Refined solution structure of the dimeric N-terminal HHCC domain of HIV-2 integrase.

The solution structure of the dimeric N-terminal domain of HIV-2 integrase (residues 1-55, named IN(1-55)) has been determined using NMR spectroscopy. The structure of the monomer, which was already reported previously [Eijkelenboom et al. (1997) Curr. Biol., 7, 739-746], consists of four alpha-helices and is well defined. Helices alpha1, alpha2 and alpha3 form a three-helix bundle that is stabilized by zinc binding to His12, His16, Cys40 and Cys43. The dimer interface is formed by the N-terminal tail and the first half of helix alpha3. The orientation of the two monomeric units with respect to each other shows considerable variation. 15N relaxation studies have been used to characterize the nature of the intermonomeric disorder. Comparison of the dimer interface with that of the well-defined dimer interface of HIV-1 IN(1-55) shows that the latter is stabilized by additional hydrophobic interactions and a potential salt bridge. Similar interactions cannot be formed in HIV-2 IN(1-55) [Cai et al. (1997) Nat. Struct. Biol., 4, 567-577], where the corresponding residues are positively charged and neutral ones.

Dissecting the role of the N-terminal domain of human immunodeficiency virus integrase by trans-complementation analysis.

The human immunodeficiency virus (HIV) integrase protein (IN) catalyzes two reactions required to integrate HIV DNA into the human genome: 3' processing of the viral DNA ends and integration. IN has three domains, the N-terminal zinc-binding domain, the catalytic core, and the C-terminal SH3 domain. Previously, it was shown that IN proteins mutated in different domains could complement each other. We now report that this does not require any overlap between the two complementing proteins; an N-terminal domain, provided in trans, can restore IN activity of a mutant lacking this domain. Only the zinc-coordinating form of the N-terminal domain can efficiently restore IN activity of an N-terminal deletion mutant. This suggests that interaction between different domains of IN is needed for functional multimerization. We find that the N-terminal domain of feline immunodeficiency virus IN can support IN activity of an N-terminal deletion mutant of HIV type 2 IN. These cross-complementation experiments indicate that the N-terminal domain contributes to the recognition of specific viral DNA ends.

Mutational scan of the human immunodeficiency virus type 2 integrase protein.

Retroviral integrase (IN) cleaves linear viral DNA specifically near the ends of the DNA (cleavage reaction) and subsequently couples the processed ends to phosphates in the target DNA (integration reaction). In vitro, IN catalyzes the disintegration reaction, which is the reverse of the integration reaction. Ideally, we would like to test the role of each amino acid in the IN protein. We mutagenized human immunodeficiency virus type 2 IN in a random way using PCR mutagenesis and generated a set of mutants in which 35% of all residues were substituted. Mutant proteins were tested for in vitro activity, e.g., site-specific cleavage of viral DNA, integration, and disintegration. Changes in 61 of the 90 proteins investigated showed no phenotypic effect. Substitutions that changed the choice of nucleophile in the cleavage reaction were found. These clustered around the active-site residues Asp-116 and Glu-152. We also found alterations of amino acids that affected cleavage and integration differentially. In addition, we analyzed the disintegration activity of the proteins and found substitutions of amino acids close to the dimer interface that enhanced intermolecular disintegration activity, whereas other catalytic activities were present at wild-type levels. This study shows the feasibility of investigating the role of virtually any amino acid in a protein the size of IN.

The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc.

BACKGROUND: Integrase mediates a crucial step in the life cycle of the human immunodeficiency virus (HIV). The enzyme cleaves the viral DNA ends in a sequence-dependent manner and couples the newly generated hydroxyl groups to phosphates in the target DNA. Three domains have been identified in HIV integrase: an amino-terminal domain, a central catalytic core and a carboxy-terminal DNA-binding domain. The amino-terminal region is the only domain with unknown structure thus far. This domain, which is known to bind zinc, contains a HHCC motif that is conserved in retroviral integrases. Although the exact function of this domain is unknown, it is required for cleavage and integration. RESULTS: The three-dimensional structure of the amino-terminal domain of HIV-2 integrase has been determined using two-dimensional and three-dimensional nuclear magnetic resonance data. We obtained 20 final structures, calculated using 693 nuclear Overhauser effects, which display a backbone root-mean square deviation versus the average of 0.25 A for the well defined region. The structure consists of three alpha helices and a helical turn. The zinc is coordinated with His 12 via the N epsilon 2 atom, with His16 via the N delta 1 atom and with the sulfur atoms of Cys40 and Cys43. The alpha helices form a three-helix bundle that is stabilized by this zinc-binding unit. The helical arrangement is similar to that found in the DNA-binding domains of the trp repressor, the prd paired domain and Tc3A transposase. CONCLUSION: The amino-terminal domain of HIV-2 integrase has a remarkable hybrid structure combining features of a three-helix bundle fold with a zinc-binding HHCC motif. This structure shows no similarity with any of the known zinc-finger structures. The strictly conserved residues of the HHCC motif of retroviral integrases are involved in metal coordination, whereas many other well conserved hydrophobic residues are part of the protein core.

DNA substrate requirements for different activities of the human immunodeficiency virus type 1 integrase protein.

The integrase protein (IN) of human immunodeficiency virus type 1 removes two nucleotides from both 3' ends of the viral DNA (donor cleavage) and subsequently couples the newly generated 3' OH groups to phosphates in the target DNA (integration). The sequence requirements of IN for cleavage as well as for integration of viral DNA substrates have previously been studied by mutational analyses and by adduct interference assays. We extended these studies by analysis of heteroduplex oligonucleotide substrates and by missing-base analysis. We found for some base pairs that mutation of only one of the two bases and not the other affected IN activity. These base pairs center around the cleavage site. Besides donor cleavage and integration, IN can also perform "intermolecular disintegration," which has been described as the reversal of the integration reaction. We found that this reaction is independent of viral DNA sequences. In addition, the optimum spacing between the integration sites in intermolecular disintegration does not reflect the spacing found in vivo. These results indicate that this reaction is not the exact reversal of integration but rather is a sequence-independent phosphoryl transfer reaction between gapped DNA duplex molecules.

Cell cycle controlled histone H1, H3, and H4 genes share unusual arrangements of recognition motifs for HiNF-D supporting a coordinate promoter binding mechanism.

Cell cycle and growth control of the DNA binding and transactivation functions of regulatory factors provides a direct mechanism by which cells may coordinate transcription of a multitude of genes in proliferating cells. The promoters of human DNA replication dependent histone H4, H3, and H1 genes interact with at least seven distinct proteins. One of these proteins is a proliferation-specific nuclear factor, HiNF-D, that interacts with a key cis-regulatory element (H4-Site II; 41 bp) present in H4 genes. Here we describe binding sites for HiNF-D in the promoters of H3 and H1 genes using cross-competition, deletion analysis, and methylation interference assays, and we show that HiNF-D recognizes intricate arrangements of at least two sequence elements (CA- and AG-motifs). These recognition motifs are irregularly dispersed and distantly positioned in the proximal promoters (200 bp) of both the H3 and H1 genes. In all cases, these motifs either overlap or are in close proximity to other established transcriptional elements, including ATF and CCAAT sequences. Although HiNF-D can interact with low affinity to a core recognition domain, auxiliary elements in both the distal and proximal portions of each promoter cooperatively enhance HiNF-D binding. Thus, HiNF-D appears to bridge remote regulatory regions, which may juxtapose additional trans-activating proteins interacting within histone gene promoters. Consistent with observations in many cell culture systems, the interactions of HiNF-D with the H4, H3, and H1 promoters are modulated in parallel during the cessation of proliferation in both osteosarcoma cells and normal diploid osteoblasts, and these events occur in conjunction with concerted changes in histone gene expression. Thus, HiNF-D represents a candidate participant in coordinating transcriptional control of several histone gene classes.

Concerted control of multiple histone promoter factors during cell density inhibition of proliferation in osteosarcoma cells: reciprocal regulation of cell cycle-controlled and bone-related genes.

Cell density-induced growth inhibition of osteosarcoma cells (ROS 17/2.8) results in the shutdown of proliferation-specific histone H4 and H2B genes and the concomitant up-regulation of several osteoblast-related genes. In several respects, this reciprocal regulatory relationship is analogous to the proliferation/differentiation transition stage during development of the bone cell phenotype in normal diploid osteoblasts. Here, we comprehensively analyzed the promoter binding activities interfacing with key regulatory elements in the cell cycle-dependent histone and bone-specific osteocalcin genes. Similarly, we examined factors interacting with a series of general transcription regulatory elements that are present in a broad spectrum of promoters. The results show that histone promoter binding activities HiNF-D, HiNF-P/H4TF-2, H4UA-1, and OCT-1, as well as AP-1 activity, are proliferation dependent. These factors decline coordinately during the cessation of proliferation in both ROS 17/2.8 bone tumor cells and normal diploid osteoblasts. Collective down-regulation of these trans-activating factors occurs in both cell types within the physiological context of constitutive regulation of ubiquitous transcription factors (Sp1, ATF, and CCAAT binding proteins). In addition, during growth inhibition of ROS 17/2.8 cells we observe a complex series of modifications in protein/DNA interactions of the osteocalcin gene. These modifications include both increased and decreased representation of promoter factor complexes occurring at steroid hormone response elements as well as tissue-specific basal promoter sequences. These results demonstrate cell growth regulation of the promoter factors binding to the proliferation-specific histone and tissue-specific osteocalcin genes during the cessation of proliferation.

Overlapping and CpG methylation-sensitive protein-DNA interactions at the histone H4 transcriptional cell cycle domain: distinctions between two human H4 gene promoters.

Transcriptional regulation of vertebrate histone genes during the cell cycle is mediated by several factors interacting with a series of cis-acting elements located in the 5' regions of these genes. The arrangement of these promoter elements is different for each gene. However, most histone H4 gene promoters contain a highly conserved sequence immediately upstream of the TATA box (H4 subtype consensus sequence), and this region in the human H4 gene FO108 is involved in cell cycle control. The sequence-specific interaction of nuclear factor HiNF-D with this key proximal promoter element of the H4-FO108 gene is cell cycle regulated in normal diploid cells (J. Holthuis, T.A. Owen, A.J. van Wijnen, K.L. Wright, A. Ramsey-Ewing, M.B. Kennedy, R. Carter, S.C. Cosenza, K.J. Soprano, J.B. Lian, J.L. Stein, and G.S. Stein, Science, 247:1454-1457, 1990). Here, we show that this region of the H4-FO108 gene represents a composite protein-DNA interaction domain for several distinct sequence-specific DNA-binding activities, including HiNF-D, HiNF-M, and HiNF-P. Factor HiNF-P is similar to H4TF-2, a DNA-binding activity that is not cell cycle regulated and that interacts with the analogous region of the H4 gene H4.A (F. LaBella and N. Heintz, Mol. Cell. Biol. 11:5825-5831, 1991). The H4.A gene fails to interact with factors HiNF-M and HiNF-D owing to two independent sets of specific nucleotide variants, indicating differences in protein-DNA interactions between these H4 genes. Cytosine methylation of a highly conserved CpG dinucleotide interferes with binding of HiNF-P/H4TF-2 to both the H4-FO108 and H4.A promoters, but no effect is observed for either HiNF-M or HiNF-D binding to the H4-FO108 gene. Thus, strong evolutionary conservation of the H4 consensus sequence may be related to combinatorial interactions involving overlapping and interdigitated recognition nucleotides for several proteins, whose activities are regulated independently. Our results also suggest molecular complexity in the transcriptional regulation of distinct human H4 genes.