Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0021051 (immunodeficiency)
 71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Integration of the human immunodeficiency virus type 1 (HIV-1) DNA into the human genome requires the virus-encoded integrase protein. The recombinant integrase protein of HIV-1 (isolate Bru) was prepared by constructing a plasmid based on pET-15b encoding the integrase gene. Integrase of HIV-1 was purified using a bacterial expression system (Escherichia coli). The main kinetic parameters of HIV-1 integrase (K(m) = (3.7 +/- 0.2).10(-10) M, k(cat) = (1.2 +/- 0.3).10(-7 )sec(-1)) were determined using an oligonucleotide duplex constructed on the basis of the U5-terminal sequence of proviral HIV-1 DNA as the substrate. Inhibition of integrase by aurintricarbonic acid ([I](50) = 6.3 +/- 0.4 microM) and dependence of integrase activity on Mg2+ and Mn2+ concentration were studied.
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PMID:Characterization of recombinant integrase of human immunodeficiency virus type 1 (isolate Bru). 1460 41

Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+. Activation of RNase H by these metal ions followed sigmoidal dose-response curves suggesting cooperative metal ion binding. Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose-response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.
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PMID:Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors. 1462 18

Bovine viral diarrhea virus (BVDV) is amongst the best-characterized members of the Flaviviridae, that includes the hepatitis C virus (HCV). The virally encoded RNA-dependent RNA polymerase (RdRp) plays a crucial role during replication and therefore represents an important target for the development of antiviral drugs. Here we studied biochemical mechanisms associated with the inhibition of BVDV RNA synthesis by 2'-hydroxyl, 3'-deoxynucleoside triphosphates (3'-dNTPs). All four nucleotide analogues are effectively incorporated and act as chain-terminators. However, relatively low, physiologically relevant concentrations of pyrophosphate (PPi) are sufficient to drive the reaction backwards, which results in primer unblocking and rescue of RNA synthesis. Metal ion requirements for nucleotide incorporation and pyrophosphorolysis are similar; the efficiency of both reactions is higher with Mn2+ as compared to Mg2+. Complexes containing chain-terminated primer strands are stable in the presence of heparin, which increases the probability that pyrophosphorolysis occurs before the enzyme can dissociate from its nucleic acid substrate. In contrast to the reverse transcriptase of the human immunodeficiency virus type-1 (HIV-1 RT), the BVDV RdRp may not recruit NTP pools as PPi donors. Conversely, we found that the efficiency of primer unblocking is severely compromised in the presence of increasing concentrations of the NTP that is complementary to the next template position. These data suggest that the incoming NTP can access its designated binding site, which, in turn, prevents the catalytically competent complexation of PPi. The results of this study provide novel insights into mechanisms involved in pyrophosphorolysis associated with viral RdRps, and suggest that the excision reaction is likely to be an important parameter that can affect susceptibility to nucleotide analogue inhibitors directed against viral RdRps.
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PMID:Excision of incorporated nucleotide analogue chain-terminators can diminish their inhibitory effects on viral RNA-dependent RNA polymerases. 1500 48

The toxicity of nucleoside reverse transcriptase inhibitors (NRTIs) is linked to altered mitochondrial DNA (mtDNA) replication and subsequent disruption of cellular energetics. This manifests clinically as elevated concentrations of lactate in plasma. The mechanism(s) underlying how the changes in mtDNA replication lead to lactic acidosis remains unclear. It is hypothesized that mitochondrial oxidative stress links the changes in mtDNA replication to mitochondrial dysfunction and ensuing NRTIs toxicity. To test this hypothesis, changes in mitochondrial function, mtDNA amplification efficiency, and oxidative stress were assessed in HepG2-cultured human hepatoblasts treated with the NRTI stavudine (2',3'-didehydro-2',3'-deoxythymidine or d4T) for 48 h. d4T produced significant mitochondrial dysfunction with a 1.5-fold increase in cellular lactate to pyruvate ratios. In addition, d4T caused a dose-dependent decrease in mtDNA amplification and a correlative increase in abundance of markers of mitochondrial oxidative stress. Manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, MnTBAP, a catalytic antioxidant, ameliorated or reversed d4T-induced changes in cell injury, energetics, mtDNA amplification, and mitochondrial oxidative stress. In conclusion, d4T treatment elevates mitochondrial reactive oxygen species (ROS), enhances mitochondrial oxidative stress, and contributes mechanistically to NRTI-induced toxicity. These deleterious events may be potentiated in acquired immunodeficiency syndrome (AIDS) by human immunodeficiency virus (HIV) infection itself, coinfection (e.g., viral hepatitis), aging, substance, and alcohol use.
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PMID:Mitochondrial oxidative stress in human hepatoma cells exposed to stavudine. 1528 86

Retroviral integrase prepares viral DNA for integration by removing 2 nucleotides from each end of unintegrated DNA in a reaction referred to as processing. However, it has been known since the processing assay was first described that avian integrases frequently nick 3 nucleotides, as well as 2 nucleotides, from viral DNA ends when reaction mixtures contain Mn2+. We now report that specificity for the biologically relevant "-2" site is enhanced when the serine at amino acid 124 of Rous sarcoma virus (RSV) integrase is replaced by alanine, valine, glycine, lysine, or aspartate. The protein with a serine-to-aspartate substitution exhibited especially high fidelity for the correct site, as evidenced by a ratio of -2 nicks to -3 nicks that was more than 40-fold greater than that for the wild-type enzyme in reactions with Mn2+. Even with Mg2+, the substituted proteins exhibited greater specificity than the wild type, especially the S124D protein. Moreover, this protein was more efficient than the wild type at processing viral DNA ends. Unexpectedly, however, the S124D protein was significantly impaired at catalyzing the insertion of viral DNA ends in reactions with Mn2+ and joining was undetectable in reactions with Mg2+. Thus, the S124D protein has separated the processing and joining activities of integrase. Similar results were found for human immunodeficiency virus integrase with the analogous substitution. No proteins with comparable properties have been described. Moreover, RSV virions containing integrase with the S124D mutation were unable to replicate in cell cultures. Together, these data suggest that integrase has evolved to have submaximal processing activity so that it can also catalyze DNA joining.
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PMID:A substitution in rous sarcoma virus integrase that separates its two biologically relevant enzymatic activities. 1579 55

Integration is a crucial step in the life cycle of human immunodeficiency virus type 1 (HIV-1); therefore, inhibitors of HIV-1 integrase are candidates for antiretroviral therapy. Two 7-hydroxytropolone derivatives (alpha-hydroxytropolones) were found to inhibit HIV-1 integrase. A structure-activity relationship investigation with several tropolone derivatives from The National Cancer Institute compound repository demonstrated that the 7-hydroxy group is essential for integrase inhibition. alpha-Hydroxytropolones preferentially inhibit strand transfer and are inhibitory both in the presence of magnesium or manganese. Lack of inhibition of disintegration in the presence of magnesium coupled with results from different cross-linking assays suggests alpha-hydroxytropolones as interfacial inhibitors. We propose that alpha-hydroxytropolones chelate the divalent metal (Mg2+ or Mn2+) in the enzyme active site. The most active compound against HIV-1 integrase in biochemical assays [2,4,6-cycloheptatrien-1-one, 2,7-dihydroxy-4-isopropyl (NSC 18806) IC50 = 4.8 +/- 2.5 microM] exhibits weak cytoprotective activity against HIV-1(IIIB) in a cell-based assay. alpha-Hydroxytropolones represent a new family of inhibitors for the development of novel drugs against HIV infection.
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PMID:Preferential inhibition of the magnesium-dependent strand transfer reaction of HIV-1 integrase by alpha-hydroxytropolones. 1641 35

The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn2+ or Mg2+ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3'-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg2+ than in the presence of Mn2+. Modifications of base pairs 7-9 affected 3'-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.
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PMID:Probing of HIV-1 integrase/DNA interactions using novel analogs of viral DNA. 1650 Aug 99

Paired metal ions have been proposed to be central to the catalytic mechanisms of RNase H nucleases, bacterial transposases, Holliday junction resolvases, retroviral integrases and many other enzymes. Here we present a sensitive assay for DNA transesterification in which catalysis by human immunodeficiency virus-type 1 (HIV-1) integrase (IN) connects two DNA strands (disintegration reaction), allowing detection using quantitative PCR (qPCR). We present evidence suggesting that the three acidic residues of the IN active site function through metal binding using metal rescue. In this method, the catalytic acidic residues were each substituted with cysteines. Mn2+ binds tightly to the sulfur atoms of the cysteine residues, but Mg2+ does not. We found that Mn2+, but not Mg2+, could rescue catalysis of each cysteine-substituted enzyme, providing evidence for functionally important metal binding by all three residues. We also used the PCR-boosted assay to show that HIV-1 IN could carry out transesterification reactions involving DNA 5' hydroxyl groups as well as 3' hydroxyls as nucleophiles. Lastly, we show that Mn2+ by itself (i.e. without enzyme) can catalyze formation of a low level of PCR-amplifiable product under extreme conditions, allowing us to estimate the rate enhancement due to the IN-protein scaffold as at least 60 million-fold.
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PMID:Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay. 1708 78

Ty1 reverse transcriptase/RNase H (RT/RH) is exquisitely sensitive to manganese concentrations. Elevated intracellular free Mn(2+) inhibits Ty1 retrotransposition and in vitro Ty1 RT-polymerizing activity. Furthermore, Mn(2+) inhibition is not limited to the Ty1 RT, as this ion similarly inhibits the activities of both avian myeloblastosis virus and human immunodeficiency virus type 1 RTs. To further characterize Mn(2+) inhibition, we generated RT/RH suppressor mutants capable of increased Ty1 transposition in pmr1 Delta cells. PMR1 codes for a P-type ATPase that regulates intracellular calcium and manganese ion homeostasis, and pmr1 mutants accumulate elevated intracellular manganese levels and display 100-fold less transposition than PMR1(+) cells. Mapping of these suppressor mutations revealed, surprisingly, that suppressor point mutations localize not to the RT itself but to the RH domain of the protein. Furthermore, Mn(2+) inhibition of in vitro RT activity is greatly reduced in all the suppressor mutants, whereas RH activity and cleavage specificity remain largely unchanged. These intriguing results reveal that the effect of these suppressor mutations is transmitted to the polymerase domain and suggest biochemical communication between these two domains during reverse transcription.
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PMID:Mn2+ suppressor mutations and biochemical communication between Ty1 reverse transcriptase and RNase H domains. 1753 63

We have carried out the study of the photochemical properties of a series of synthetic meso-tetraphenylsulfonated porphyrins (TPPMS4) bonded to several metal ions such as: Cu(II), Zn(II), Pd(II), Mn(II), Fe(III), Ni(II) and Co(II) for the optimization of their clinical applications as antiviral agents against the human immunodeficiency virus (HIV-1) as well as the study of the in vitro antiviral photoinactivation mechanisms with future application in blood sterilization. A selective inhibition has been determined in the viral growth (HIV-1) when this is irradiated in the presence of the complex TPPFeS4 and TPPMnS4 (photosensitizer-mediated Type I reaction) as well as in the 1O2-mediated (Type II reaction) in the presence of TPPPdS4 and TPPZnS4, remaining cellular viability unaltered in each case.
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PMID:Comparative antiviral (HIV) photoactivity of metalized meso- tetraphenylsulfonated porphyrins. 1833 33


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