Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR.

Human immunodeficiency virus type 1 (HIV-1) gene expression is activated by Tat, a virally encoded protein. Tat trans-activation requires viral (trans-activation--responsive; TAR) RNA sequences located in the R region of the long terminal repeat (LTR). Existing evidence suggests that Tat probably cooperates with cellular factors that bind to TAR RNA in the overall trans-activation process. A HeLa complementary DNA was isolated and characterized that encodes a TAR RNA-binding protein (TRBP). TRBP activated the HIV-1 LTR and was synergistic with Tat function.

Relatedness of an RNA-binding motif in human immunodeficiency virus type 1 TAR RNA-binding protein TRBP to human P1/dsI kinase and Drosophila staufen.

TRBP is a human cellular protein that binds the human immunodeficiency virus type 1 TAR RNA. Here, we show that the intact presence of amino acids 247 to 267 in TRBP correlates with its ability to bind RNA. This region contains a lysine- and arginine-rich motif, KKLAKRNAAAKMLLRVHTVPLDAR. A 24-amino-acid synthetic peptide (TR1) of this sequence bound TAR RNA with affinities similar to that of the entire TRBP, thus suggesting that this short motif contains a sufficient RNA-binding activity. Using RNA probe-shift analysis, we determined that TR1 does not bind all double-stranded RNAs but prefers TAR and other double-stranded RNAs with G+C-rich characteristics. Immunoprecipitation of TRBP from human immunodeficiency virus type 1-infected T lymphocytes recovered TAR RNA. This is consistent with a TRBP-TAR ribonucleoprotein during viral infection. Computer alignment revealed that TR1 is highly homologous to the RNA-binding domain of human P1/dsI protein kinase and two regions within Drosophila Staufen. We suggest that these proteins are related by virtue of sharing a common RNA-binding moiety.

An Arg/Lys-rich core peptide mimics TRBP binding to the HIV-1 TAR RNA upper-stem/loop.

TRBP is a cellular protein that binds to the HIV-1 leader RNA, TAR. Circular dichroism experiments have shown that a 24 amino acid peptide (TR1), located within a dsRNA binding domain (dsRBD) of TRBP, binds TAR with a 3:1 stoichiometry, eliciting a conformational change involving base unstacking. The binding characteristics of synthetic structural variants of TAR indicate that guanine residues play a key role in the TR1-RNA interaction and that binding sites exist in the upper-stem/loop and lower stem region of TAR. Deletion analysis of TR1 has led to the identification of a 15 amino acid subpeptide (TR13) which is necessary and sufficient to bind to the high affinity upper-stem/loop binding site of TAR. Alanine scanning of TR13 has revealed that mutations in either Lys or Arg residues result in altered TAR-binding, and molecular modelling/docking experiments have shown that the two Arg residues of TR13 can interact with two appropriately spaced guanine residues in the upper-stem/loop of TAR. The TR13 lysine residues appear to be essential for maintaining structural integrity and the correct positioning of the Arg side-chains. We propose that TRBP binds TAR by means of a "2-G hook" motif and that the binding specificity of this particular member of the family of double-stranded RNA-binding proteins lies within the highly conserved dsRBD core motif. Finally, our results also suggest that TRBP may function in vivo by modifying the tertiary structure of TAR RNA.

Cloning of Saccharomyces cerevisiae promoters using a probe vector based on phleomycin resistance.

Vectors that confer high levels of phleomycin (Ph) resistance to Saccharomyces cerevisiae have been constructed with the TEF1 and ENO1 promoters, the Tn5 ble gene and the CYC1 terminator. They are able to transform yeast cells grown on rich glucose medium containing a moderate level of Ph (10 micrograms/ml, corresponding to 100-fold the minimal inhibitory concentration). Frequencies of transformation are identical to those obtained with the URA3 marker on a defined medium. A promoter probe vector, based on the same ble marker, enabled us to isolate sequences from chromosomal yeast DNA that had promoter activities. These DNA fragments have been sequenced and those which promote the highest levels of Ph resistance have been found to be either A + T-rich or have a potentially new and more efficient translation start site.

Bleomycin resistance conferred by a drug-binding protein.

The protein coded by a bleomycin-resistance gene (ble) cloned from producing actinomycetes was purified from a culture of a recombinant E. coli strain and its action on bleomycin was determined by in vitro assays. The protein binds reversibly in a one to one ratio to bleomycin which can no longer cleave DNA. The bleomycin resistance of cells harboring a ble gene could be accounted for by a sequestering effect of the bleomycin-binding protein.

Expression of TAR RNA-Binding Protein in Baculovirus and Co-Immunoprecipitation with Insect Cell Protein Kinase.

TAR RNA-binding protein TRBP was originally isolated by its binding affinity for radiolabeled HIV-1 leader RNA. Subsequent studies have suggested that this protein is one member of a family of double-stranded RNA-binding proteins. Recent findings indicate that TRBP might function to antagonize the translational inhibitory effect that can be mediated through cellular protein kinase, PKR. Here, we report on the over-expression of a cDNA coding for TRBP in eukaryotic SF9 cells using baculovirus. We characterized the nuclear localization of TRBP in insect cells, and we demonstrate that TRBP co-immunoprecipitates with a protein in these cells antigenically related to human PKR. Copyright 1995 S. Karger AG, Basel

Sequential steps in Tat trans-activation of HIV-1 mediated through cellular DNA, RNA, and protein binding factors.

The regulation of HIV expression is controlled by the activity of the Long Terminal Repeat (LTR). Trans-activation by the virally encoded Tat protein is one of the main mechanisms of LTR activation. Tat binds to its target, TAR RNA, and cellular proteins that bind the LTR, Tat, or TAR RNA are important components of the trans-activation process. We will review the factors that have been characterized for a possible involvement in this mechanism. Whereas LTR binding proteins consist of Sp1 and TBP, a large number of factors that bind TAR RNA have been isolated. We have previously cloned two of them by RNA probe recognition: TRBP and La. We have shown that the in vitro and in vivo binding of TRBP to TAR RNA correlates with a constant expression of the protein during HIV-1 infection. Several proteins that interact with Tat have mainly positive, but some negative, effects on trans-activation. Genetic studies have defined that human chromosome 12 encodes a protein that will allow trans-activation in rodent cells. The binding and the functional data about these proteins suggest sequential steps for the Tat trans-activation mechanism. Each of these intracellular molecular events could be the target for molecular intervention against the virus.

Ribosomal proteins L5 and L11 co-operatively inactivate c-Myc via RNA-induced silencing complex.

Oncogene MYC is highly expressed in many human cancers and functions as a global regulator of ribosome biogenesis. Previously, we reported that ribosomal protein (RP) L11 binds to c-Myc and inhibits its transcriptional activity in response to ribosomal stress. Here, we show that RPL5, co-operatively with RPL11, guides the RNA-induced silencing complex (RISC) to c-Myc mRNA and mediates the degradation of the mRNA, consequently leading to inhibition of c-Myc activity. Knocking down of RPL5 induced c-Myc expression at both mRNA and protein levels, whereas overexpression of RPL5 suppressed c-Myc expression and activity. Immunoprecipitation revealed that RPL5 binds to 3'UTR of c-Myc mRNA and two subunits of RISC, TRBP (HIV-1 TAR RNA-binding protein) and Ago2, mediating the targeting of c-Myc mRNA by miRNAs. Interestingly, RPL5 and RPL11 co-resided on c-Myc mRNA and suppressed c-Myc expression co-operatively. These findings uncover a mechanism by which these two RPs can co-operatively suppress c-Myc expression, allowing a tightly controlled ribosome biogenesis in cells.

Phleomycin resistance encoded by the ble gene from transposon Tn 5 as a dominant selectable marker in Saccharomyces cerevisiae.

Phleomycin, a water-soluble antibiotic of the bleomycin family is as effective against Saccharomyces cerevisiae cells as against Escherichia coli cells. The ble gene of transposon Tn5, which confers resistance to phleomycin, was inserted in place of the iso-1-cytochrome C (CYC1) gene on an autonomously replicative multicopy E. coli-yeast shuttle plasmid. Higher resistance levels are obtained in S. cerevisiae when the region immediately upstream from the initiation codon conforms to the nucleotide sequence stringencies observed in almost every yeast gene. The expected regulation pattern of the whole CYC1 promoter confers different phleomycin resistance levels to the cell under varying physiological conditions. Partial deletions in the CYC1 promoter lead to changes in the resistance level of cells which are mostly accounted for by the removal of known positive and negative regulatory elements. Some of the vector constructions allow direct selection of phleomycin-resistant transformants on rich media.

Identification of cellular proteins that bind to the human immunodeficiency virus type 1 trans-activation-responsive TAR element RNA.

Human immunodeficiency virus type 1 (HIV-1) trans-activator protein Tat activates the expression of its viral long terminal repeat (LTR) through a target transactivation-responsive element termed TAR. We have constructed cell lines that constitutively express the HIV-1 Tat protein. Analyses of nuclear proteins from these cells and from matched control cells that do not express Tat have identified three proteins that bind to a radiolabeled HIV-1 TAR RNA probe. These polypeptides are 100 kDa, 62 kDa, and 46 kDa in size. Competition experiments using a wild-type TAR RNA sequence, a biologically inactive mutant sequence of TAR, and an unrelated RNA species demonstrated that these proteins show higher binding affinity to wild-type TAR than to the other two non-trans-activatable sequences. We hypothesize that these cellular proteins may mediate a function necessary in Tat-dependent activation of the LTR. The fact that no differences were seen in the binding profiles of nuclear proteins to TAR RNA in Tat-producing and Tat-nonproducing cells suggests that Tat does not directly interact with TAR.

Phleomycin resistance as a dominant selectable marker in CHO cells.

The Tn5 and the Streptoalloteichus hindustanus (Sh) ble genes conferring resistance to bleomycin-phleomycin antibiotics have been cloned into a mammalian vector under the RSV-LTR promoter. The resulting plasmids, pUT506 and pUT507 respectively, were used to transfect CHO cells by either the calcium phosphate or the recently described polybrene-DMSO method. Phleomycin- or bleomycin-resistant clones arose with a higher frequency after transfection with pUT507, and pUT507 transfectants were more resistant to both antibiotics than pUT506 transfectants. Phleomycin resistance in pUT507 transfectants was stable and associated with integration of plasmid sequences in genomic DNA. The Sh ble gene, which confers a dominant phleomycin-resistance phenotype, should provide a useful transferable selectable marker in CHO cells as well as in other animal cell lines.

Identification of limiting steps for efficient trans-activation of HIV-1 promoter by Tat in Saccharomyces cerevisiae.

Cellular context is an important determinant for the activity of Tat, the trans-activator of human immunodeficiency virus (HIV). We have investigated HIV-1 promoter expression and trans-activation in Saccharomyces cerevisiae to provide clues about the limiting steps for Tat activity in this organism. A minimal 43-nucleotide HIV promoter (HIV43) has the activity of a weak yeast promoter in the presence or absence of various enhancer binding sites (bs), whereas the entire long terminal repeat is not expressed. None of these constructs could be trans-activated by Tat. Fusion proteins Gal4 binding domain (BD)-Tat48 and Gal4BD-Tat72 are active with different efficiencies on various yeast promoters that have Gal4 bs. They have 70 and 50% of Gal4 wild type activity on hybrid HIV promoters fused to Gal4 bs only in the presence of AP1 bs. This study shows that trans-activation of the HIV-1 promoter by Tat occurs in yeast when Tat is targeted to the promoter and a functional enhancer activity is present. Sp1 function and Tat transfer from the RNA to the promoter are two major elements for in vivo trans-activation of HIV-1 that are defective in S. cerevisiae but can be replaced by functional equivalents.

Efficient trans-activation by the HIV-2 Tat protein requires a duplicated TAR RNA structure.

Human immunodeficiency viruses HIV-1 and HIV-2 encode a Tat protein that activates transcription from the long terminal repeats. The target for transactivation is termed the trans-acting responsive (TAR) element. TAR has an extensively folded RNA secondary structure and is present at the 5' end of all viral mRNAs. Considerable similarities exist between both Tat and TAR of the two viruses. The TAR element of HIV-2 (TAR-2) resembles a tandem duplication of the TAR-1 hairpin structure. Tat-2 conserves many of the protein domains in Tat-1, although it is slightly larger than its counterpart. Given the similarity between the two Tat proteins, it is somewhat unexpected that HIV-2 Tat (Tat-2) only poorly activates the heterologous TAR-1 element. Here, we tested whether the duplicated structure of TAR-2 is required for full Tat-2 activity. We show that the addition of a second TAR hairpin to TAR-1 increased its Tat-2 responsiveness by 3-fold.

Genetic mapping in human and mouse of the locus encoding TRBP, a protein that binds the TAR region of the human immunodeficiency virus (HIV-1).

Productive infection with HIV-1, the virus responsible for AIDS, requires the involvement of host cell factors for completion of the replicative cycle, but the identification of these factors and elucidation of their specific functions has been difficult. A human cDNA, TRBP, was recently cloned and characterized as a positive regulator of gene expression that binds to the TAR region of the HIV-1 genome. Here we demonstrate that this factor is encoded by a gene, TARBP2, that maps to human chromosome 12 and mouse chromosome 15, and we also identify and map one human pseudogene (TARBP2P) and two mouse TRBP-related sequences (Tarbp2-rs1, Tarbp2-rs2). The map location of the expressed gene identifies it as a candidate for the previously identified factor encoded on human chromosome 12 that has been shown to be important for expression of HIV-1 genes. Western blotting indicates that despite high sequence conservation in human and mouse, the TARBP2 protein differs in apparent size in primate and rodent cells.

Analysis of a binding difference between the two dsRNA-binding domains in TRBP reveals the modular function of a KR-helix motif.

Double-stranded RNA-binding proteins constitute a large family with conserved domains called dsRBDs. One of these, TRBP, a protein that binds HIV-1 TAR RNA, has two dsRBDs (dsRBD1 and dsRBD2), as indicated by computer sequence homology. However, a 24-amino-acid deletion in dsRBD2 completely abolishes RNA binding, suggesting that only one domain is functional. To analyse further the similarities and differences between these domains, we expressed them independently and measured their RNA-binding affinities. We found that dsRBD2 has a dissociation constant of 5.9 x 10-8 M, whereas dsRBD1 binds RNA minimally. Binding analysis of 25-amino-acid peptides in TRBP and other related proteins showed that only one peptide in TRBP and one in Drosophila Staufen bind TAR and a GC-rich TAR-mimic RNA. Whereas a 25-mer peptide derived from dsRBD2 (TR5) bound TAR RNA, the equivalent peptide in dsRBD1 (TR6) did not. Molecular modelling indicates that this difference can mainly be ascribed to the replacement of Arg by His residues. Mutational analyses in homologous peptides also show the importance of residues K2 and L3. Analysis of 15-amino-acid peptides revealed that, in addition to TR13 (from TRBP dsRBD2), one peptide in S6 kinase has RNA-binding properties. On the basis of previous and the present results, we can define, in a broader context than that of TRBP, the main outlines of a modular KR-helix motif required for binding TAR. This structural motif exists independently from the dsRBD context and therefore has a modular function.

Induction of RNA-binding proteins in mammalian cells by DNA-damaging agents.

A technique to detect RNA-binding proteins (RBP) involving hybridization of RNA probe to proteins transferred to a membrane was used to study RBP in different mammalian cells and in cells after genotoxic stress. With this approach, up to 13 proteins of different sizes were detected in crude nuclear extracts by using a viral RNA probe consisting of the trans-activation-responsive (TAR) element of human immunodeficiency virus type 1 (HIV-1). The TAR RNA probe contains a stem-loop structure found in nascent HIV-1 transcripts. A G+C-rich probe with similar structure also bound to many of these RBP. Only a 102-kDa protein strongly bound to other RNA probes lacking this structure, while a probe with an A+U-rich stem-loop structure fail to bind most RBP, thus indicating a RNA secondary structure preference. The expression of these RBP varied substantially in nine different human and hamster cell lines, with no detectable RBP in two human myeloid lines. Evidence for induction of these RBP was found in six of seven lines after treatment with DNA-damaging agents; UV radiation was the most effective agent. In Chinese hamster ovary cells, which showed the strongest response, all five RBP present in untreated cells rapidly increased in activity after UV irradiation, and eight additional RBP were detected. The induction of these RBP by DNA-damaging agents indicates one or more possible roles for these proteins in the cellular response to genotoxic stress and in viral activation after such stress.

Direct interactions between autoantigen La and human immunodeficiency virus leader RNA.

We have characterized the in vivo and in vitro binding of human La protein to the human immunodeficiency virus type 1 (HIV-1) leader RNA, the trans-activation response element (TAR). In immunoprecipitation studies using anti-La serum, La-TAR ribonucleoproteins were recovered from HIV-1-infected lymphocytes. Further characterization of this interaction revealed that La has preference for the TAR stem. However, TAR RNA recognition tolerated changes in the primary sequence of the stem as long as the secondary structure was conserved. This structural aspect of La-TAR recognition was confirmed in competition studies in which certain homopolymers influenced complex formation while other single-stranded and double-stranded RNAs had no effect. Deletion mutants of recombinant La protein were used to demonstrate that the residues responsible for binding to polymerase III precursor transcripts overlapped the binding domain for the TAR leader RNA. This finding of a direct interaction between La and TAR has functional implications for translational regulation of HIV-1 mRNAs as demonstrated in the accompanying report (Y. V. Svitkin, A. Pause, and N. Sonenberg, J. Virol. 68:7001-7007, 1994).