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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The single-stranded DNA containing the Moloney murine leukemia virus origin for plus-strand synthesis was cloned in M13mp2 and used as a template for avian myeloblastosis virus reverse transcriptase in the presence of Moloney RNA which had been treated with pancreatic RNase A. The RNA pieces containing the polypurine stretch near the plus-strand origin were processed, presumably by RNase H, to generate primers for DNA synthesis which initiated both at the correct origin site and at one nucleotide downstream from the correct site. Approximately 50% of the labeled DNA fragments synthesized under these conditions retained the priming RNA on their 5' ends. When the isolated fragments were hybridized back to the template DNA and again treated with the reverse transcriptase, all of the RNA was removed from the labeled DNA. By using 5'-end-labeled pancreatic RNase A-resistant fragments, it was possible to show that the RNA primers were removed intact. It appears from these results that the RNase H activity associated with the enzyme shows a preference for cutting at the junction between the RNA and DNA moieties of such complexes and therefore is ideally suited for removing RNA primers.
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PMID:Mechanism of RNA primer removal by the RNase H activity of avian myeloblastosis virus reverse transcriptase. 619 10

A 190-base-pair DNA-RNA hybrid containing the Moloney murine leukemia virus origin of plus-strand DNA synthesis was constructed and used as a source of template-primer for the reverse transcriptase in vitro. Synthesis was shown to initiate precisely at the known plus-strand origin. The observation that some of the origin fragments retained ribonucleotide residues on their 5' ends suggests that the primer for chain initiation is an RNA molecule left behind by RNase H during the degradation of the RNA moiety of the DNA-RNA hybrid. If the RNase H is responsible for creating the correct primer terminus, then it must possess a specific endonucleolytic activity capable of recognizing the sequence in the RNA where plus strands are initiated. The 16-base RNase A-resistant fragment which spans the plus-strand origin can also serve as a source of the specific plus-strand primer RNA. Evidence is presented that some of the plus-strand origin fragments synthesized in the endogenous reaction contain 5' ribonucleotides, suggesting that specific RNA primers for plus-strand initiation may be generated during reverse transcription in vivo as well.
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PMID:RNA-primed initiation of Moloney murine leukemia virus plus strands by reverse transcriptase in vitro. 620 82

In the presence of Mn2+, reverse transcriptase of both human immunodeficiency virus and murine leukemia virus hydrolyzes duplex RNA. However, designating this novel activity RNase D conflicts with Escherichia coli RNase D, which participates in tRNA processing. On the basis of its location in the RNase H domain, we propose that this novel retroviral activity be redesignated RNase H*.
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PMID:Redesignation of the RNase D activity associated with retroviral reverse transcriptase as RNase H. 750 4

The natural product of the Red Sea sponge Verongia sp., identified as 3,5,8-trihydroxy-4-quinolone, was found to be a potent inhibitor of the RNA-directed DNA synthesis of the reverse transcriptases (RTs) of human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2, respectively). This inhibition was unaffected by the nature of the primer template used for DNA synthesis. The DNA-dependent DNA polymerase activity was inhibited to a lesser extent, whereas the ribonuclease H (RNase H) function associated with both HIV RTs was only slightly inhibited. The inhibition by the trihydroxyquinolone is reversible and noncompetitive with respect to both substrates--dTTP and the template primer poly(rA)n.oligo(dT)12-18. The inhibitor binds HIV-1 RT with a high affinity (Ki = 0.46 microM). This compound was shown also to inhibit the catalytic activities of the RT of murine leukemia virus, establishing the general inhibitory effect on retroviral RTs. Introductions of acetyl or methoxy moieties at positions with potential activity have generated three synthetic analogs of the natural compound. Only one analog, 5,8-dimethoxy-4-quinolone, exhibited an inhibition potency similar to that of the unmodified compound. Analysis of the three analogs has led us to the conclusion that the hydroxyl group at the ortho position to the carbonyl group in the pyridinone ring is a key structural element for the inhibitory activity. Thus, it could well be that the inhibitor interacts with the enzyme through a hydrogen bond of this hydroxyl group. We hope that the identification of the inhibitory site of the compound might be an important step toward the rational design of new potent anti-HIV RT drugs.
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PMID:3,5,8-Trihydroxy-4-quinolone, a novel natural inhibitor of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2. 751 Sep 44

Plus strand priming during retroviral reverse transcription requires specific cleavage within the polypurine tract of the viral genome by the reverse transcriptase-associated RNase H. Previously it has been shown that a 190-base RNA-DNA hybrid containing the Moloney murine leukemia virus polypurine tract can serve as a substrate for the priming reaction. To investigate the structural requirements for the reaction, a series of DNA oligonucleotides was hybridized to the 190-base single-stranded RNA and tested as substrates for RNase H. At low enzyme concentrations, the sites of cleavage are located 17-23 nucleotides from the 3'-end of the DNA oligonucleotide, consistent with the observations of others that binding of the DNA polymerase at a primer terminus fixes the position of cleavage by RNase H. At higher enzyme concentrations, additional cleavages are observed in the RNA 3' of these sites, but there is no preference for cleavage at the plus strand origin. In contrast to the results with DNA oligonucleotides, hybridization of RNA oligonucleotides containing the polypurine tract to the 190-base single-stranded DNA generates substrates that are cleaved at the origin and efficiently extended into DNA. An RNA oligonucleotide hybridized downstream of the polypurine tract is cleaved but not extended. These results support the view that RNase H cleavage to generate the plus strand primer is uncoupled from minus strand DNA synthesis.
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PMID:The use of DNA and RNA oligonucleotides in hybrid structures with longer polynucleotide chains to probe the structural requirements for moloney murine leukemia virus plus strand priming. 751 53

The accepted model of retroviral reverse transcription includes a circular DNA intermediate which requires strand displacement synthesis for linearization and creation of an integration-competent, long terminal repeat-flanked DNA product. We have used an in vitro model of this last step of reverse transcription to examine the role of the viral enzyme, reverse transcriptase (RT), in displacement synthesis. We show that Moloney murine leukemia virus RT possesses an activity which allows for displacement synthesis through a minimum of 1,334 bp of duplex DNA--an extent much greater than that required during in vivo reverse transcription and over 25-fold greater than has been previously demonstrated for a viral RT. RT does not function as a helicase in the classical sense but appears to closely couple duplex DNA melting with synthesis-driven translocation of the enzyme. In the absence of synthesis, the unwound region created by a primer-positioned RT appears to be no greater than 2 bp and does not advance along the template. Additionally, RT does not utilize ATP or any deoxynucleoside triphosphate not directly encoded by the template strand to catalyze processive duplex unwinding at a nick; nor does binding of the enzyme unwind duplex DNA in the absence of a 3' terminus. The approximate maximum chain elongation rate during strand displacement synthesis by Moloney murine leukemia virus RT falls between 0.73 and 1.5 nucleotides per s at 37 degrees C. The RNase H activity of RT does not appear to play a role in displacement synthesis; however, a 181-amino-acid C-terminal truncation of RT displays a dramatically reduced ability to catalyze synthesis through duplex DNA.
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PMID:Strand displacement synthesis capability of Moloney murine leukemia virus reverse transcriptase. 751 25

Specific, high-affinity RNA ligands to avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases were isolated from a combinatorial RNA library using the SELEX (systematic evolution of ligands by exponential enrichment) procedure. The selected RNA ligands bound their respective reverse transcriptases with approximately nanomolar dissociation constants. The ligands did not exhibit primary sequence conservation from selections against different target enzymes. Moreover, the selected ligands competed with the binding of template/primer complex and inhibited both the RNA-dependent DNA polymerase and the RNase H activities of the cognate reverse transcriptase. SELEX can yield both high-affinity and high-specificity oligonucleotide antagonists against specific members of a protein family.
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PMID:Selection of high-affinity RNA ligands to reverse transcriptase: inhibition of cDNA synthesis and RNase H activity. 751 91

Alpha-beta chimeric 17-mer oligodeoxyribonucleotides containing either 5, 10 or 15 beta nucleotides were synthesized. The stability of the RNA/chimera hybrids was only slightly affected by the alpha stretch and by the alpha-beta link, as was the affinity of the Moloney Murine Leukemia Virus reverse transcriptase for the duplexes. All chimeras inhibited in vitro cDNA synthesis in a cell-free system to various extent, via the degradation of the RNA target by RNase H.
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PMID:Chimeric alpha-beta oligonucleotides as antisense inhibitors of reverse transcription. 753 37

The RNase H domain of murine leukemia virus (MuLV) reverse transcriptase (RT) was replaced with Escherichia coli RNase H, and the effect on RNase H activity and processive DNA synthesis was studied, using RNA-DNA hybrids containing sequences from the MuLV polypurine tract (PPT). Two chimeric RTs, having the entire polymerase domain or all but the last 19 amino acids, were expressed. In both cases, these RTs made multiple cuts in PPT-containing substrates, whereas wild-type cleavages occurred primarily at sites consistent with the distance between the polymerase and RNase H active sites. Primer extension assays performed with the chimeric RTs, an RNase H-minus RT, and wild-type showed that the presence of a wild-type viral RNase H domain facilitates processive DNA synthesis. When wild-type RT was bound to primer-template, two retarded bands could be detected in band-shift assays. In the absence of primer extension, a high proportion of enzyme-bound primer-template was associated with the faster-migrating band, whereas with DNA synthesis, more of the bound radioactivity was in the super-shifted complex. This suggests that the super-shifted complex contains the active form of RT. The mutant RTs were deficient in formation of this complex, but the chimeric RTs were somewhat less defective than the RNase H-minus mutant. Our results demonstrate that in the wild-type enzyme, the RNase H domain is required to stabilize the interaction between RT and primer-template.
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PMID:Defects in primer-template binding, processive DNA synthesis, and RNase H activity associated with chimeric reverse transcriptases having the murine leukemia virus polymerase domain joined to Escherichia coli RNase H. 753 33

To determine the various roles of RNase H in reverse transcription, we generated a panel of mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase based on sequence alignments and the crystal structures of Escherichia coli and human immunodeficiency virus type 1 RNases H (S. W. Blain and S. P. Goff, J. Biol. Chem. 268:23585-23592, 1993). These mutations were introduced into a full-length provirus, and the resulting genomes were tested for infectivity by transient transfection assays or after generation of stable producer lines. Several of the mutant viruses replicated normally, some showed significant delays in infectivity, and others were noninfectious. Virions were collected, and the products of the endogenous reverse transcription reaction were examined to determine which steps might be affected by these mutations. Some mutants left their minus-strand strong-stop DNA in RNA-DNA hybrid form, in a manner similar to that of RNase H null mutants. Some mutants showed increased polymerase pausing. Others were impaired in first-strand translocation, independently of their wild-type ability to degrade genomic RNA, suggesting a new role for RNase H in strand transfer. DNA products synthesized in vivo by the wild-type and mutant viruses were also examined. Whereas wild-type virus did not accumulate detectable levels of minus-strand strong-stop DNA, several mutants were blocked in translocation and did accumulate this intermediate. These results suggest that in vivo wild-type virus normally translocates minus-strand strong-stop DNA efficiently.
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PMID:Effects on DNA synthesis and translocation caused by mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase. 753 10


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