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)

During the course of reverse transcription, human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) initiates plus-strand DNA synthesis from two highly conserved, purine-rich RNA segments of the viral genome referred to as the 3' and central polypurine tracts (3' and cPPTs). Processing of these elements occurs in several sequential steps including (1) minus-strand DNA synthesis over the PPT(s), (2) ribonuclease H (RNase H) mediated cleavage at the PPT 3' terminus, (3) plus-strand DNA synthesis from the nascent RNA primer(s), and (4) primer removal. Completing each of these steps precisely and specifically is essential, as failure to do so can result in reduced virus replication and/or impaired integration of viral DNA into the host cell genome. In this review, plus-strand primer processing in HIV-1 is discussed from biochemical, structural, and historical perspectives. A comparative analysis of PPT-processing in different LTR-containing retroelements is also presented.
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PMID:'Binding, bending and bonding': polypurine tract-primed initiation of plus-strand DNA synthesis in human immunodeficiency virus. 1518 42

The RNase H cleavages that generate and remove the polypurine tract (PPT) primer during retroviral reverse transcription must be specific in order to create a linear viral DNA that is suitable for integration. Lentiviruses contain a highly conserved sequence consisting of six guanine residues at the 3' end of the PPT (hereafter referred to as the G tract). We introduced mutations into the G tract of a human immunodeficiency virus type 1-based vector and determined the effects on the virus titer and RNase H cleavage specificity. Most mutations in the G tract had little or no effect on the virus titer. Mutations at the second and fifth positions of the G tract increased the proportion of two-long-terminal-repeat (2-LTR) circle junctions with one or two nucleotide insertions. The second and fifth positions of the G tract make specific contacts with amino acids in the RNase H domain that are important for RNase H cleavage specificity. These complementary data define protein-nucleic acid interactions that help control the specificity of RNase H cleavage. When the G-tract mutants were analyzed in a viral background that was deficient in integrase, in most cases the proportion of consensus 2-LTR circle junctions increased. However, in the case of a mutant with Ts at the second and fifth positions of the G tract, the proportion of 2-LTR circle junctions containing the one-nucleotide insertion increased, suggesting that linear viral DNAs containing an extra base are substrates for integration. This result is consistent with the idea that the 3' end-processing reactions of retroviral integrases may help to generate defined ends from a heterogenous population of linear viral DNAs.
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PMID:Effects of mutations in the G tract of the human immunodeficiency virus type 1 polypurine tract on virus replication and RNase H cleavage. 1554 82

ZAM is an LTR-retrotransposon from Drosophila melanogaster that belongs to the genus errantivirus, viruses similar in structure and replication cycle to vertebrate retroviruses. A key component to its lifecycle is its reverse transcriptase which copies single-stranded genomic RNA into DNA. Here, we provide a detailed characterization of the enzymatic activities of the reverse transcriptase encoded by ZAM. When expressed in vitro, the reverse transcriptase domain associated with the RNase H domain encoded by the ZAM pol gene forms homodimers and displays an efficient RNA-dependent DNA-polymerase activity. It requires either Mg2+ or Mn2+ divalent cations, and works in basic pH, with a peak at around pH9. The so-called [RT-RH] polypeptide displays an optimal activity at 22 degrees C, a property that makes it well-adapted to the temperature of its host. This study contributes to our understanding of the general structures and functions of retroviral reverse transcriptases, a necessary process in the search for novel inhibitors.
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PMID:Functional characteristics of a reverse transcriptase encoded by an endogenous retrovirus from Drosophila melanogaster. 1576 68

Retrotransposons commonly encode a reverse transcriptase (RT), but other functional domains are variable. The acquisition of new domains is the dominant evolutionary force that brings structural variety to retrotransposons. Non-long-terminal-repeat (non-LTR) retrotransposons are classified into two groups by their structure. Early branched non-LTR retrotransposons encode a restriction-like endonuclease (RLE), and recently branched non-LTR retrotransposons encode an apurinic/apyrimidinic endonuclease-like endonuclease (APE). In this study, we report a novel non-LTR retrotransposon family Dualen, identified from the Chlamydomonas reinhardtii genome. Dualen encodes two endonucleases, RLE and APE, with RT, ribonuclease H, and cysteine protease. Phylogenetic analyses of the RT domains revealed that Dualen is positioned at the midpoint between the early-branched and the recently branched groups. In the APE tree, Dualen was branched earlier than the I group and the Jockey group. The ribonuclease H domains among the Dualen family and other non-LTR retrotransposons are monophyletic. Phylogenies of three domains revealed the monophyly of the Dualen family members. The domain structure and the phylogeny of each domain imply that Dualen is a retrotransposon conserving the domain structure just after the acquisition of APE. From these observations, we discuss the evolution of domain structure of non-LTR retrotransposons.
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PMID:An extraordinary retrotransposon family encoding dual endonucleases. 1607 10

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

Retroviral polypurine tracts (PPTs) serve as primers for plus-strand DNA synthesis during reverse transcription. The generation and removal of the PPT primer requires specific cleavages by the RNase H activity of reverse transcriptases; removal of the PPT primer defines the left end of the linear viral DNA. We replaced the endogenous PPT from RSVP(A)Z, a replication-competent shuttle vector based on Rous sarcoma virus (RSV), with alternate retroviral PPTs and the duck hepatitis B virus "PPT." Viruses in which the endogenous RSV PPT was replaced with alternate PPTs had lower relative titers than the wild-type virus. 2-LTR circle junction analysis showed that the alternate PPTs caused significant decreases in the fraction of viral DNAs with complete (consensus) ends and significant increases in the insertion of part or all of the PPT at the 2-LTR circle junctions. The last two nucleotides in the 3' end of the RSV PPT are GA. Examination of the (mis)cleavages of the alternate PPTs revealed preferential cleavages after GA dinucleotide sequences. Replacement of the terminal 3' A of the RSV PPT with G caused a preferential miscleavage at a GA sequence spanning the PPT-U3 boundary, resulting in the deletion of the terminal adenine normally present at the 5' end of the U3. A reciprocal G-to-A substitution at the 3' end of the murine leukemia virus PPT increased the relative titer of the chimeric RSV-based virus and the fraction of consensus 2-LTR circle junctions.
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PMID:Alternate polypurine tracts (PPTs) affect the rous sarcoma virus RNase H cleavage specificity and reveal a preferential cleavage following a GA dinucleotide sequence at the PPT-U3 junction. 1622 89

To probe structural features of a polypurine tract (PPT) that mediate its specific recognition and processing, a model 20 bp RNA/DNA hybrid duplex, which includes the full PPT sequence of the Saccharomyces cerevisiae LTR-retrotransposon Ty3, has been investigated using solution NMR spectroscopy. While homonuclear NOESY and DQF-COSY analyses indicate that this PPT-containing RNA/DNA hybrid adopts an overall A-form-like helical geometry, an unexpected sugar pucker switch has been detected for the ribose at position +1, relative to the cleavage site, on the RNA strand. A model of the conformational changes induced by the A- to B-type sugar pucker switch shows a significant change in the backbone trajectory of the RNA strand, which alters the presentation of backbone phosphate and 2' hydroxyl groups 3' of this residue. This observation implies that part of the mechanism governing RNase H fidelity may be through distortion of the RNA/DNA helix one base ahead of the scissile bond.
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PMID:A ribose sugar conformational switch in the LTR-retrotransposon Ty3 polypurine tract-containing RNA/DNA hybrid. 1630 91

We reported previously that substitutions F61L, F61W, F61Y and F61A in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase affect strand displacement synthesis [T. S. Fisher, T. Darden and V. R. Prasad (2003) J. Mol. Biol., 325, 443-459]. We have now determined the effect of these mutations on HIV replication. All mutant viruses were replication defective. Measuring replication intermediates in infected cells did not reveal a specific block as all mutants displayed reduced DNA synthesis (wild-type>F61L>F61W>F61Y>F61A). Analysis of 2-LTR circle junctions revealed that F61W and F61Y mutants generated increased aberrant circle junctions. Circle junctions corresponding to F61Y included 3'-PPT insertions suggesting ribonuclease H defect. In vitro assays mimicking PPT primer generation indicated that F61L, F61W and F61Y mutant RTs were unaffected, while F61A mutant cleaved both at PPT/U3 junction and at +6 with similar efficiencies. In assays measuring cleavage at the RNA/DNA junction to remove the PPT primer, all mutants were significantly affected with F61Y and F61A being most severely impaired. Our results show that (i) replication block of most mutants is due to more than one biochemical defect; (ii) mutations in polymerase domain can affect the function of a distal domain; and (iii) virological analyses of RT mutations can yield insight into structure-function relationship that is otherwise not obvious.
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PMID:Analysis of HIV-1 replication block due to substitutions at F61 residue of reverse transcriptase reveals additional defects involving the RNase H function. 1672 31

Twenty-five repetitive elements are first described in the genomes of the arbuscular mycorrhizal (AM) fungi Gigaspora margarita, Gig. rosea and Glomus mosseae. Nineteen repetitive DNA sequences isolated by genomic library screening and four by self-priming PCR had no homology to known DNA sequences, except for two Gig. margarita sequences and one Gig. rosea sequence which showed amino acid similarity to retrotransposons. Part of the Gig. rosea sequence was also similar to a DNA transposon. Two other retrotransposon sequences were isolated using PCR targeting of reverse transcriptase and ribonuclease H domains. Evidence is provided for three gypsy-like LTR retrotransposon and two non-LTR retrotransposon sequences in the AM fungal genomes. Four contain stop codons indicating that they cannot be active. Expression of three retrotransposons was not detected in germinating spores or intraradical hyphae of Gig. margarita. Southern blot analyses indicated that these three sequences are dispersed in the genome and that two are methylated. Sequence analysis of different GmarLTR1 copies showed they have undergone mutations by transitions, which may have been induced by cytosine methylation. Transposable elements may have played a major role in shaping genome structure and size during evolution of the Glomeromycota.
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PMID:Repetitive DNA sequences include retrotransposons in genomes of the Glomeromycota. 1702 73

The recognition and precise cleavage of the polypurine tract (PPT) of the human immunodeficiency virus type 1 (HIV-1) is an essential step in HIV-1 reverse transcription. The accurate cleavage, and the subsequent removal, of the PPT by the RNase H activity of HIV-1 RT defines the left end of the double-stranded viral DNA genome, the substrate for integration into the host genome. Previous analyses have shown that mutations in the 3'-end (G-tract) of the PPT cause alterations in RNase H cleavage specificity. In particular, mutations at positions 2 and 5 in the G-tract increased the frequency of retention of PPT sequences in the 2-LTR circle junction. To better understand why these mutations affected PPT cleavage in vivo, we analyzed the cleavage of PPT substrates in vitro that contained altered sequences and unusual base substitutions. Our results, herein, confirm that mutations at positions 2 and 5 of the G-tract do significantly alter the cleavage specificity at the PPT/U3 junction, and further suggest that the miscleavages observed in vivo were due to an improper generation of the PPT primer, as opposed to its improper removal. Finally, our results point to the structure of the PPT, rather than the base-specific contacts between the PPT and HIV-1 RT, as the primary determinants of RNase H cleavage specificity at the PPT/U3 junction.
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PMID:In vitro analysis of the effects of mutations in the G-tract of the human immunodeficiency virus type 1 polypurine tract on RNase H cleavage specificity. 1712 64


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