Oral immune dysfunction is associated with the expansion of FOXP3+PD-1+Amphiregulin+ T cells during HIV infection.

Residual systemic inflammation and mucosal immune dysfunction persist in people living with HIV, despite treatment with combined anti-retroviral therapy, but the underlying immune mechanisms are poorly understood. Here we report that the altered immune landscape of the oral mucosa of HIV-positive patients on therapy involves increased TLR and inflammasome signaling, localized CD4+ T cell hyperactivation, and, counterintuitively, enrichment of FOXP3+ T cells. HIV infection of oral tonsil cultures in vitro causes an increase in FOXP3+ T cells expressing PD-1, IFN-γ, Amphiregulin and IL-10. These cells persist even in the presence of anti-retroviral drugs, and further expand when stimulated by TLR2 ligands and IL-1ß. Mechanistically, IL-1ß upregulates PD-1 expression via AKT signaling, and PD-1 stabilizes FOXP3 and Amphiregulin through a mechanism involving asparaginyl endopeptidase, resulting in FOXP3+ cells that are incapable of suppressing CD4+ T cells in vitro. The FOXP3+ T cells that are abundant in HIV-positive patients are phenotypically similar to the in vitro cultured, HIV-responsive FOXP3+ T cells, and their presence strongly correlates with CD4+ T cell hyper-activation. This suggests that FOXP3+ T cell dysregulation might play a role in the mucosal immune dysfunction of HIV patients on therapy.

RNA recognition by the human immunodeficiency virus Tat and Rev proteins.

The human immunodeficiency virus (HIV-1) regulatory proteins, Tat and Rev, are important potential targets for the development of new drug therapies against HIV infection. Both proteins are highly specific RNA-binding proteins that recognize cis-acting regulatory elements in the viral mRNAs. These interactions are fascinating paradigms of a new principle of RNA recognition in which the protein makes contact with functional groups displayed in a distorted major groove of an RNA duplex.

Control of human immunodeficiency virus replication by the tat, rev, nef and protease genes.

Immediately after infection, human immunodeficiency virus directs the synthesis of three regulatory proteins tat, rev and nef that together allow the synthesis of the structural proteins of the virus after a delay of several hours. Viral mRNA production is controlled by the tat gene, which appears to stimulate elongation by RNA polymerase II, and the rev gene, which allows the accumulation of unspliced or partially spliced mRNAs in the cytoplasm. The nef gene is dispensible for virus growth but may limit virus spread by downregulating the levels of cellular surface proteins such as the CD4 receptor. Virus maturation also depends critically on the protease gene which allows the orderly rearrangement of the viral core structures in newly budded virions as well as the vpu and vif genes which allow efficient production of mature envelope glycoprotein.

Transformation of growth factor-dependent myeloid stem cells with retroviral vectors carrying c-myc.

Myeloid progenitor cells and macrophages derived from bone marrow and spleen were efficiently transformed in vitro by infection with Moloney-based retroviral vectors carrying a human c-myc gene. Infected cells were plated in agar in the presence of combinations of the murine lymphokines CSF-1, IL-3, GM-CSF and IL-1. Between 20% and 100% of the colony-forming cells in the initial bone marrow or spleen population could be infected and gave rise to drug-resistant colonies. A large fraction of the infected cells showed continued proliferation after transfer to liquid media and we have derived over 200 growth factor-dependent cell lines. These include adherent and non-adherent CSF-1 or GM-CSF dependent macrophages and macrophage precursors and cell lines which require complex combinations of growth factors for optimal growth. Each of the cell lines displays a unique pattern of expression of surface markers specific for the myeloid lineage including the Mac-1, Mac-2, Mac-3, Ser-4 and F4/80 antigens. Surface markers not specifically associated with the myeloid lineage such as the MHC class II antigens and the Fc-receptor; and surface markers normally associated with the B-cell and T-cell lineages such as B220, L3T4 and Thy1.2 are also found on these cell lines.

Regulation of cell cycle duration by c-myc levels.

Early passage murine fibroblasts infected with retroviral vectors carrying human c-myc 'minigenes' express high levels of c-myc and have a dramatically shortened G1-phase of the cell cycle. Cells infected with viruses where c-myc is expressed from the viral LTR (MSN-4 virus) express more c-myc protein than cells infected with viruses where c-myc is expressed from the SV40 early promoter (NSM-7 virus). Populations of cells were infected with high titre viruses, selected for drug-resistance, pulse labelled with bromodeoxyuridine (BrdUrd) and chased in BrdUrd free media. This allows accurate, simultaneous, measurement of the rate of exit of unlabelled cells from G1 and progression of BrdUrd-labelled cells through S-phase. The length of the G1-phase in cell populations infected with the MSN-4 virus is 4.65 h, a reduction of nearly 30% compared to the G1-phase length of 6.50 h seen in cells infected with the VSN-2 control virus. Cells infected with NSM-7 virus show an intermediate phenotype and have a G1-phase of 5.25 h. The lengths of the S-phase (4.50 to 4.75 h) and G2 + M phases (2.75 h) were not significantly altered by exogenous c-myc expression. When chases are performed in growth-factor free media, the G1-phase of infected and non-infected cells is extended by approximately 2 h. Cells infected with the c-myc viruses continue to cycle more rapidly than uninfected cells. Growth factor-deprived cells, restimulated with serum, show similar alterations of the cell cycle kinetics. MSN-4 and NSM-7 infected cells, expressing high levels of c-myc, enter S-phase 2 to 4 h earlier, but less synchronously, than control cells, and sustain subsequent rounds of DNA synthesis, while control cells do not. However, cells carrying activated c-myc genes have nearly-normal morphologies and are not tumourgenic in syngenic mice. These results demonstrate that c-myc levels are rate limiting for events in G1, and the length of G1 varies proportionally with the level of exogenous c-myc expression.

Cyclic nucleotide phosphodiesterases of Hyalophora cecropia silkmoth fat body.

Two soluble forms of 3':5'-cyclic-nucleotide phosphodiesterase (o':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) were found in the larval fat body of the silkmoth Hyalophora cecropia. These differ in elution profile on Sephadex G-200, solubility in ammonium sulfate, metal ion requirements and kinetic properties. Phosphodiesterase I has Km values of 11 muM and 1.8 muM for cyclic AMP and cyclic GMP, respectively, has 5-fold greater maximal activity with cyclic AMP than with cyclic GMP, and is activated by Mg2+ and Co2+, and inhibited by EDTA. phosphodiesterase II has Km values of 625 muM and 125 muM for cyclic AMP and cyclic GMP, respectively, has similar maximal activity with both substrates, and is not activated by divalent metal ions or inhibited by EDTA. Cyclic nucleotides and methylxanthines competitively inhibit both enzymes. Phosphodiesterase is found in both soluble and particulate fractions of homogenates. Total activity is highest during the larval stage of the insect, drops markedly following pupation, and rises again during pharate adult development.

Tackling Tat.

Activation of cellular genes typically involves control of transcription initiation by DNA-binding regulatory proteins. The human immunodeficiency virus transactivator protein, Tat, provides the first example of the regulation of viral gene expression through control of elongation by RNA polymerase II. In the absence of Tat, initiation from the long terminal repeat is efficient, but transcription is impaired because the promoter engages poorly processive polymerases that disengage from the DNA template prematurely. Activation of transcriptional elongation occurs following the recruitment of Tat to the transcription machinery via a specific interaction with an RNA regulatory element called TAR, a 59-residue RNA leader sequence that folds into a specific stem-loop structure. After binding to TAR RNA, Tat stimulates a specific protein kinase called TAK (Tat-associated kinase). This results in hyperphosphorylation of the large subunit of the RNA polymerase II carboxyl- terminal domain. The kinase subunit of TAK, CDK9, is analogous to a component of a positive acting elongation factor isolated from Drosophila called pTEFb. Direct evidence for the role of TAK in transcriptional regulation of the HIV long terminal repeat comes from experiments using inactive mutants of the CDK9 kinase expressed in trans to inhibit transcription. A critical role for TAK in HIV transcription is also demonstrated by selective inhibition of Tat activity by low molecular mass kinase inhibitors. A second link between TAK and transactivation is the observation that the cyclin component of TAK, cyclin T1, also participates in TAR RNA recognition. It has been known for several years that mutations in the apical loop region of TAR RNA abolish Tat activity, yet this region of TAR is not required for binding by recombinant Tat protein in vitro, suggesting that the loop region acts as a binding site for essential cellular co-factors. Tat is able to form a ternary complex with TAR RNA and cyclin T1 only when a functional loop sequence is present on TAR.

Direct evidence that HIV-1 Tat stimulates RNA polymerase II carboxyl-terminal domain hyperphosphorylation during transcriptional elongation.

The human immunodeficiency virus type-1 (HIV-1) Tat protein regulates transcription by stimulating RNA polymerase processivity. Using immobilised templates, we have been able to study the effects of Tat on protein kinase activity during the pre-initiation and elongation stages of HIV-1 transcription. In pre-initiation complexes formed at the HIV-1 LTR, the C-terminal domain (CTD) of RNA polymerase II is rapidly phosphorylated by transcription factor IIH (TFIIH). Addition of Tat does not affect either the rate or the extent of CTD phosphorylation in the pre-initiation complexes. By contrast, Tat is able to stimulate additional CTD phosphorylation in elongation complexes. This reaction creates a novel form of the RNA polymerase that we have called RNA polymerase IIo*. Formation of the RNA polymerase IIo* occurs only after transcription of templates carrying a functional TAR RNA element and is strongly inhibited by low concentrations of 5,6-dichloro-1-beta- D -ribofuranosyl benzimidazole (DRB), a potent inhibitor of CDK9, the protein kinase subunit of the Tat-associated kinase (TAK). Immunoblotting experiments have shown that CDK9 and its associated cyclin, cyclin T1, are present at equivalent levels in both the pre-initiation and elongation complexes. We conclude that activation of the CDK9 kinase, leading to CTD phosphorylation, occurs only in elongation complexes that have transcribed through the Tat-recognition element, TAR RNA.

Synergistic stimulation of HIV-1 rev-dependent export of unspliced mRNA to the cytoplasm by hnRNP A1.

The structural and accessory proteins of human immunodeficiency virus type 1 are expressed by unspliced or partially spliced mRNAs. Efficient transport of these mRNAs from the nucleus requires the binding of the viral nuclear transport protein Rev to an RNA stem-loop structure called the RRE (Rev response element). However, the RRE does not permit Rev to stimulate the export of unspliced mRNAs from the efficiently spliced beta-globin gene in the absence of additional cis-acting RNA regulatory signals. The p17gag gene instability (INS) element contains RNA elements that can complement Rev activity. In the presence of the INS element and the RRE, Rev permits up to 30 % of the total beta-globin mRNA to be exported to the cytoplasm as unspliced mRNA. Here, we show that a minimal sequence of 30 nt derived from the 5' end of the p17 gag gene INS element (5' INS) is functional and permits the export to the cytoplasm of 14% of the total beta-globin mRNA as unspliced pre-mRNA. Gel mobility shift assays and UV cross-linking experiments have shown that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and a cellular RNA-binding protein of 50 kDa form a complex on the 5' INS. Mutants in the 5' INS that prevent hnRNP A1 and 50 kDa protein binding are inactive in the transport assay. To confirm that the hnRNP A1 complex is responsible for INS activity, a synthetic high-affinity binding site for hnRNP A1 was also analysed. When the high affinity hnRNP A1 binding site was inserted into the beta-globin reporter, Rev was able to increase the cytoplasmic levels of unspliced mRNAs to 14%. In contrast, the mutant hnRNP A1 binding site, or binding sites for hnRNP C and L are unable to stimulate Rev-mediated RNA transport. We conclude that hnRNP A1 is able to direct unspliced globin pre-mRNA into a nuclear compartment where it is recognised by Rev and then transported to the cytoplasm.

Periodic features in the amino acid sequence of nematode myosin rod.

Properties of the amino acid sequence of the nematode myosin rod region, deduced from cloned DNA, are analysed. The rod sequence of 1117 residues contains a regular region of 1094 residues, which has features typical of an alpha-helical coiled coil, followed by a short non-helical tailpiece at the carboxyl end. The hydrophobic amino acids show the expected seven-residue pattern a, b, c, d, e, f, g, which is modulated by a longer repeat of 28-residue zones. In addition, there are four one-residue insertions, or skip residues, at the ends of zones, at positions 351, 548, 745 and 970. Myosin is considerably less hydrophobic than tropomyosin or alpha-keratin and the outer surface of the coiled coil is covered by clusters of positive and negatively charged amino acid side-chains. Molecular models suggest that the coiled coil is continuous throughout the rod, with an approximately uniform left-handed twist, except for a few turns of helix near each skip region, where the twist flattens out to accommodate the extra residue. Fourier transforms of the amino acid profiles show strong periodicities based on repeats of seven residues (7/2 and 7/3) and 28 residues (especially 28/3 and 28/9). The positive and negative charges each have strong 28/3-residue periodicities that are out of phase with one another. The negative charges also show a 196/9-residue modulation frequency, which may reflect the presence of a 196-residue structural unit in muscle, approximately 2 X 143 A long. The distribution of charged amino acids suggests that electrostatic forces are dominant in forming the thick filament structure. Models that allow regular patterns of interacting charges are restricted and the simplest types are discussed.

The structure of the human immunodeficiency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein.

The human immunodeficiency virus type-1 (HIV-1) Tat protein stimulates transcriptional elongation. Tat is introduced to the transcription machinery by binding to the transactivation response region (TAR) RNA stem-loop encoded by the 5' leader sequence found on all HIV-1 mRNAs. We have used multidimensional heteronuclear NMR to determine the structure of the TAR RNA in the presence of the ADP-1 polypeptide, a 37-mer that carries the minimal RNA recognition region of the Tat protein and closely mimics Tat binding specificity. In the presence of a variety of ligands, including ADP-1, related basic peptides and the amino acid derivative argininamide, the bulge region of TAR undergoes a local conformational rearrangement and forms a more stable structure. The structure of TAR in the bound form has been determined from over 1000 NMR-derived constraints. The U23 residue at the 5' end of the bulge is positioned near G26 and A27 in the major groove, rather than stacked on A22 as in the free TAR. U23 and G26 are brought into close proximity by contacts to the guanidinium group and side-chain amide group of a common arginine residue. However, the interaction of this guanidinium group with TAR is not the only source of binding specificity. Besides NOEs to the arginine residue participating in the conformational change, ADP-1 shows additional intermolecular NOEs to TAR, suggesting that there are multiple points of contacts between TAR RNA and residues from the basic and core regions of Tat. These structural results provide important clues towards the identification of small molecular mass and/or peptidomimetic inhibitors of the essential Tat-TAR interaction.

Sequence analysis of the complete Caenorhabditis elegans myosin heavy chain gene family.

The sequences of three myosin heavy chain (MHC) genes from Caenorhabditis elegans, myo-1, 2 and 3, are presented. These genes, together with unc-54, comprise the entire nematode sacromeric MHC family. Comparison of nematode MHC sequences and sarcomeric, smooth and non-muscle MHCs from other organisms highlights conserved sequence features of the MHC rod believed to be important for thick filament assembly. These include: conservation of sequence differences between individual 28 amino acid repeats; invariant placements of large aromatic residues, such as tryptophan, in the rod sequences; conservation of "weak spots" in the hydrophobic seam; and conservation of non-uniform charge distributions along the length of the rod. The rod sequences of the body wall isoforms A and B are more closely related to each other than to the pharyngeal isoforms C and D, suggesting that structural constraints have been imposed by their location within the same thick filament. We have also identified the major transcriptional start site for gene unc-54. Surprisingly, there are no TATA or other known transcription factor elements immediately upstream from the unc-54 start site, or in the upstream regions of the other genes of the C. elegans MHC gene family.

The human immunodeficiency virus long terminal repeat includes a specialised initiator element which is required for Tat-responsive transcription.

The effects of mutations in human immunodeficiency virus type-1 (HIV-1) long terminal repeat on initiation and on Tat-mediated trans-activation were studied using cell-free transcription assays. All the elements that are necessary for efficient transcription initiation in vitro are included in the core promoter. This region contains three tandem Sp1 binding sites, a TATA element and an initiator (INR) sequence. Although the HIV-1 INR element overlaps the trans-activation response region (TAR), it forms an integral part of the promoter. The HIV-1 INR element was characterised in detail using a template that carries a complete HIV-1 promoter and a displaced TAR RNA element. The results demonstrate that the sequence G+1GGTCT is essential for HIV-1 INR function. RNase protection experiments show that Tat acts exclusively to stimulate transcriptional elongation. Mutations in the core promoter elements reduce initiation rates dramatically but do not block Tat activity. For each mutation studied, the total level of transcription in the presence of Tat is proportional to the rate of initiation in the absence of Tat. Furthermore the rate of initiation remains constant in the presence or absence of Tat. We conclude that the elements of the HIV-1 core promoter act in concert to simulate initiation. By contrast, Tat acts independently of the core promoter elements and stimulates elongation. The data strongly suggest that Tat is recruited to the elongating transcription complex during its transit through TAR.

Paramyosin gene (unc-15) of Caenorhabditis elegans. Molecular cloning, nucleotide sequence and models for thick filament structure.

Paramyosin is a major structural component of thick filaments isolated from many invertebrate muscles. The Caenorhabditis elegans paramyosin gene (unc-15) was identified by screening with specific antibodies an "exon-expression" library containing lacZ/nematode gene fusions. Short probes recovered from the library were used to identify bacteriophage lambda and cosmid clones that encompass the entire paramyosin (unc-15) gene. From these clones, numerous subclones containing epitopes reacting with anti-paramyosin sera were obtained, providing strong evidence that the initial cloned fragment was, in fact, derived from the structural gene for paramyosin. The complete nucleotide sequence of a 12 x 10(3) base-pair region spanning the gene was obtained. The gene is composed of ten short exons encoding a protein of 866 [corrected] amino acid residues. Paramyosin is highly similar to residues 267 to 1089 of myosin heavy chain rods. For most of its length, paramyosin appears to form an alpha-helical coiled-coil and shows the expected heptad repeat of hydrophobic amino acid residues and the 28-residue repeat of charged amino acids characteristic of myosin heavy chain rods. However, paramyosin differs from myosin in having non-helical extensions at both the N and C termini and an additional "skip" residue that interrupts the 28-residue repeat. The distribution of charges along the length of the paramyosin rod is also significantly different from that of myosin heavy chain rods. Potential charge-mediated interactions between paramyosin rods and between paramyosin and myosin rods were calculated using a model successfully applied previously to the analysis of the myosin rod sequences. Myosin rods aligned in parallel show optimal charge-charge interactions at multiples of 98 residue staggers (i.e. at axial displacements of multiples of 143 A). Paramyosin rods, in contrast, appear to interact optimally at parallel staggers of 493 residues (i.e. at axial displacements of 720 A) but show only weak interaction peaks at 98 or 296 residues. Similar calculations suggest optimal interactions between paramyosin molecules and myosin rods and in their anti-parallel alignments. The implications of these results for the structure of the bare zone and the assembly of nematode thick filaments are discussed.

Interactions of INS (CRS) elements and the splicing machinery regulate the production of Rev-responsive mRNAs.

The human immunodeficiency virus type (HIV-1) Rev protein stimulates the export to the cytoplasm of unspliced HIV-1 mRNAs carrying the Rev response element (RRE). However, simple addition of the RRE to beta-globin pre-mRNA does not confer a Rev response on this heterologous transcript. In this paper, we demonstrate that a strong Rev response is conferred on beta-globin pre-mRNA when an inhibitory (INS) element is inserted into the gene together with the RRE. In the presence of INS element, Rev was able to stimulate the export to the cytoplasm of unspliced mRNA 10 to 15-fold. INS elements from the HIV-1 p17 gag and pol genes were equally active in complementing Rev-dependent nuclear export of unspliced mRNA. By contrast, mutated p17 gag INS element, known to be inactive in gag mRNA instability assays, was unable to complement the Rev/RRE system and stimulate nuclear export. Similarly, AUUUA-instability elements from the granulocyte-macrophage colony stimulating factor mRNA (GM-CSF) destabilised beta-globin mRNA but could not substitute for the HIV INS elements. Complementation between the Rev/RRE system and the INS elements was only observed when splicing was efficient. When splicing of the beta-globin gene receptor is impaired by mutations in the 5' splice donor, the 3' splice acceptor sequence, or the polypyrimidine tract, the majority of the unspliced mRNA is exported from the nucleus in the absence of Rev. In the presence of splice site mutations, Rev is able to act independently of a functional INS element and increase the export of unspliced mRNA three to fivefold. We propose that nuclear factor(s) binding to INS elements separate unspliced beta-globin pre-mRNA from the splicing apparatus. Pre-mRNA in this "INS compartment" remains accessible to Rev. Thus, there is a synergy between the INS elements and Rev which leads to enhanced nuclear export of unspliced mRNA.

Flexible regions of RNA structure facilitate co-operative Rev assembly on the Rev-response element.

The oligomerisation of Rev on the Rev-response element (RRE) was studied using a series of model substrates. Only a monomer of Rev is able to bind efficiently to a high affinity site that is flanked by perfect duplex RNA. Addition of a bulge or a second stem structure adjacent to the high affinity site permits the co-operative incorporation of a second Rev molecule to the RNA. Model RREs carrying bulges can bind Rev with a higher degree of co-operativity than the native structure. Oligomerisation was efficient when the bulge was moved to the opposite strand of the duplex, but was severely impaired when the distance between the bulge and the high affinity site was increased by more than 8 bp. Rev can oligomerise at either end of the RNA-protein complex formed at the high affinity site; when the duplex flanking a high affinity site is disrupted by a bulge or a stem, oligomerisation proceeds in the direction of the disruption regardless of the orientation of the high affinity site. The results are consistent with the "molecular rheostat" model for RRE function, which suggests that Rev binding to the RRE is highly distributive and provides a sensitive measurement of intracellular Rev concentrations.

Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the unc-54 myosin heavy chain of Caenorhabditis elegans.

We have sequenced 11 representative mutations of the unc-54 myosin heavy chain gene of Caenorhabditis elegans that affect the synthesis, assembly or enzymatic activity of the encoded myosin heavy chain. Six of the sequenced unc-54 mutations cause premature termination of protein synthesis. Four mutations (e1092, e1115, e1213, e1328) were ochre mutations, one mutation (e903) was a frameshift, which caused premature termination at a nearby UGA terminator, and one mutation (e190) was a deletion that altered the reading frame and caused termination at an ochre codon. Two mutations (e675 and s291) were inphase deletions, which resulted in a shortened myosin rod segment. These aberrant myosins fail to assemble into normal thick filaments. The sequence alterations of the missense mutations (e1152, s74, s95) indicated amino acid residues that are critical for myosin function. The mutation e1152 causes the production of a myosin heavy chain that fails to assemble into thick filaments. It had two adjacent amino acid substitutions at the extreme amino terminus of the rod, indicating a role for subfragment-2 in thick filament assembly. Mutants homozygous for s74 or s95 are very slow-moving, although they make myosin heavy chains that assemble normally. The encoded amino acid substitutions of s95 and s74 are in the 23 X 10(3) Mr and 50 X 10(3) Mr domains of the myosin head, flanking the ATP binding site. The sequenced mutations are distributed throughout the gene in the order predicted from genetic fine-structure mapping experiments. Seven of eight point mutations isolated following ethylmethane sulphonate mutagenesis were G X C to A X T transitions. A single X-ray-induced allele proved to be a deletion of two adjacent thymidine residues. The three deletion mutations were found in a region of the myosin rod with numerous direct and inverted nucleotide sequence repeats, but their origin cannot be accounted for by homologous recombination. Instead, a comparison of the deletion junctions suggests that the deletions arose by a site-specific mechanism.

Regulatory and essential light-chain-binding sites in myosin heavy chain subfragment-1 mapped by site-directed mutagenesis.

Site-directed mutagenesis of the cloned subfragment-1 (S-1) region of the unc-54 gene, encoding the myosin heavy chain B (MHC B) from Caenorhabditis elegans, has been used to locate binding sites for the regulatory and essential light chains. MHC B S-1 synthesized in Escherichia coli co-migrated with rabbit skeletal muscle myosin S-1 (Mr 90,000), was recognized by anti-nematode myosin antiserum on immunoblots, and specifically bound to 125I-labelled regulatory and essential light chains in a gel overlay assay. Deletion of 102 residues from the C terminus (mutant 655) reduced regulatory and essential light-chain binding to about 30% and 20% of wild-type levels, respectively. Similar reductions in relative binding of the two light chains were seen with mutant 534, in which 38 residues were deleted from the C terminus. Potential binding sites within 75 residues of the C terminus of S-1 were mapped by construction of five other mutant S-1 clones (398, 399, 400, 409 and 411) containing internal deletions of ten to 12 amino acid residues. These showed up to 30% reductions in their ability to bind essential light chains, but did not differ significantly from wild-type in their ability to bind regulatory light chains. Another mutant, 415, containing a deletion of a conserved acidic hexapeptide, E-D-I-R-D-E, showed enhancement of binding of regulatory and essential light chains to 150% and 165% of wild-type levels. Hence, the major binding sites for both light chains are within 38 amino acid residues of the C terminus.

A molecular rheostat. Co-operative rev binding to stem I of the rev-response element modulates human immunodeficiency virus type-1 late gene expression.

The complete biologically active human immunodeficiency virus type-1 (HIV-1) rev-response element (RRE) RNA is 351 nucleotides (nt) in length, and includes an extra 58 nt on the 5' end and 59 nt on the 3' end beyond the sites proposed in the original models for the RRE secondary structure. The extra sequences are able to form a duplex structure which extends Stem I. The presence of an elongated Stem I structure in the RRE RNA was confirmed by nuclease mapping experiments. Nuclease protection experiments have shown that rev binds to restricted regions of the RRE, including the high affinity site located at the base of Stem IIb and along the length of the Stem I region. The three large stem-loop structures which protrude from Stem I and Stem IIb (Stems IIc, III+IV and V) remain accessible to nucleases even in the presence of a large excess of protein. Gel-retardation experiments show that the truncations of Stem I reduced the total number of rev molecules that can bind co-operatively and with high affinity to the RRE RNA. To test whether the elongated Stem I structure is required for maximal rev activity, a series of truncations which progressively reduced the length of Stem I was introduced into an HIV-1 derived reporter plasmid. In the presence of rev and a functional RRE, there is an increase in the levels of gag and env mRNA in the cytoplasm and a decrease in levels of tat and rev mRNAs. Each of the truncations in Stem I reduced the rev responses, with the longest truncations producing the greatest losses of activity. The data suggest that the RRE acts as a "molecular rheostat" designed to detect rev levels during the early stages of the HIV growth cycle.

Hydrogen-bonding contacts in the major groove are required for human immunodeficiency virus type-1 tat protein recognition of TAR RNA.

The binding site for tat on TAR RNA was analysed by preparing a series of model RNA substrates carrying site-specific functional group modifications. The test RNAs were prepared by annealing two short synthetic oligoribonucleotides to form a duplex structure with a U-rich bulge and flanking sequences identical to TAR RNA. Tat binds these duplex RNAs with approximately half the affinity for wild-type TAR RNA. Substitution at positions U23 or U25 by the base analogue, O4-methyl-dT, which is deficient in its ability to hydrogen-bond at the N3 position reduces tat affinity more than 20-fold. Modifications to purines in the stem of TAR RNA that affect hydrogen-bonding ability in either the major or the minor groove of duplex RNA were also tested. Removal of the nitrogen atom at either the N7 position of G26 or at the N7 position of A27 reduces tat affinity 10- to 20-fold. By contrast removal of the exocyclic amino group in the minor groove at position G26, by substitution with inosine, does not affect tat binding significantly. A single methylphosphonate substitution at the phosphate bond between A22 and U23 also leads to a significant loss of tat binding ability, whereas all other methylphosphonate substitutions in the U-rich bulge are not harmful to tat binding. We conclude that tat forms multiple specific hydrogen bonds to a series of dispersed sites displayed in the major groove of the TAR RNA molecule. These include the N3-H of U23, the N7 of G26, the N7 of A26 and the phosphate between A22 and U23.