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

We have analyzed the effects of several natural compounds related to avarols and avarones on the catalytic functions of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). The most potent substances, designated as avarone A,B and E and avarol F, inhibited indiscriminately the enzymatic activities of HIV-1 RT, namely the RNA-dependent and DNA-dependent DNA polymerase as well as the ribonuclease H. The inhibition of the DNA polymerase activity was found to be non-competitive with respect to either the template-primer or the deoxynucleotidetriphosphate. These studies suggest that the hydroxyl group at the ortho position to the carbonyl group at the quinone ring is involved in blocking the RT activity. The identification of the active site of the inhibitors will hopefully lead to the rational design of new potent anti-HIV drugs.
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PMID:The inhibition of human immunodeficiency virus type 1 reverse transcriptase by avarol and avarone derivatives. 169 11

The sequence of the LTR-LTR circle junction of human immunodeficiency virus type 1 (HIV-1) was determined. The circle junction sequences were amplified by the polymerase chain reaction and cloned into M13 sequencing vectors. The circle junction contains 4 base pairs that are not present in the integrated provirus. We show that reverse transcription in HIV-1 initiates with the addition of a dC to the tRNA primer, suggesting that the tRNA used to initiate reverse transcription ends with the consensus CCA triplet. This indicates that the source of one of the four bases in the circle junction is probably the terminal A of the tRNA primer used to initiate reverse transcription. We propose that, in HIV-1, removal of the tRNA primer by RNase H cleavage shows an unusual specificity such that cleavage occurs between the terminal rA and the adjacent rC of the tRNA primer. These data also imply that the HIV-1 integration protein removes two bases from each end of the linear viral DNA during integration as has been described for other well-studied retroviruses.
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PMID:Sequence of the circle junction of human immunodeficiency virus type 1: implications for reverse transcription and integration. 169 9

Substitution of the conserved Asp-443 residue of HIV-1 reverse transcriptase by asparagine specifically suppressed the ribonuclease H activity of the enzyme without affecting the reverse transcriptase activity, suggesting involvement of this ionizable residue at the ribonuclease H active site. An analogous asparagine substitution of the Asp-498 residue yielded an unstable enzyme that was difficult to enzymatically characterize. However, the instability caused by the Asn-498 mutation was relieved by the introduction of a second distal Asn-443 substitution, yielding an enzyme with wild type reverse transcriptase activity, but lacking ribonuclease H activity.
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PMID:Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase. 169 2

The ribonuclease H (RNase H) domain of human immuno-deficiency virus (HIV-1) reverse transcriptase has been produced with the aim of providing sufficient amounts of protein for biophysical studies. A plasmid vector is described which directs high level expression of the RNase H domain under the control of the lambda PL promoter. The domain corresponds to residues 427-560 of the 66 kDa reverse transcriptase. The protein was expressed in Escherichia coli and was purified using ion-exchange and size exclusion chromatography. The purified protein appears to be in a native-like homogeneous conformational state as determined by 1H-NMR spectroscopy and circular dichroism measurements. HIV-protease treatment of the RNase H domain resulted in cleavage between Phe-440 and Tyr-441.
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PMID:Purification and characterization of the RNase H domain of HIV-1 reverse transcriptase expressed in recombinant Escherichia coli. 169 94

We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a polypeptide with an apparent molecular weight of 68 kDa. The HIV-2 reverse transcriptase (RT) made in E. coli is soluble in bacterial extracts and possesses both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activities typical of retroviral RTs. The HIV-2 RT expression clone was used to generate mutations in HIV-2 RT. There is a strong correlation between the effects of individual mutations on the DNA polymerase and RNase H activities. Mutations that profoundly affect the two catalytic functions are not clustered in any particular region of the polypeptide. Those few mutations that selectively affect either the RNase H or the DNA polymerase suggest that, like other retroviral RTs, the DNA polymerase is associated with the amino-terminal portion of HIV-2 RT and the RNase H with the carboxy-terminal portion. Genetically, the HIV-2 RT resembles the HIV-1 RT more closely than it resembles Moloney murine leukemia virus RT. The two catalytic functions of Moloney murine leukemia virus RT can be separately expressed in active form by molecular cloning; those of HIV-1 and HIV-2 RT cannot.
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PMID:Mutational analysis of the DNA polymerase and ribonuclease H activities of human immunodeficiency virus type 2 reverse transcriptase expressed in Escherichia coli. 170 48

The mode of action of the RNase H activity from HIV-1 was analyzed with a purified recombinant p66/p51 reverse transcriptase RT/RNase H protein and RNA-DNA hybrid consisting of RNA harboring the polypurine tract (ppt) and three complementary synthetic DNA oligonucleotides. Upon incubation of this preformed RNA-DNA hybrid with the p66/p51 RT/RNase H, a cleavage pattern is observed that indicates endonucleolytic RNase H activity with some sequence specificity for the next to last nucleotide of the 3'-end of the ppt RNA and one cut within the ppt. The RNase H avoids cleavage of G or A stretches. During RNA-directed DNA synthesis the RNA is hydrolyzed in a concerted action of RT and RNase H whereby the RNase H exhibits endonuclease as well as 3'-5'-exonuclease activity. The distance between the active centers of the RT and RNase H corresponds to 18 base pairs of the RNA-DNA hybrid. Plus-strand DNA-directed DNA synthesis initiates exactly at the next to last nucleotide of the 3'-end of the ppt RNA by means of the RNase H activity.
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PMID:Interaction of HIV-1 ribonuclease H with polypurine tract containing RNA-DNA hybrids. 170 2

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 DNA polymerase and RNase H activities of HIV reverse transcriptase are both essential for HIV replication. Although the two activities are both catalyzed by a single polypeptide, they are physically separate; i.e., the DNA polymerase resides in the N-terminal domain whereas the RNase H is localized in the C-terminal domain. The present study was undertaken to characterize the enzymatic properties of these two activities and to determine whether the two catalytic sites are also functionally distinct. We have observed that EGTA specifically stimulates, whereas CaCl2 selectively inhibits, the RNA-dependent DNA polymerase activity but that neither compound has any effect on the RNase H activity of a recombinant HIV reverse transcriptase. The stimulation of the DNA polymerase activity by EGTA is dependent on the Mg2+ concentration; the greatest stimulation is observed at low Mg2+ concentrations. Similarly, the inhibition of DNA polymerase activity by Ca2+ is influenced by Mg2+ concentration. Ca2+ inhibition can be reversed by increasing Mg2+ concentrations, suggesting the possibility that CaCl2 inhibits the reverse transcriptase activity by competing for a metal-binding site on the enzyme. The pyrophosphate analogue phosphonoformate selectively inhibits the polymerase activity but not the RNase H activity of HIV reverse transcriptase. In contrast, the RNase H activity can be selectively inhibited by deoxyadenosine 5'-monophosphate, whereas the DNA polymerase activity is not inhibited. These results suggest that the DNA polymerase and RNase activities are not only physically separate but that they are also functionally distinct.
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PMID:Functional characterization of RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase. 170 16

A phosphorothioate homocytidine 10-mer containing a cholesteryl moiety covalently linked to the 5'-end (Chol-SdC10) inhibited syncytium formation in susceptible T cells induced by HIV-1 and HIV-2. The syncytium inhibition effect was minimal with unmodified cytidine homopolymer of the same net charge. Chol-SdC10 was shown to protect CEM cells against infection by cell-free HIV-1 particles without any apparent toxicity to the growth of CD4+ T cells. The DNA polymerase activity of the purified reverse transcriptase (RT) of HIV-1 was markedly inhibited by Chol-SdC10 but the effect on the RNase H activity of RT was minimal. Analysis of the kinetics of reverse transcriptase inhibition mediated by the drug revealed that the inhibition at a higher concentration was competitive with respect to template primer binding and noncompetitive at lower concentrations. Chol-SdC10 also partially blocked the binding of gp120 to CD4 in a solid-phase ELISA. These results confirm that the anti-HIV activity of phosphorothioate cytidine homopolymers increases markedly by covalent modification with the cholesteryl moiety at the 5'-end and demonstrates that the cytoprotective effect is manifested at multiple steps in the virus life cycle. These steps include inhibition of retroviral replication activity as well as the binding and fusion of HIV with CD4+ T cells.
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PMID:Mode of action of 5'-linked cholesteryl phosphorothioate oligodeoxynucleotides in inhibiting syncytia formation and infection by HIV-1 and HIV-2 in vitro. 170 17

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


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