Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two constituent protein domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase were expressed separately and purified to homogeneity. The N-terminal domain (p51) behaves as a monomeric protein exhibiting salt-sensitive DNA polymerase activity. The C-terminal domain (p15) on its own has no detectable RNase H activity. However, the combination of both isolated p51 and p15 in vitro leads to reconstitution of RNase H activity on a defined substrate. These results demonstrate that domains of HIV-1 reverse transcriptase are functionally interdependent to a much higher degree than in the case of reverse transcriptase from Moloney murine leukemia virus.
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PMID:Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 reverse transcriptase. 170 27

The crystal structure of the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT) has been determined at a resolution of 2.4 A and refined to a crystallographic R factor of 0.20. The protein folds into a five-stranded mixed beta sheet flanked by an asymmetric distribution of four alpha helices. Two divalent metal cations bind in the active site surrounded by a cluster of four conserved acidic amino acid residues. The overall structure is similar in most respects to the RNase H from Escherichia coli. Structural features characteristic of the retroviral protein suggest how it may interface with the DNA polymerase domain of p66 in the mature RT heterodimer. These features also offer insights into why the isolated RNase H domain is catalytically inactive but when combined in vitro with the isolated p51 domain of RT RNase H activity can be reconstituted. Surprisingly, the peptide bond cleaved by HIV-1 protease near the polymerase-RNase H junction of p66 is completely inaccessible to solvent in the structure reported here. This suggests that the homodimeric p66-p66 precursor of mature RT is asymmetric with one of the two RNase H domains at least partially unfolded.
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PMID:Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase. 184 17

HIV reverse transcriptase (RT) is the target of the most widely used treatments for AIDS. Biochemical and mutagenesis studies performed on HIV-1 RT are reviewed in light of the enzyme's structure and functions. Features described include domain arrangement, dimerization, proteolytic processing, and specific recognition of the priming tRNA. Possible regions of functional importance as determined by comparative amino acid sequence analysis and by site-directed mutagenesis are identified. Among the conclusions of the analysis is the unexpected realization that the substrate for proteolytic maturation of the HIV-1 RT p66/p66 homodimer to the p66/p51 heterodimer is most likely an unfolded RNase H domain. In addition, the current progress in crystallization and structure determination of HIV-1 RT is described. Finally, a functional-model of the active reverse transcription complex is presented.
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PMID:HIV reverse transcriptase structure-function relationships. 171 68

Purified recombinant reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) was used to raise 21 monoclonal antibodies with anti-RT specificities. The antibodies were characterized using Western blotting against native virus and recognized either the p66 or p66, p51 components of RT. Further immunoblotting using either cyanogen bromide fragmented RT or truncated mutants of RT along with cross-competition studies enabled the location of various immunogenic regions of RT to be identified. Three antibodies recognized a linear epitope in the N-terminal region (amino acids 128-176). Also, a neutralizing RT antibody recognized a conformational epitope in this region. Three monoclonals had epitopes mapped to linear sequences in the RNase H region at the C-terminus of the RT. Another neutralizing antibody, also requiring folding of the RT protein had its epitope more centrally located (231-353). Of the remaining 13 monoclonals, 7 were roughly located in the C-terminal region and required folding of the protein for epitope recognition and only three of the remaining six could be mapped to conformational epitopes in N-terminal and central regions of the RT. None of the antibodies tested recognized HIV-2 RT products p68 and p55 in Western blot.
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PMID:Monoclonal antibodies define linear and conformational epitopes of HIV-1 pol gene products. 171 17

The reverse transcriptase/RNase H of HIV-1 is composed of a p66/p51 heterodimer when analyzed from virus particles. A recombinant reverse transcriptase (RT)/RNase H which after purification consisted mainly of p66 was analyzed as substrate of the purified recombinant HIV-1 protease p9 in vitro. The p66 protein if treated with the protease is processed to a stable p66/p51 heterodimer. A p15 protein is a prominent cleavage product which was identified as the carboxyterminal portion of p66 by means of a monoclonal antibody. It exhibits RNase H activity when tested by activated gel analysis. Presence of SDS during the incubation allowed complete degradation of p66 depending on the conditions, which indicates that conformation of a substrate is relevant for cleavage by the HIV-1 protease. A synthetic heptapeptide AET-FYVD derived from the region between RT and RNase H is cleaved efficiently in vitro by the HIV-1 protease at the F'Y junction, and may mimick a natural cleavage site. P66/p51 heterodimers exhibit higher RT and RNase H activities than p66 when renatured from polyacrylamide gels.
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PMID:Cleavage of the HIV-1 p66 reverse transcriptase/RNase H by the p9 protease in vitro generates active p15 RNase H. 171 81

Poly(rA).oligo(dT)n binding to human immunodeficiency virus type-1 reverse transcriptase heterodimer (p66-p51) was primer length-dependent. The estimated Kd for (n = 10-14) was 20-30 nM and for (n = 16-20) was 0.11-0.14 nM. Gel electrophoretic analysis of the patterns of primer extension was consistent with an abrupt change in the Kd between a primer length of 14 and 16 nucleotides. Further, the rate constant for dissociation of the reverse transcriptase-template-primer complex was determined from steady state kinetics and enzyme-template-primer trapping experiments to be independent of primer length. Thus, the abrupt change in Kd was most likely due to a change in the rate constant for formation of the reverse transcriptase-template-primer complex. A similar shift in the Kd for template-primer binding was observed with poly(dA).oligo(dT)n. Reverse transcriptase homodimer (p66) catalyzed the incorporation of dTMP into poly(rA).oligo(dT)n with the same primer length dependence observed for the heterodimer. In contrast, binding of the p51 homodimer to poly(rA).oligo(dT)n was independent of primer length. Thus, the RNase H domain may contribute to reverse transcriptase heterodimer or p66 homodimer binding to template-primers in which the primer length is greater than 14 nucleotides.
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PMID:Human immunodeficiency virus reverse transcriptase. Effect of primer length on template-primer binding. 171 16

We have purified and determined functional parameters of reconstituted, recombinant HIV-1 reverse transcriptase (RT) heterodimers within which either the p66 or p51 polypeptide was selectively mutated in one or both aspartic acid residues constituting the proposed polymerase active site (-Y-M-D-D-). Heterodimers containing a mutated p51 polypeptide retain almost wild type levels of both RNA-dependent DNA polymerase and ribonuclease H (RNaseH) activity. In contrast, heterodimers whose p66 polypeptide was likewise mutated exhibit wild type RNaseH activity but are deficient in RNA-dependent DNA polymerase activity. These results indicate that in heterodimer RT, the p51 component cannot compensate for active site mutations eliminating the activity of p66, indirectly implying that solely the p66 aspartic acid residues of heterodimer are crucial for catalysis.
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PMID:Subunit-selective mutagenesis indicates minimal polymerase activity in heterodimer-associated p51 HIV-1 reverse transcriptase. 171 45

Cytoplasmic extracts prepared from cells infected with metabolically radiolabeled virions of human immunodeficiency virus type 1 contain viral DNA in association with labeled viral proteins. Viral DNA can be purified from these extracts by gel filtration chromatography and sucrose gradient sedimentation as a part of a nucleoprotein complex containing integrase as the only viral protein detectable by immunoprecipitation and gel electrophoretic analysis. The purified complex contains no detectable gag gene products, including p17, p24, p7, or p6, and contains no additional pol gene products, including the p10 protease, p66 and p51 polymerase, or the p15 RNase H. Nearly all of the purified nucleoprotein complexes are capable of integrating into heterologous DNA targets in vitro. These observations demonstrate that integrase is a component of the human immunodeficiency virus type 1 preintegration complex and suggest that integrase may be the only viral protein necessary for the integration of retroviral DNA.
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PMID:Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. 200 49

Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-Leu*Phe-) and (b) between the p51 and RNase H domain (-Phe*Tyr-) within p66 of RT in the HIV-1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a beta-branched (Ile) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a beta-branched (Val) residue in the P2 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.
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PMID:Mutating P2 and P1 residues at cleavage junctions in the HIV-1 pol polyprotein. Effects on hydrolysis by HIV-1 proteinase. 204 56

Human immune deficiency virus (HIV) replicates by conversion of the RNA genome into the double-stranded DNA provirus. The reverse transcriptase is not the only enzymatic function crucial in DNA-provirus synthesis. A viral-coded RNase H activity which specifically degrades RNA in RNA-DNA hybrids has been shown to be essential as well. Here we demonstrate that the HIV-reverse transcriptase which consists of a two-polypeptide complex, p66 and p51, copurifies with an RNase H activity which exhibits properties of a processive exonuclease. Only the p66 molecule, not p51, is active as polymerase as evidenced by activated gel analysis. p66 exhibits RNase H activity when precipitated as immune complex by a monoclonal antibody raised against a bacterially expressed carboxy-terminal portion of p66. The monoclonal antibody which does not interfere with enzyme activity also precipitates a second population of molecules with RNase H activity which is of low mol. wt, p15. This RNase H appears therefore to be derived from the carboxy terminus of p66 during processing to the p51 polypeptide. It exhibits low template-binding ability and is of a non-processing mode of action which may be due to the absence of the reverse transcriptase domain. These results lend experimental support to the hypothesis that the RNase H gene maps at the carboxy terminus of the reverse transcriptase. Since both RNase H populations are virus-coded they may be essential for retrovirus replication in general and useful targets for chemotherapeutic agents.
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PMID:Identification and characterization of HIV-specific RNase H by monoclonal antibody. 245 83


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