Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A deletion derivative of the integrase protein from human immunodeficiency virus type-1 (HIV-1) consisting of the central core domain (amino acids 50-212) has been characterized biophysically and biochemically. This deletion mutant is of particular interest for structural studies as it can carry out the disintegration reaction suggesting the presence of an active site and, under certain conditions, is more soluble than full-length integrase. The circular dichroism and fluorescence of the deletion mutant and the 288-residue full-length integrase were similar, indicating that the core residues maintain similar overall conformations in both proteins. The deletion mutant is approximately 10% more alpha-helical than the full-length protein. Analytical centrifugation demonstrated that both proteins undergo monomer-dimer association although the truncated protein showed slightly less tendency to dimerize; the dissociation constants were 2.5 x 10(-5) M for the full-length protein and 8.0 x 10(-5) M for the truncated protein. The disintegration activity of both proteins was also compared. Although a higher concentration of the truncation mutant was required for optimal activity, the mutant did not have altered pH or Mn2+ requirements relative to the full-length protein. The combined biophysical and enzymatic studies suggest that this truncated form of HIV-1 integrase is likely to be useful for structural studies.
 ...
PMID:Biophysical and enzymatic properties of the catalytic domain of HIV-1 integrase. 796 98

We present evidence suggesting that the 3'-processing activity of HIV-1 integrase is dramatically affected by electrostatic and/or steric perturbations 3' to the conserved CA dinucleotide. When the phosphodiester bond 3' to the scissile phosphodiester is replaced by a methylphosphonodiester linkage, 3'-processing decreases by two orders of magnitude. This block of cleavage can be somewhat overcome by increasing the pH of the reaction. Labeling of the substrates at the 3'-end revealed blockage of water and glycerol, but stimulation of the viral DNA 3'-hydroxyl, acting as the nucleophile with the methylphosphonodiester substrate. Interestingly, a circular trinucleotide was formed using the phosphodiester and methylphosphonodiester substrates when the terminal nucleotide was 3'-deoxyadenosine but not 2'-deoxyadenosine. Mutagenesis of the enzyme active site has previously been shown to alter the choice of nucleophile in the 3'-processing reaction. Taken together, the results in this study suggest that 'mutagenesis' of the DNA backbone can also alter the choice of nucleophile.
 ...
PMID:Methylphosphonodiester substitution near the conserved CA dinucleotide in the HIV LTR alters both extent of 3'-processing and choice of nucleophile by HIV-1 integrase. 797 Dec 74

Integrase is the only viral protein necessary for integration of retroviral DNA into chromosomal DNA of the host cell. Biochemical analysis of human immunodeficiency virus type 1 (HIV-1) integrase with purified protein and synthetic DNA substrates has revealed extensive information regarding the mechanism of action of the enzyme, as well as identification of critical residues and functional domains. Since in vitro reactions are carried out in the absence of other viral proteins and they analyze strand transfer of only one end of the donor substrate, they do not define completely the process of integration as it occurs during the course of viral infection. In an effort to further understand the role of integrase during viral infection, we initially constructed a panel of 24 HIV-1 mutants with specific alanine substitutions throughout the integrase coding region and analyzed them in a human T-cell line infection. Of these mutant viruses, 12 were capable of sustained viral replication, 11 were replication defective, and 1 was temperature sensitive for viral growth. The replication defective viruses express and correctly process the integrase and Gag proteins. Using this panel of mutants and an additional set of 18 mutant viruses, we identified nine amino acids which, when replaced with alanine, destroy integrase activity. Although none of the replication-defective mutants are able to integrate into the host genome, a subset of them with alterations in the catalytic triad are capable of Tat-mediated transactivation of an indicator gene linked to the viral long terminal repeat promoter. We present evidence that integration of the HIV-1 provirus is essential not only for productive infection of T cells but also for virus passage in both cultured peripheral blood lymphocytes and macrophage cells.
 ...
PMID:Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells. 798 32

Several previously unnoticed genes in the human immunodeficiency virus type 1 (HIV-1), potentially encoding selenoproteins, have been discovered by analyzing the genomic RNA structure and its relation to novel open reading frames. We have found a number of new potential RNA pseudoknots, including one in the long terminal repeat, several that coincide with highly conserved enzyme active site sequences in the pol coding region, and one in the env coding region. These pseudoknots can potentially direct the synthesis of selenocysteine (SeC) containing--1 frameshift fusion proteins. This is possible because we have found potential SeC insertion sequences (SECIS) in the RNA of HIV and other retroviruses; such structures are known to be necessary and sufficient for the incorporation of SeC at UGA "stop" codons anywhere in a eukaryotic mRNA. In several locations, UGA codons in the -1 reading frame are highly conserved across a broad spectrum of primate immunodeficiency viruses. Due to the degeneracy of the genetic code, this conservation cannot be explained by evolutionary selection of the pol gene protein sequence alone. Such observations, combined with the conservation of the associated reading frames, strongly suggest that these are real genes, and thus that the pseudoknots are also real. A protease pseudoknot-directed -1 frameshift fusion protein contains a highly conserved SeC codon and has significant similarities to a number of DNA binding proteins, including papillomavirus E2 proteins, suggesting it may be a virally encoded repressor of HIV transcription when cleaved by protease from the rest of the gag-pol gene product. A reverse transcriptase (RT) frameshift fusion protein replaces the RT active site with a highly conserved SeC-containing module. An integrase frameshift fusion protein contains the N-terminal integrase DNA-binding domain and a potential ATP-binding "GKS" motif; it has significant similarities to several helicases, but no SeC codons. A potential frameshift fusion protein from env has one SeC codon, but not in a highly conserved position. SeC incorporation could extend the nef gene product by 33 residues through the C-terminal UGA codon without frameshifting, potentially leading to substantial SeC utilization in infected cells.(ABSTRACT TRUNCATED AT 400 WORDS)
 ...
PMID:A basis for new approaches to the chemotherapy of AIDS: novel genes in HIV-1 potentially encode selenoproteins expressed by ribosomal frameshifting and termination suppression. 806 94

Protein folding conditions were established for human immunodeficiency virus integrase (IN) obtained from purified bacterial inclusion bodies. IN was denatured by 6 M guanidine.HCl-5 mM dithiothreitol, purified by gel filtration, and precipitated by ammonium sulfate. The reversible solvation of precipitated IN by 6 M guanidine.HCl allowed for wide variation of protein concentration in the folding reaction. A 6-fold dilution of denatured IN by 1 M NaCl buffer followed by dialysis produced enzymatically active IN capable of 3' OH end processing, strand transfer, and disintegration using various human immunodeficiency virus-1 (HIV-1) long terminal repeat DNA substrates. The specific activities of folded IN preparations for these enzymatic reactions were comparable to those of soluble IN purified directly from bacteria. The subunit composition and enzymatic activities of IN were affected by the folding conditions. Standard folding conditions were defined in which monomers and protein aggregates sedimenting as dimers and tetramers wree produced. These protein aggregates were enzymatically active, whereas monomers had reduced strand transfer activity. Temperature modifications of the folding conditions permitted formation of mainly monomers. Upon assaying, these monomers were efficient for strand transfer and disintegration, but the oligomeric state of IN under the conditions of the assay is determinate. Our results suggest that monomers of the multidomain HIV-1 IN are folded correctly for various catalytic activities, but the conditions for specific oligomerization in the absence of catalytic activity are undefined.
 ...
PMID:Folding of the multidomain human immunodeficiency virus type-I integrase. 806 20

We have used the molecular dynamics (MD) simulation package AMBER4 to search the conformation of a peptide predicted as a leucine zipper motif for the human immunodeficiency virus type 1 integrase protein (HIV IN-LZM). The peptide is composed of 22 amino acid residues and its location is from Val 151 to Leu 172. The searching procedure also includes two known alpha-helices that served as positive controls--namely, a 22-residue GCN4-p1 (LZM) and a 20-residue poly (L-alanine) (PLA). A 21-residue peptide extracted from a cytochrome C crystal (CCC-t) with determined conformation as a beta-turn is also included as a negative control. At the beginning of the search, two starting conformations--namely, the standard right-handed alpha-helix and the fully stretched conformations--are generated for each peptide. Structures generated as standard alpha-helix are equilibrated at room temperature for 90 ps while structures generated as a fully stretched one are equilibrated at 600 K for 120 ps. The CCC-t and PLA helices are nearly destroyed from the beginning of equilibration. However, for both the HIV IN-LZM and the GCN4-p1 LZM structures, there is substantial helicity being retained throughout the entire course of equilibration. Although helix propagation profiles calculated indicate that both peptides possess about the same propensity to form an alpha-helix, the HIV IN-LZM helix appears to be more stable than the GCN4-p1 one as judged by a variety of analyses on both structures generated during the equilibration course. The fact that predicted HIV IN-LZM can exist as an alpha-helix is also supported by the results of high temperature equilibration run on the fully stretched structures generated. In this run, the RMS deviations between the backbone atoms of the structures with the lowest potential energy (PE) identified within every 2 ps and the structure with the lowest PE searched in the same course of simulation are calculated. For both the HIV IN-LZM and the GCN4-p1 LZM, these rms values decrease with the decrease of PE, which indicates that both structures are closer in conformations as their PEs are moved deeper into the PE well.
 ...
PMID:Molecular dynamics simulation of a leucine zipper motif predicted for the integrase of human immunodeficiency virus type 1. 807 85

The synthesis of a 36-residue fragment of HIV integrase by standard Fmoc-polyamide chemistry was unsuccessful (Trt-protected Cys) or difficult (Acm-protected Cys); after 10-12 couplings, deprotection became hindered and a major portion (30%-50%) of the growing peptide chains became inaccessible to reagents. The synthesis was repeated using N-(2-hydroxy-4-methoxybenzyl) (Hmb) backbone protection at two strategic positions in the sequence, and Acm protection of Cys. The synthesis was trouble-free and progressed smoothly to completion. Electrospray mass spectrometric analysis at each step of the cleavage and purification showed that Hmb groups become O-acetylated during treatment of the peptide with acetic anhydride in the presence of tertiary base. The acetylated Hmb was refractory to cleavage with TFA/scavenger mixtures but could be readily removed following deacetylation with piperidine. The target peptide was purified to > 98% purity and an overall yield of 15%.
 ...
PMID:Roles of electrospray mass spectrometry, counterion distribution monitoring and N-(2-hydroxy-4-methoxybenzyl) backbone protection in peptide synthesis. 808 Oct 67

Retroviral DNA integration requires the activity of at least one viral protein, the integrase (IN) protein. We cloned and expressed the integrase gene of feline immunodeficiency virus (FIV) in Escherichia coli as a fusion to the malE gene and purified the IN fusion protein by affinity chromatography. The protein is active in site-specific cleavage of the viral DNA ends, DNA strand transfer, and disintegration. FIV IN has a relaxed viral DNA substrate requirement: it cleaves and integrates FIV DNA termini, human immunodeficiency virus DNA ends, and Moloney murine leukemia virus DNA ends with high efficiencies. In the cleavage reaction, IN exposes a specific phosphodiester bond near the viral DNA end to nucleophilic attack. In vitro, either H2O, glycerol, or the 3' OH group of the viral DNA terminus can serve as nucleophile in this reaction. We found that FIV IN preferentially uses the 3' OH ends of the viral DNA as nucleophile, whereas HIV IN protein preferentially uses H2O and glycerol as nucleophiles.
 ...
PMID:Activities of the feline immunodeficiency virus integrase protein produced in Escherichia coli. 810 10

Single-amino-acid changes in a highly conserved central region of the human immunodeficiency virus type 1 (HIV-1) integrase protein were analyzed for their effects on viral protein synthesis, virion morphogenesis, and viral replication. Alteration of two amino acids that are invariant among retroviral integrases, D116 and E152 of HIV-1, as well as a mutation of the highly conserved amino acid S147 blocked viral replication in two CD4+ human T-cell lines. Mutations of four other highly conserved amino acids in the region had no detectable effect on viral replication, whereas mutations at two positions, N117 and Y143, resulted in viruses with a delayed-replication phenotype. Defects in virion precursor polypeptide processing, virion morphology, or viral DNA synthesis were observed for all of the replication-defective mutants, indicating that changes in integrase can have pleiotropic effects on viral replication.
 ...
PMID:Genetic analysis of the human immunodeficiency virus type 1 integrase protein. 810 24

We report the activities of HIV integrase protein on a novel DNA substrate, consisting of a pair of gapped duplex molecules. Integrase catalyzed an intermolecular disintegration reaction that requires positioning of a pair of the gapped duplexes in a configuration that resembles the intgration intermediate. However, the major reaction resulted from an intramolecular reaction involving a single gapped duplex, giving rise to a hairpin. Surprisingly, a deletion mutant of integrase that lacks both the amino and carboxyl terminal regions still catalyzed the intermolecular disintegration reaction, but supported only a very low level of the intramolecular reaction. The central core region of integrase is therefore sufficient to both bind the gapped duplex DNA and juxtapose a pair of such molecules through protein-protein interactions. We suggest that the branched DNA structures of the previously reported disintegration substrate, and the intermolecular disintegration substrate described here, assist in stabilizing protein-protein interactions that otherwise require the amino and carboxy terminal regions of integrase.
 ...
PMID:Intermolecular disintegration and intramolecular strand transfer activities of wild-type and mutant HIV-1 integrase. 815 8


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>