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)

The multifunctional enzyme human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer composed of a 66-kDa (p66) subunit and a p66-derived 51-kDa (p51) subunit. p66/p51 HIV-1 RT contains 1 functional DNA polymerase and 1 ribonuclease H (RNase H) active site, which both reside in the p66 subunit at spatially distinct regions. In this study, we have investigated the relationship between the heterodimeric structure of HIV-1 RT and its enzymatic properties by introducing mutations at RT codon W401 that inhibit the formation of p66/p51 heterodimers. We demonstrate a striking correlation between abrogation of both HIV-1 RT dimerization and DNA polymerase activity. In contrast, the p66 monomers exhibited only moderately slowed catalytic rates of DNA polymerase-dependent and DNA polymerase-independent RNase H cleavage activity compared with the wild-type (WT) enzyme. Furthermore, no major changes in the unique cleavage patterns were observed between the WT and mutant enzymes for the different substrates used in the RNase H cleavage assays. Based on these results, and on our current understanding of HIV-1 RT structure, we propose that the p66 monomer can adopt an open tertiary conformation that is similar to that observed for the subunit in the heterodimeric enzyme. We also propose that the formation of intersubunit interactions in HIV-1 RT regulates the establishment of a functional DNA polymerase active site.
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PMID:Relationship between enzyme activity and dimeric structure of recombinant HIV-1 reverse transcriptase. 1585 4

The reverse transcriptase (RT) of all retroviruses is required for synthesis of the viral DNA genome. The human immunodeficiency virus type 1 (HIV-1) RT exists as a heterodimer made up of 51-kDa and 66-kDa subunits. The crystal structure and in vitro biochemical analyses indicate that the p66 subunit of RT is primarily responsible for the enzyme's polymerase and RNase H activities. Since both the p51 and p66 subunits are generated from the same coding region, as part of the Pr160(Gag-Pol) precursor protein, there are inherent limitations for studying subunit-specific function with intact provirus in a virologically relevant context. Our lab has recently described a novel system for studying the RT heterodimer (p51/p66) wherein a LTR-vpr-p51-IRES-p66 expression cassette provided in trans to an RT-deleted HIV-1 genome allows precise molecular analysis of the RT heterodimer. In this report, we describe in detail the specific approaches, alternative strategies, and pitfalls that may affect the application of this novel assay for analyzing RT subunit structure/function in infectious virions and human target cells. The ability to study HIV-1 RT subunit structure/function in a physiologically relevant context will advance our understanding of both RT and the process of reverse transcription. The study of antiretroviral drugs in a subunit-specific virologic context should provide new insights into drug resistance and viral fitness. Finally, we anticipate that this approach will help elucidate determinants that mediate p51-p66 subunit interactions, which is essential for structure-based drug design targeting RT heterodimerization.
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PMID:Analysis of human immunodeficiency virus type 1 reverse transcriptase subunit structure/function in the context of infectious virions and human target cells. 1612 51

Each of the human immunodeficiency virus type 1 (HIV-1) pol-encoded enzymes, protease (PR), reverse transcriptase (RT), and integrase (IN), is active only as a dimer (or higher-order oligomer in the case of IN), but only RT comprises subunits of different masses. RT is a heterodimer of 66-kDa and 51-kDa subunits. The latter is formed by HIV PR-catalyzed cleavage of p66 during virion maturation, resulting in the removal of the RNase H (RNH) domain of a p66 subunit. In order to study the apparent need for RT heterodimers in the context of the virion, we introduced a variety of mutations in the RT p51-RNH protease cleavage site of an infectious HIV-1 molecular clone. Surprisingly, rather than leading to virions with increased RT p66 content, most of the mutations resulted in significantly attenuated virus that contained greatly decreased levels of RT that in many cases was primarily p51 RT. IN levels were also reduced in several mutants. However, most mutants showed normal levels of the Pr160(gag-pol) precursor polyprotein, suggesting that reduced virion RT arose from proteolytic instability rather than decreased incorporation. Mutant virion p24 Gag levels were equivalent to wild type, indicating that Gag incorporation and processing were not affected. Repeated passage of MT-2 cells exposed to mutant viruses led to the appearance of virus with improved replication capacity; these virions contained normally processed RT at near-wild-type levels. These results imply that additional proteolytic processing of RT to the p66/p51 heterodimer is essential to provide proteolytic stability of RT during HIV-1 maturation.
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PMID:Virion instability of human immunodeficiency virus type 1 reverse transcriptase (RT) mutated in the protease cleavage site between RT p51 and the RT RNase H domain. 1614 Jul 71

It is not known whether the low infectivity and low virion-associated polymerase activity of human T-cell lymphotropic virus type-1 (HTLV-1) are due to the quantity or quality of the reverse transcriptase (RT), because the protein has not yet been fully characterized. We have developed anti-RT antibodies and constructed HTLV-1 expression plasmids that express truncated or hemagglutinin-tagged Pol polyproteins to examine the maturation and composition of HTLV-1 RT. We detected virion-associated proteins corresponding to RT-integrase (IN) (pr98) and RT (p62) as well as smaller proteins containing the polymerase (p49) or RNase H domains. We have identified the amino acid sequences in the Pol polyprotein that are cleaved by HTLV-1 protease to yield RT and IN. We have also identified the cleavage sites within RT that give rise to the p49 polymerase fragment. Immunoprecipitation of an epitope-tagged p62 subunit coprecipitated p49, indicating that the HTLV-1 RT complex can exist as a p62/p49 heterodimer analogous to the RT of HIV-1 (p66/p51).
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PMID:Synthesis, processing, and composition of the virion-associated HTLV-1 reverse transcriptase. 1636 88

Deletions, insertions, and amino acid substitutions in the beta3-beta4 hairpin loop-coding region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been associated with resistance to nucleoside RT inhibitors when appearing in combination with other mutations in the RT-coding region. In this work, we have measured the in vivo fitness of HIV-1 variants containing a deletion of 3 nucleotides affecting codon 69 (Delta69) of the viral RT as well as the replication capacity (RC) ex vivo of a series of recombinant HIV-1 variants carrying an RT bearing the Delta69 deletion or the T69A mutation in a multidrug-resistant (MDR) sequence background, including the Q151M complex and substitutions M184V, K103N, Y181C, and G190A. Patient-derived viral clones having RTs with Delta69 together with S163I showed increased RCs under drug pressure. These data were consistent with the viral population dynamics observed in a long-term-treated HIV-1-infected patient. In the absence of drugs, viral clones containing T69A replicated more efficiently than those having Delta69, but only when patient-derived sequences corresponding to RT residues 248 to 527 were present. These effects could be attributed to a functional interaction between the C-terminal domain of the p66 subunit (RNase H domain) and the DNA polymerase domain of the RT. Finally, recombinant HIV-1 clones bearing RTs with MDR-associated mutations, including deletions at codon 69, showed increased susceptibilities to protease inhibitors in phenotypic assays. These effects correlated with impaired Gag cleavage and could be attributed to delayed maturation and decreased production of active protease in those variants.
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PMID:Relative fitness and replication capacity of a multinucleoside analogue-resistant clinical human immunodeficiency virus type 1 isolate with a deletion of codon 69 in the reverse transcriptase coding region. 1731 58

Pausing by reverse transcriptase (RT) during retroviral replication increases the frequency of homologous strand transfer, nucleotide misincorporation, and non-templated nucleotide addition. Pausing frequency increases at sites of DNA damage or upon incorporation of nucleotide analogs with steric barriers. These lesions thus likely stimulate mutations leading to resistant viral strains that escape drug treatments or immune surveillance. To study the response of retroviral RTs to bulky 2' adducts, a ribozyme-catalyzed reaction was used to generate an RNA template strand containing a thiophosphate adduct at a specific 2'-hydroxyl located upstream from a polyadenosine sequence. Subsequent alkylation increased the size of the adduct. Polymerization readthrough efficiencies were compared for mature RTs derived from HIV-1 (p66/p51), AMV (p95/p63), MMLV (p80 monomer), and a truncated version of HIV-1 RT lacking the RNase H domain (p51/p51 homodimer). Readthrough at the 2' lesion was markedly greater for the p51/p51 homodimer of HIV-1 RT than for the other enzymes, suggesting that the presence of the RNase H domain increases the probability that the modified primer/template will encounter a barrier to translocation. Comparison to published structures suggests potential unfavorable interactions between the 2' adduct and W24, F61, I63, D76, and R78 in the fingers domain of the RT. We propose that the enhanced readthrough observed upon RNase H domain deletion alters the trajectory of the primer/template in this region that diminishes steric and electrostatic clash with these residues. The template also included a penta-adenosine sequence that induced pausing in the order MMLV > HIV-1 (p66/p51) > AMV ~ HIV-1 (p51/p51).
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PMID:HIV-1 reverse transcriptase pausing at bulky 2' adducts is relieved by deletion of the RNase H domain. 1739 57

The enzymatic activities of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are strictly correlated with the dimeric forms of this vital retroviral enzyme. Accordingly, the development of inhibitors targeting the dimerization of RT represents a promising alternative antiviral strategy. Based on mutational studies, we applied a structure-based ligand design approach generating pharmacophoric models of the large subunit connection subdomain to possibly identify small molecules from the ASINEX database, which might interfere with the RT subunit interaction. Docking studies of the selected compounds identified several candidates, which were initially tested in an in vitro subunit association assay. One of these molecules (MAS0) strongly reduced the association of the two RT subunits p51 and p66. Most notably, the compound simultaneously inhibited both the polymerase as well as the RNase H activity of the retroviral enzyme, following preincubation with t(1/2) of about 2 h, indicative of a slow isomerization step. This step most probably represents a shift of the RT dimer equilibrium from an active to an inactive conformation. Taken together, to the best of our knowledge, this study represents the first successful rational screen for a small molecule HIV RT dimerization inhibitor, which may serve as attractive hit compound for the development of novel therapeutic agents.
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PMID:Small molecule inhibitors targeting HIV-1 reverse transcriptase dimerization. 1831 36

Previous studies have demonstrated that nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) act as chemical enhancers of human immunodeficiency virus type 1 (HIV-1) RT dimerization. In the current study, we sought to define the role of key residues (101, 103, 108, 181, 188, 190, 225 and 318) in the NNRTI-binding pocket on HIV-1 RT heterodimer stability. Thirteen mutant RTs were constructed and evaluated for p66/p51 RT heterodimer formation using the well-established yeast two-hybrid assay. We found that the mutations K101A, P225H, Y318F and Y318W decreased RT heterodimer stability whereas K103N, V108I, V108W, Y181C, Y188L, G190A, G190E, G190W and P225W increased RT heterodimer stability. While these results demonstrate that residues that comprise the NNRTI-binding pocket contribute to the stability of p66/p51 HIV-1 RT, they did not suggest any obvious correlation between RT dimer stability and the extent of NNRTI resistance. Remarkably, mutations at residue G190 (A, E, W) in the p66 RT subunit were found to dramatically increase heterodimer stability. Notably, the G190W mutation increased RT dimer stability almost to the same extent as did 5 microM efavirenz. In light of these findings, we characterized the in vitro activity of recombinant RT expressing mutations at G190 in the p66 subunit only and compared them with a wild-type enzyme complexed with efavirenz. We found that while mutations at G190 had a significant effect on both the DNA polymerase and ribonuclease H activity of the enzyme, their phenotypic effects did not mirror those induced by efavirenz-binding to RT.
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PMID:Impact of residues in the nonnucleoside reverse transcriptase inhibitor binding pocket on HIV-1 reverse transcriptase heterodimer stability. 1833 60

High-throughput screening of National Cancer Institute libraries of synthetic and natural compounds identified the vinylogous ureas 2-amino-5,6,7,8-tetrahydro-4 H-cyclohepta[ b]thiophene-3-carboxamide (NSC727447) and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (NSC727448) as inhibitors of the ribonuclease H (RNase H) activity of HIV-1 and HIV-2 reverse transcriptase (RT). A Yonetani-Theorell analysis demonstrated that NSC727447, and the active-site hydroxytropolone RNase H inhibitor beta-thujaplicinol were mutually exclusive in their interaction with the RNase H domain. Mass spectrometric protein footprinting of the NSC727447 binding site indicated that residues Cys280 and Lys281 in helix I of the thumb subdomain of p51 were affected by ligand binding. Although DNA polymerase and pyrophosphorolysis activities of HIV-1 RT were less sensitive to inhibition by NSC727447, protein footprinting indicated that NSC727447 occupied the equivalent region of the p66 thumb. Site-directed mutagenesis using reconstituted p66/p51 heterodimers substituted with natural or non-natural amino acids indicates that altering the p66 RNase H primer grip significantly affects inhibitor sensitivity. NSC727447 thus represents a novel class of RNase H antagonists with a mechanism of action differing from active site, divalent metal-chelating inhibitors that have been reported.
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PMID:Vinylogous ureas as a novel class of inhibitors of reverse transcriptase-associated ribonuclease H activity. 1883 89

Crystal structures and simulations suggest that conformational changes are critical for the function of HIV-1 reverse transcriptase. The enzyme is an asymmetric heterodimer of two subunits, p66 and p51. The two subunits have the same N-terminal sequence, with the p51 subunit lacking the C-terminal RNase H domain. We used hydrogen exchange mass spectrometry to probe the structural dynamics of RT. H/D exchange revealed that the fingers and palm subdomains of both subunits form the stable core of the heterodimer. In the crystal structure, the tertiary fold of the p51 subunit is more compact than that of the polymerase domain of the p66 subunit, yet both subunits show similar flexibility. The p66 subunit contains the polymerase and RNase H catalytic sites. H/D exchange indicated that the RNase H domain of p66 is very flexible. The beta-sheet beta12-beta13-beta14 lies at the base of the thumb subdomain of p66 and contains highly conserved residues involved in template/primer binding and NNRTI binding. Using the unique ability of hydrogen exchange mass spectrometry to resolve slowly interconverting species, we found that beta-sheet beta12-beta13-beta14 undergoes slow cooperative unfolding with a t(1/2) of <20 s. The H/D exchange results are discussed in relation to existing structural, simulation, and sequence information.
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PMID:Solution structural dynamics of HIV-1 reverse transcriptase heterodimer. 1959 35


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