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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate whether human immunodeficiency virus type 1 pol gene mutations are selected during prolonged 2',3'-dideoxycytidine (ddC) therapy, we used the polymerase chain reaction to amplify a portion of the reverse transcriptase segment of the pol gene from the peripheral blood mononuclear cell DNA of a patient with AIDS before and after an 80-week course of ddC therapy. The consensus sequence from the second sample contained a unique double mutation (ACT to GAT) in the codon for reverse transcriptase amino acid 69, causing substitution of aspartic acid (Asp) for the wild-type threonine (Thr). A mutation (ACA to ATA) also occurred in the codon for position 165, causing substitution of isoleucine (Ile) for Thr. The GAT (Asp) codon was introduced into the pol gene of a molecular clone of human immunodeficiency virus via site-directed mutagenesis. Following transfection, mutant and wild-type viruses were tested for susceptibility to ddC by a plaque reduction assay. The mutant virus was fivefold less susceptible to ddC than the wild type; cross-resistance to 3'-azido-3'-deoxythymidine or 2'3'-dideoxyinosine was not found. The Ile-165 mutation did not confer additional ddC resistance. The Asp-69 substitution may have contributed to the generation of resistant virus in this patient.
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PMID:Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2',3'-dideoxycytidine. 131 43

We have purified and determined functional parameters of reconstituted, recombinant HIV-1 reverse transcriptase (RT) heterodimers within which either the p66 or p51 polypeptide was selectively mutated in one or both aspartic acid residues constituting the proposed polymerase active site (-Y-M-D-D-). Heterodimers containing a mutated p51 polypeptide retain almost wild type levels of both RNA-dependent DNA polymerase and ribonuclease H (RNaseH) activity. In contrast, heterodimers whose p66 polypeptide was likewise mutated exhibit wild type RNaseH activity but are deficient in RNA-dependent DNA polymerase activity. These results indicate that in heterodimer RT, the p51 component cannot compensate for active site mutations eliminating the activity of p66, indirectly implying that solely the p66 aspartic acid residues of heterodimer are crucial for catalysis.
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PMID:Subunit-selective mutagenesis indicates minimal polymerase activity in heterodimer-associated p51 HIV-1 reverse transcriptase. 171 45

Selected conserved amino acids in the putative RNase H domain of reverse transcriptase (RT) were modified in a molecularly cloned infectious provirus and in a Moloney murine leukemia virus RT expression vector by site-directed mutagenesis. Substitution of either of two conserved aspartic acid residues in proviral DNA prevented production of infectious particles in transfected NIH 3T3 cells, and the same modifications depressed RT-associated RNase H activity by more than 25-fold with little or no effect on polymerase activity.
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PMID:Inhibition of RNase H activity and viral replication by single mutations in the 3' region of Moloney murine leukemia virus reverse transcriptase. 246 6

During senescence in the filamentous fungus Podospora anserina, specific regions of the mitochondrial genome, termed senDNA are excised, ligated and amplified. We have cloned in their entirety three such autonomously replicating plasmids, alpha, beta and epsilon senDNA. None of these plasmids displayed cross-hybridization nor did we detect any significant DNA homology by computer analysis. The complete DNA sequence of the 2.5 kb alpha, the 5.5 kb epsilon and about 3.4 kb of the 9.8 kb beta senDNA is presented (kb = 10(3) base-pairs). These sequences were analyzed for the presence of consensus sequences common to introns, and it was found that alpha senDNA has the characteristics of a group II intron, epsilon senDNA contains three group I introns, and beta senDNA did not show relevant sequences in the 3.4 kb examined. Comparison of the 5' and 3'-flanking sequences of alpha senDNA with oxi 3 (Co I) amino acid sequences from Neurospora crassa and Saccharomyces cerevisiae revealed significant homology and provided strong support that the excised alpha senDNA itself consists entirely of an intron. Upstream from the oxi 3 gene a transfer RNA cysteine sequence was detected. beta senDNA contained four tRNA sequences, aspartic acid, serine, valine and tryptophan, and sequences homologous to URFC (untranslated reading frame C) as well as two new URFs. epsilon senDNA contained sequences homologous to ATPase 8 and URFl; URFl was interrupted by three group I introns. The excision site sequences, as located by S1 nuclease mapping were unique for each senDNA. Analysis for repeated units showed that each plasmid contained elements which could be involved in secondary structure required for the alignment of distal ends preparatory to excision. These results are interpreted in terms of the structural requirements of mobile elements including the possible involvement of reverse transcriptase in the excision-ligation-amplification process.
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PMID:Excision-amplification of mitochondrial DNA during senescence in Podospora anserina. DNA sequence analysis of three unique "plasmids". 299 55

Uteroglobin is a protein that is synthesized in large quantities by the rabbit uterine endometrial cells and secreted into the uterine lumen around the time of implantation of the developing blastocysts. The protein is also synthesized constitutively at a low level in the lung. In the uterus, synthesis of the protein is induced by progesterone but repressed by estradiol; whereas in the lung, it is not hormonally responsive. Using a full-length cDNA clone, we have established the nucleotide sequence of uteroglobin mRNA and have determined its levels in uterus and lung during early pregnancy. The clone, pUG617, contains all but 24 nucleotides at the 5' untranslated region of the structural gene. To establish the full mRNA sequence, we isolated a 5' end-labeled DNA fragment from pUG617 and extended its length using reverse transcriptase after hybridization with uterine poly(A)-containing RNA. The 5'-terminal sequence of uteroglobin mRNA was established by sequencing the extended DNA fragment. The nucleotide sequence of the peptide-coding portion of the gene has resolved some previously reported discrepancies in the amino acid sequence of the mature protein and those in the signal peptide. By comparison of sequences with a partial uteroglobin cDNA clone isolated by another laboratory, a polymorphic nucleotide at position 246 of the gene has been identified, where a G-A transition has caused an amino acid substitution from aspartic acid to asparagine at residue 46 of the mature protein. Analysis of steady-state RNA levels in the uterus has shown that the induction and repression of uteroglobin synthesis during early pregnancy is the result of accumulation and depletion of its mRNA, respectively. During the same period in the lung, no consistent changes in uteroglobin mRNA level were evident, reflecting the constitutive levels of the protein in this tissue.
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PMID:Hormonally regulated mammalian gene expression: steady-state level and nucleotide sequence of rabbit uteroglobin mRNA. 629 63

The structure of unliganded HIV-1 reverse transcriptase has been determined at 2.35 A resolution and refined to an R-factor of 0.219 (for all data) with good stereochemistry. The unliganded structure was produced by soaking out a weak binding non-nucleoside inhibitor, HEPT, from pregrown crystals. Comparison with the structures of four different RT and non-nucleoside inhibitor complexes reveals that only minor domain rearrangements occur, but there is a significant repositioning of a three-stranded beta-sheet in the p66 subunit (containing the catalytic aspartic acid residues 110, 185 and 186) with respect to the rest of the polymerase site. This suggests that NNIs inhibit RT by locking the polymerase active site in an inactive conformation, reminiscent of the conformation observed in the inactive p51 subunit.
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PMID:Mechanism of inhibition of HIV-1 reverse transcriptase by non-nucleoside inhibitors. 754 Sep 35

The conserved aspartic acid residue 488 in the RNase H domain of HIV-1 reverse transcriptase (RT) was mutated to alanine. RT was expressed in Escherichia coli alone or with the entire pol-gene polyprotein consisting of proteinase, RT, and integrase and processed by the HIV-1 proteinase in the bacterial cell. Expression of mutant RT together with the proteinase resulted in an overproduction of RT p51 vs p66. The mutation also altered the conformation of the RT p66/p51 heterodimer as shown by the loss of binding of monoclonal antibodies to mutant RT in ELISA. Crystallographic data shows that a salt bridge exists between Asp 488 and Lys 465 of RNase H which stabilizes the uncleavable form of RT p66, and that substitution of Asp for Ala would prevent the formation of this salt bridge. Our results indicate that disruption of this salt bridge through mutation of Asp 488 interferes with the conformational changes that regulate the limited processing of p66 to 51 by the virus proteinase. Homology data suggest that such a bridge may be present in other lentiviruses. The mutation introduced caused a moderate decrease in both the RNase H activity and the polymerase activity of RT, indicating that the proper folding of the RNase H domain of RT is necessary to achieve full polymerase activity.
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PMID:Disruption of a salt bridge between Asp 488 and Lys 465 in HIV-1 reverse transcriptase alters its proteolytic processing and polymerase activity. 769 May 4

A previous genetic analysis of the human immunodeficiency virus type 1 integrase protein failed to identify single amino acid substitutions that only block the integration of viral DNA (C.-G. Shin, B. Taddeo, W.A. Haseltine, and C.M. Farnet, J. Virol. 68:1633-1642, 1994). Additional substitutions of amino acids that are highly conserved among retroviral integrases were constructed in human immunodeficiency virus type 1 and analyzed for their effects on viral protein synthesis and processing, virion morphology, and viral DNA synthesis and integration in an attempt to identify mutants with a specific defect in integration. Four single amino acid substitutions resulted in replication defective viruses. Conservative, single amino acid substitutions of the two invariant aspartic acid residues found in all retroviral integrases prevented the integration of viral DNA and had no detectable effect on the other stages in the viral replication cycle, indicating that these mutants exhibited a specific defect in integration. Mutations at two positions, S-81 and P-109, blocked the integration of viral DNA but also resulted in the production of viral particles that exhibited reduced reverse transcriptase activity, suggesting additional defects in viral replication. Substitution of the highly conserved amino acid T66 had no effect on viral replication in a CD4+ human T-cell line. This analysis extends the range of possible phenotypes that may be produced by single amino acid substitutions in conserved residues of the integrase protein.
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PMID:Integrase mutants of human immunodeficiency virus type 1 with a specific defect in integration. 796 34

The expression of the pol gene of human immunodeficiency virus type 1 (HIV-1) occurs by a ribosomal frameshift between the gag and the pol genes. The Gag-Pol polyprotein is produced at levels of 5 to 10% of that of the Gag protein, and is incorporated into virions to provide the viral protease, reverse transcriptase, and integrase which are essential for replication. The mechanism(s) by which the Gag-Pol polyprotein are targeted to the HIV virion is unknown, although it is believed to be via an interaction with the Gag protein. To further explore the mechanism by which the Gag-Pol polyprotein is incorporated into virions, we have constructed a mutation which changes an aspartic acid in the protease active site to asparagine (pHXB2pro-); a four-amino-acid insertion into the protease gene (pHXB2Smal); and insertion of translational termination codons in the protease gene following the gag gene (pHXB55). Transfection of these proviral genomes into COS-1 cells resulted in intracellular expression of only Pr55gag, demonstrating the inactivation of the viral protease. The expression of Pr55gag was evident in cells transfected with pHXB2pro- during a short pulse and first 3 hr of chase period, whereas at later times the intracellular levels of Pr55gag were greatly reduced. In contrast, the intracellular Pr55gag expressed from transfection of pHXB2Smal or pHXB55 were evident even after 6- or 12-hr chase times. To ascertain the effects of the mutations on the assembly and release of viruslike particles, the supernatants from the transfected cells were analyzed for the presence of Pr55gag. The release of Pr55gag from cells transfected with pHXB2pro- occurred as early as 1 hr following chase period, and increased for up to 3 hr. In contrast, reduced levels of Pr55gag were detected in the medium from cells transfected with pHXB2Smal or pHXB55. Subcellular fractionation studies demonstrated that the Pr55gag expressed from transfection of pHXB2pro- was rapidly targeted to intracellular membranes, while the majority of the Pr55gag expressed from transfection of pHXB2Smal or pHXB55 was distributed evenly between the cytoplasm and membrane fractions. Finally, the released viruslike particles obtained from the transfection of proviral genome pHXB2pro- were stable to mild detergent treatment, whereas particles obtained from transfection of pHXB2Smal and pHXB55 were relatively unstable. These results demonstrate that subtle changes in the Gag-Pol polyprotein of HIV-1 can have significant effects on the assembly and physical stability of the released virus.
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PMID:Mutations in the protease gene of human immunodeficiency virus type 1 affect release and stability of virus particles. 850 89

The high error rates characteristic of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) are a presumptive source of the viral hypermutability that impedes prevention and therapy of acquired immunodeficiency syndrome (AIDS). We have analyzed two mutants of HIV-1 RT by conducting a comparative study of the accuracy of DNA synthesis. Each mutant bears a single amino acid substitution adjacent to the two aspartic acid residues at positions 185 and 186 in the highly conserved DNA polymerase active site. The first mutant, Met 184-->Leu (M184L), displays a marked reduction in both misinsertion and mispair extension, suggesting a fidelity of DNA synthesis significantly higher than that of the wild-type HIV-1 RT. The second mutant, Tyr 183-->Phe (Y183F), shows a decrease in mispair extension with no significant change in misincorporation. Thus, the overall pattern of error-proneness of DNA synthesis is: wild-type HIV-1 RT > Y183F > M184L. Taken together, it is possible that residues 183 and 184 contribute to the low fidelity of DNA synthesis characteristic of the reverse transcriptases of HIV-1, HIV-2 and possibly, of other lentiviruses. Our observations may bear on the nature of potential mutations responsible for resistance to the nucleoside analogs used in chemotherapy of AIDS.
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PMID:Mutational studies of human immunodeficiency virus type 1 reverse transcriptase: the involvement of residues 183 and 184 in the fidelity of DNA synthesis. 876 85


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