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)

The stimulatory effect of Mg2+ and Mn2+ on the ribonuclease H (RNase H) functions of HIV-1 reverse transcriptase (RT) has been evaluated using a model 90-nt RNA template/36-nt DNA primer. Wild type enzyme exhibits similar endonuclease and directional processing activities in response to both cations, while RNase H activity (hydrolysis of double-stranded RNA) is only evident in the presence of Mn2+. Enzyme altered at the p66 residue Glu478 (Glu478-->Gln478), which participates in metal ion binding, is completely inactive in Mg2+. However, Mn2+ restores specifically its endoribonuclease activity. In the presence of Mn2+, mutant RT also catalyzes specific removal of the tRNA replication primer, eliminating the possibility of contaminating Escherichia coli RNase H in our recombinant enzyme. However, the efficiency with which mutant RT catalyzes transfer of nascent DNA between RNA templates (an event mandating RNase H activity) is severely reduced. These findings raise the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retroviral replication.
 ...
PMID:Divalent cation modulation of the ribonuclease functions of human immunodeficiency virus reverse transcriptase. 754 83

Retroviral integrase (IN) functions to insert retroviral DNA into the host cell chromosome in a highly coordinated manner. IN catalyzes two biochemically separable reactions: processing of the viral DNA ends and joining of these ends to the host DNA. Previous studies suggested that these two reactions are chemically similar and are carried out by a single active site that is characterized by a highly conserved constellation of carboxylate residues, the D,D(35)E motif. We report here the crystal structure of the isolated catalytic domain of avian sarcoma virus (ASV) IN, solved using multiwavelength anomalous diffraction data for a selenomethionine derivative and refined at 1.7 A resolution. The protein is a crystallographic dimer with each monomer featuring a five-stranded mixed beta-sheet region surrounded by five alpha-helices. Based on the general fold and the arrangement of catalytic carboxylate residues, it is apparent that ASV IN is a member of a superfamily of proteins that also includes two types of nucleases, RuvC and RNase H. The general fold and the dimer interface are similar to those of the analogous domain of HIV-1 IN, whose crystal structure has been determined at 2.5 A resolution. However, the ASV IN structure is more complete in that all three critical carboxylic acids, Asp64, Asp121 and Glu157, are ordered. The ordered active site and the considerably higher resolution of the present structure are all important to an understanding of the mechanism of retroviral DNA integration, as well as for designing antiviral agents that may be effective against HIV.
 ...
PMID:High-resolution structure of the catalytic domain of avian sarcoma virus integrase. 756 93

Short oligonucleotides that can bind to adjacent sites on target mRNA sequences are designed and evaluated for their binding affinity and biological activity. Sequence-specific binding of short tandem oligonucleotides is compared with a full-length single oligonucleotide (21mer) that binds to the same target sequence. Two short oligonucleotides that bind without a base separation between their binding sites on the target bind cooperatively, while oligonucleotides that have a one or two base separation between the binding oligonucleotides do not. The binding affinity of the tandem oligonucleotides is improved by extending the ends of the two oligonucleotides with complementary sequences. These extended sequences form a duplex stem when both oligonucleotides bind to the target, resulting in a stable ternary complex. RNase H studies reveal that the cooperative oligonucleotides bind to the target RNA with sequence specificity. A short oligonucleotide (9mer) with one or two mismatches does not bind at the intended site, while longer oligonucleotides (21mers) with one or two mismatches still bind to the same site, as does a perfectly matched 21mer, and evoke RNase H activity. HIV-1 inhibition studies reveal an increase in activity of the cooperative oligonucleotide combinations as the length of the dimerization domain increases.
 ...
PMID:Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides. 756 72

We have investigated two regions of the viral RNA of human immunodeficiency virus type 1 (HIV-1) as potential targets for antisense oligonucleotides. An oligodeoxynucleotide targeted to the U5 region of the viral genome was shown to block the elongation of cDNA synthesized by HIV-1 reverse transcriptase in vitro. This arrest of reverse transcription was independent of the presence of RNase H activity associated with the reverse transcriptase enzyme. A second oligodeoxynucleotide targeted to a site adjacent to the primer binding site inhibited reverse transcription in an RNase H-dependent manner. These two oligonucleotides were covalently linked to a poly(L-lysine) carrier and tested for their ability to inhibit HIV-1 infection in cell cultures. Both oligonucleotides inhibited virus production in a sequence- and dose-dependent manner. PCR analysis showed that they inhibited proviral DNA synthesis in infected cells. In contrast, an antisense oligonucleotide targeted to the tat sequence did not inhibit proviral DNA synthesis but inhibited viral production at a later step of virus development. These experiments show that antisense oligonucleotides targeted to two regions of HIV-1 viral RNA can inhibit the first step of viral infection--i.e., reverse transcription--and prevent the synthesis of proviral DNA in cell cultures.
 ...
PMID:Sequence-specific inhibition of human immunodeficiency virus (HIV) reverse transcription by antisense oligonucleotides: comparative study in cell-free assays and in HIV-infected cells. 756 37

The crystal structure of the core domain of bacteriophage Mu transposase, MuA, has been determined at 2.4 A resolution. The first of two subdomains contains the active site and, despite very limited sequence homology, exhibits a striking similarity to the core domain of HIV-1 integrase, which carries out a similar set of biochemical reactions. It also exhibits more limited similarity to other nucleases, RNase H and RuvC. The second, a beta barrel, connects to the first subdomain through several contacts. Three independent determinations of the monomer structure from two crystal forms all show the active site held in a similar, apparently inactive configuration. The enzymatic activity of MuA is known to be activated by formation of a DNA-bound tetramer of the protein. We propose that the connections between the two subdomains may be involved in the cross-talk between the active site and the other domains of the transposase that controls the activity of the protein.
 ...
PMID:Structure of the bacteriophage Mu transposase core: a common structural motif for DNA transposition and retroviral integration. 762 12

An oligonucleotide-peptide conjugate, having dual binding capability for a designated RNA, was designed. The peptide portion of the conjugate interacts with a folded domain in the RNA, whereas the oligonucleotide portion hybridizes with a nearby single-stranded region in the RNA. The dual specificity was proven in a model HIV-1 TAR RNA system using an RNase H cleavage assay to assess antisense binding to this RNA. The peptide portion of the conjugate was shown to confer increased specificity on the oligonucleotide.
 ...
PMID:Dual-specificity interaction of HIV-1 TAR RNA with Tat peptide-oligonucleotide conjugates. 763 1

Site-directed mutagenesis has been used to assess the importance of lysine 263 in substrate binding of human immunodeficiency virus-1 (HIV-1) reverse transcriptase. Previous studies have indicated that lysine 263 functions in the binding of 2'-deoxynucleoside 5'-triphosphate (dNTP) substrates (Basu, A., Tirumalai, R. S., and Modak, M. J. (1989) J. Biol. Chem. 264, 8746-8752). We studied this interaction directly by using site-specific mutagenesis to change lysine 263 to a serine. Highly purified mutant enzyme K263S bound natural dNTP substrates and primed polynucleic acid substrates with equal affinity when compared to the wild type reverse transcriptase. No difference was observed in the binding of 3'-azido-2',3'-dideoxythymidine 5'-triphosphate to the mutant reverse transcriptase on the basis of Km and Ki determinations. The serine substitution had no effect on RNase H activity. These results indicate that lysine 263 is not essential in the binding of substrates to HIV-1 reverse transcriptase.
 ...
PMID:Biochemical analysis of human immunodeficiency virus-1 reverse transcriptase containing a mutation at position lysine 263. 767 98

The reverse transcriptase (RT) of HIV-1 has been mutagenized within the carboxyl-terminal domain which harbors the RNase H. Two amino acids highly conserved among all 14 known RT sequences but not in the bacterial RNase H have been mutagenized resulting in the mutant proteins N494D and Q475E. They were expressed as recombinant proteins, purified, and analyzed for their in vitro properties in comparison to the p66 homodimeric wild-type and a previously described H539N mutant. The N494D mutant closely resembles the wild-type RNase H, exhibits an endonuclease activity and a processive RNase H activity, gives rise to small RNA hydrolysis products, and acts in concert with the RT. The Q475E mutant is more defective and resembles the H539N mutant, exhibits a retarded endonuclease activity and an impaired 3'-->5' processive RNA cleavage activity, gives rise to predominantly larger RNA hydrolysis products, is less processive in the presence of competitor substrate, and is defective in its ability to hydrolyze the polypurine tract and homopolymeric hybrids. Short homopolymeric stretches cause a pausing of the RT of wild-type and mutants which results in a coordinated action of the RNase H. Pausing of the RT correlates with RNase H cleavages about 20 nucleotides behind the point of synthesis. The defects of the mutant enzymes can be interpreted on the basis of the known crystallography data.
 ...
PMID:Enzymatic analysis of two HIV-1 reverse transcriptase mutants with mutations in carboxyl-terminal amino acid residues conserved among retroviral ribonucleases H. 767

The multifunctional HIV-1 RT (human immunodeficiency virus type 1-reverse transcriptase) enzyme possesses three main functions including the RNA- and DNA-directed DNA polymerases and the RNase H. The bisheteroarylpiperazine U-87201E inhibits the two polymerase functions but not the RNase H. Enzymatic kinetic studies of the HIV-1 RT-catalyzed RNA- and DNA-directed DNA polymerase activities were carried out in order to determine if the inhibitor interferes with either the template:primer or the deoxyribonucleotide triphosphate (dNTP)-binding sites of the enzyme. The data were analyzed using steady-state kinetics, considering that the polymerase reaction is ordered in that the template:primer is added first, followed by the dNTP and that the enzyme functions processively. The data were consistent with the model. The steady-state rate constants for the forward and backward reactions were of similar magnitude for both the RNA- and DNA-catalyzed DNA polymerases and suggest that both functions share the same substrate-binding sites. The dissociation constants for the enzyme-inhibitor and enzyme-substrate-inhibitor complexes were somewhat higher for the DNA-directed DNA polymerase function as compared to the RNA directed one. This indicates that U-87201E is a more potent inhibitor for the RNA-directed DNA polymerase than the DNA-directed DNA polymerase. The pattern of inhibition exerted by U-87201E was noncompetitive with respect to both the nucleic acid and nucleotide-binding sites of the RT enzyme for both the RNA- and DNA-directed DNA polymerases. Hence, U-87201E inhibits these functions by interacting with a site distinct from the template:primer and dNTP-binding sites. HIV-2 RT was insensitive to U-87201E, demonstrating the unique sensitivity of HIV-1 RT to this inhibitor.
 ...
PMID:Steady-state kinetic studies with the non-nucleoside HIV-1 reverse transcriptase inhibitor U-87201E. 768 Oct 60

A specific cleavage by the reverse transcriptase-associated RNase H activity generates the RNA primer for plus strand DNA synthesis during reverse transcription. Previously, we used site-directed mutagenesis to define the sequence features of the polypurine tract (PPT) required for correct plus strand priming by the Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Rattray, A. J., and Champoux, J. J. (1989) J. Mol. Biol. 208, 445-456). Although the sequences of human immunodeficiency virus type 1 (HIV-1) and M-MuLV diverge completely outside a 20-base region encompassing the PPT, within this region there are only three differences between the two viruses. Here we show that the HIV-1 reverse transcriptase will utilize the M-MuLV PPT as an origin for plus strand initiation in vitro. This finding enabled us to use the set of PPT mutants previously generated in M-MuLV, in conjunction with a small set of newly derived mutations within the HIV-1 PPT, to study plus strand priming by the HIV-1 reverse transcriptase. Despite the similarity between the two PPT regions, the sequence features important for positioning RNase H for the cleavage reaction that generates the plus strand primer are different for the two viruses. For M-MuLV, the -7A residue is a critical specificity determinant in the priming reaction, whereas for HIV-1, the -2G and -4G residues play key roles in determining the specificity of priming.
 ...
PMID:The sequence features important for plus strand priming by human immunodeficiency virus type 1 reverse transcriptase. 768 Oct 62


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