification of Rev. Rev protein was expressed and purified as described (7) or further purified by chromatography on heparin-Sepharose. Rev was applied to heparin columns in a buffer containing 200 mM NaCl/50 mM Tris-HCl, pH 8.0/1 mM dithiothreitol/0.1 mM EDTA/0.1% Triton X-100 and eluted in buffer containing 2 M NaCl. After gel filtration on Superose 6 (prep grade) columns equilibrated with 200 mM NaCl/50 mM Tris HCl, pH 8.0/1 mM dithiothreitol/0.1 mM EDTA, the rev protein appeared homogeneous on SDS/polyacrylamide gels and free of RNA contaminants. Rev concentrations were determined by amino acid analysis of the purified protein.RNA-Binding Assays. DNA inserts containing RRE-related sequences were cloned between the EcoRI (5') and HindIII (3') sites of pGEM-1, either by cloning PCR products or by cloning hybridized pairs of synthetic oligonucleotides. RNA transcripts for binding exper...