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)

We present evidence that the integrases (INs) of HIV types 1 and 2 are inhibited in vitro by the reverse transcriptases (RTs) of HIV-1, HIV-2 and murine leukaemia virus. Both 3'-end processing and 3'-end joining (strand transfer) activities of IN were affected by the RTs. Full inhibitions were accomplished with most RT and IN combinations tested at around equimolar RT/IN ratios. The disintegration activity of IN was also inhibited by RTs. Neither DNA synthesis nor the ribonuclease H (RNase H) domain of RT were involved in IN inhibition, since specific DNA polymerase inhibitors did not affect the level of IN inhibition, and the p51 isoform of HIV-1 RT (which lacks the RNase H domain) is as effective in inhibiting IN as the heterodimeric p66/p51 isoform. On the other hand, the catalytic activities of HIV RTs were not affected by the INs, showing that RTs can inhibit IN activities, whereas INs do not inhibit RTs. We postulate that sequences and/or three-dimensional protein structures common to RTs interact with INs and inhibit their activities. We show evidence for this hypothesis and discuss the possible sites of IN involved in this interaction.
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PMID:Inhibition of the integrases of human immunodeficiency viruses type 1 and type 2 by reverse transcriptases. 1180 85

Understanding the molecular mechanisms of HIV-1 reverse transcriptase (RT) action and drug inhibition is essential for designing effective antiretroviral therapies. Although comparisons of the different crystal forms of RT give insights into the flexibility of different domains, a direct computational assessment of the effect of inhibitor binding on the collective dynamics of RT is lacking. A structure-based approach is used here for exploring the dynamics of RT in unliganded and inhibitor-bound forms. Non-nucleoside RT inhibitors (NNRTI) are shown to interfere directly with the global hinge-bending mechanism that controls the cooperative motions of the p66 fingers and thumb subdomains. The net effect of nevirapine binding is to change the direction of domain movements rather than suppress their mobilities. The second generation NNRTI, efavirenz, on the other hand, shows the stronger effect of simultaneously reorienting domain motions and obstructing the p66 thumb fluctuations. A second hinge site controlling the global rotational reorientations of the RNase H domain is identified, which could serve as a target for potential inhibitors of RNase H activity.
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PMID:Inhibitor binding alters the directions of domain motions in HIV-1 reverse transcriptase. 1221 Oct 16

Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RT of human immunodeficiency virus type-1 (HIV-1) is composed of two subunits. The p51, which is the smaller subunit, shares with the larger p66 subunit the same amino-terminal part (which encompasses the DNA polymerase domain) and lacks the carboxyl-terminal segment of the p66 (which is the RNase H domain). The structure of the polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains) linked to the RNase H domain. Chemical modifications by thiol-specific reagents of cysteine 280, located in alpha helix I in the thumb subdomain of the polymerase domain, affect substantially only the RNase H activity. Also, the substitution of a serine for C280 did not alter any of the RT activities. Here we have systematically modified the C280 residue to either of the following residues: W, P, H, L, M, Y, Q, E or R. Only the first two mutations lead to a marked reduction in the RNase H activity, whereas none of the mutations affected the polymerase function to a significant extent. As expected, due to their impaired RNase H, the C280W and C280P mutants also had a very low DNA strand-transfer activity. It is also apparent from subunit-directed mutagenesis that each of the RT subunits contributes to the level of RNase H activity, yet the contribution of the p51 subunit to this activity is somewhat higher than that of the p66. Steady-state kinetic analyses have indicated that the RNase H activity was reduced mainly due to the sharp increase in the K(m) rather than changes in the k(cat) values. This suggests that the modifications of C280 lead to an impaired affinity of HIV-1 RT towards the RNA-DNA substrate.
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PMID:Mutagenesis of cysteine 280 of the reverse transcriptase of human immunodeficiency virus type-1: the effects on the ribonuclease H activity. 1261 5

Human immunodeficiency virus type 2 (HIV-2) infection is a serious problem in West Africa and Asia. However, there have been relatively few studies of HIV-2 reverse transcriptase (RT), a potential target for antiviral therapy. Detailed knowledge of HIV-2 RT activities is critical for development of specific high-throughput screening assays of potential inhibitors. Here, we have conducted a systematic evaluation of HIV-2 RT function, using assays that model specific steps in reverse transcription. Parallel studies were performed with HIV-1 RT. In general, under standard assay conditions, the polymerase and RNase H activities of the two enzymes were comparable. However, when the RT concentration was significantly reduced, HIV-2 RT was less active than the HIV-1 enzyme. HIV-2 RT was also impaired in its ability to catalyze secondary RNase H cleavage in assays that mimic tRNA primer removal during plus-strand transfer and degradation of genomic RNA fragments during minus-strand DNA synthesis. In addition, initiation of plus-strand DNA synthesis was much less efficient with HIV-2 RT than with HIV-1 RT. This may reflect architectural differences in the primer grip regions in the p66 (HIV-1) and p68 (HIV-2) palm subdomains of the two enzymes. The implications of our findings for antiviral therapy are discussed.
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PMID:Human immunodeficiency virus type 2 reverse transcriptase activity in model systems that mimic steps in reverse transcription. 1280 62

Both x-ray crystallography and chemical footprinting indicate that bases of the HIV type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid deviate from standard Watson-Crick base pairing. However, the contribution of these structural anomalies to the accuracy of plus-strand primer selection by HIV-1 reverse transcriptase is not immediately clear. To address this issue, DNA templates harboring single and pairwise non-hydrogen-bonding isosteres of cytosine (2-fluoro-4-methylbenzene deoxyribonucleoside) and thymine (2,4-difluoro-5-methylbenzene deoxyribonucleoside) were synthesized and hybridized to PPT-containing RNA primers as a means of locally removing hydrogen bonding and destabilizing paired structure. Cleavage of these hybrids was examined with p66/p51 HIV-1 reverse transcriptase and a mutant carrying an alteration in the p66 RNase H primer shown to specifically impair PPT processing. Analog insertion within the PPT (rG):(dC) and central (rA):(dT) tracts repositioned the RNase H domain such that the RNA/DNA hybrid was cleaved 3-4 bp from the site of insertion, a distance corresponding closely to the spatial separation between the catalytic center and RNase H primer grip. However, PPT processing was significantly impaired when the junction between these tracts was substituted. Substitutions within the upstream (rA):(dT) tract, where maximum distortion had previously been observed, destroyed PPT processing. Collectively, our scanning mutagenesis approach implicates multiple regions of the PPT in the accuracy with which it is excised from (+) U3 RNA and DNA, and also provides evidence for close cooperation between the RNase H primer grip and catalytic center in achieving this cleavage.
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PMID:Hydrolysis of RNA/DNA hybrids containing nonpolar pyrimidine isosteres defines regions essential for HIV type 1 polypurine tract selection. 1297 38

Reverse transcriptase (RT) and integrase (IN) are two key catalytic enzymes encoded by all retroviruses. It has been shown that a specific interaction occurs between the human immunodeficiency virus type 1 (HIV-1) RT and IN proteins (X. Wu, H. Liu, H. Xiao, J. A. Conway, E. Hehl, G. V. Kalpana, V. R. Prasad, and J. C. Kappes, J. Virol. 73:2126-2135, 1999). We have now further examined this interaction to map the binding domains and to determine the effects of interaction on enzyme function. Using recombinant purified proteins, we have found that both a HIV-1 RT heterodimer (p66/p51) and its individual subunits, p51 and p66, are able to bind to HIV-1 IN. An oligomerization-defective mutant of IN, V260E, retained the ability to bind to RT, showing that IN oligomerization may not be required for interaction. Furthermore, we report that the C-terminal domain of IN, but not the N-terminal zinc-binding domain or the catalytic core domain, was able to bind to heterodimeric RT. Deletion analysis to map the IN-binding domain on RT revealed two separate IN-interacting domains: the fingers-palm domain and the carboxy-terminal half of the connection subdomain. The carboxy-terminal domain of IN alone retained its interaction with both the fingers-palm and the connection-RNase H fragments of RT, but not with the half connection-RNase H fragment. This interaction was not bridged by nucleic acids, as shown by micrococcal nuclease treatment of the proteins prior to the binding reaction. The influences of IN and RT on each other's activities were investigated by performing RT processivity and IN-mediated 3' processing and joining reactions in the presence of both proteins. Our results suggest that, while IN had no influence on RT processivity, RT stimulated the IN-mediated strand transfer reaction in a dose-dependent manner up to 155-fold. Thus, a functional interaction between these two viral enzymes may occur during viral replication.
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PMID:Interaction between human immunodeficiency virus type 1 reverse transcriptase and integrase proteins. 1511 87

The C-terminus of the HIV-1 reverse transcriptase heterodimer was reconstructed into a single polypeptide. The construct encodes the p51 thumb (T) and connection (C) subdomains joined through a linker region to the p66 connection (C) and RNase H (R) domain. The TCCR protein was purified from insoluble fractions of Escherichia coli lysates. The TCCR construct maintains Mn(2+)-dependent RNase H activity and specifically cleaves the substrate mimicking the tRNA removal required for second-strand transfer reactions.
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PMID:Expression of the C-terminus of HIV-1 reverse transcriptase p66 and p51 subunits as a single polypeptide with RNase H activity. 1533 74

A highly efficient cell-free translation system has been combined with suppressor tRNA technology to substitute nor-Tyr and 3-fluoro-Tyr in place of Tyr183 at the DNA polymerase active site of p66 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Supplementing the wild-type HIV-1 p51 RT subunit into this translation system permitted reconstitution of the biologically relevant p66/p51 heterodimer harboring Tyr analogs exclusively on the catalytically competent p66 subunit. Addition of an affinity tag at the p66 C-terminus allowed rapid, one-step purification of reconstituted and selectively mutated heterodimer HIV-1 RT via strep-Tactin-agarose affinity chromatography. The purified enzyme was demonstrated to be free of contaminating nucleases, allowing characterization of the DNA polymerase and ribonuclease H activities associated with HIV-1 RT. Preliminary characterization of HIV-1 RT(nor-Tyr) and HIV-1 RT(m-fluoro-Tyr) is presented. The success of this strategy will facilitate detailed molecular analysis of structurally and catalytically critical amino acids via their replacement with closely related, unnatural analogs.
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PMID:Site- and subunit-specific incorporation of unnatural amino acids into HIV-1 reverse transcriptase. 1547 80

Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3'-azido-3'-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.
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PMID:Dissecting the effects of DNA polymerase and ribonuclease H inhibitor combinations on HIV-1 reverse-transcriptase activities. 1568 43

A variety of biochemical and structural studies indicate that two regions of the human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid deviate from standard Watson-Crick geometry. However, it is unclear whether and how these regions cooperate to ensure PPT primer selection by reverse transcriptase-associated ribonuclease H and subsequent removal from nascent (+)-DNA. To address these issues, we synthesized oligonucleotides containing abasic lesions in either the PPT (+)-RNA primer or (-)-DNA template to locally remove nucleobases, although retaining the sugar-phosphate backbone. KMnO(4) footprinting indicates such lesions locally alter duplex structure, whereas thermal melting studies show significantly reduced stability when lesions are positioned around the scissile bond. Substituting the (-)-DNA template between positions -15 and -13 altered cleavage specificity, whereas equivalent substitutions of the (+)-RNA had almost no effect. The unpaired base of the DNA template observed crystallographically (-11C) could also be removed without significant loss of cleavage specificity. With respect to the scissile -1/+1 phosphodiester bond, template nucleobases could be removed without loss of cleavage specificity, whereas equivalent lesions in the RNA primer were inhibitory. Our data suggest an interaction between the p66 thumb subdomain of HIV-1 reverse transcriptase, and the DNA template in the "unzipped" portion of the RNA/DNA hybrid could aid in positioning the ribonuclease H catalytic center at the PPT/U3 junction and also provides insights into nucleic acid geometry around the scissile bond required for hydrolysis.
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PMID:Investigating HIV-1 polypurine tract geometry via targeted insertion of abasic lesions in the (-)-DNA template and (+)-RNA primer. 1577 25


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