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)

We have generated by site-directed mutagenesis plasmids that induce the synthesis of specific mutants of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). These recombinant mutants of HIV-1 RT, designed on the basis of our previous studies of HIV-1 and HIV-2 RTs, were analyzed for structure-function relationship by assessing their RNA-dependent and DNA-dependent DNA polymerase as well as the ribonuclease H activities. Three groups of mutants were studied. 1) We have investigated the importance of the only two sets of highly conserved double prolines found in the sequence of HIV-1 RT. The results indicate that the conversion of either one or both prolines (at positions 225 and 226) to threonines have no significant effect on all catalytic activities of the enzyme. The mutants in which prolines 419 and 420 were individually modified to threonines exhibit full activities, whereas the double proline 419/420 mutant lost most of its RNase H activity (although the DNA polymerase function was fully retained). 2) We have deleted phenylalanine 346 from HIV-1 RT, which is absent in wild type HIV-2 RT. This mutant of HIV-1 RT lost practically all catalytic activities. 3) A mutant of HIV-1 RT in which a cysteine residue substituted for alanine 446, was found to be slightly hyperactive for both DNA polymerase and RNase H activities.
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PMID:Functional analysis of novel selective mutants of the reverse transcriptase of human immunodeficiency virus type 1. 138 52

Reverse transcriptases contain a highly conserved YXDD amino acid motif believed to be important in enzyme function. The second amino acid is not strictly conserved, with a methionine, valine or alanine occupying the second position in reverse transcriptases from various retroviruses and retroelements. Recently, a 3.5-A (0.35-nm) resolution electron density map of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase positioned the YMDD motif within an antiparallel beta-hairpin structure which forms a portion of its catalytic site. To further explore the role of methionine of the conserved YMDD motif in HIV-1 reverse transcriptase function, we have substituted methionine with a valine, alanine, serine, glycine, or proline, reflecting in some cases sequence motifs of other related reverse transcriptases. Wild-type and mutant enzymes were expressed in Escherichia coli, partially purified by phosphocellulose chromatography, and assayed for the capacity to polymerize TTP by using a homopolymeric template [poly(rA)] with either a DNA [oligo(dT)] or an RNA [oligo(U)] primer. With a poly(rA).oligo(dT) template-primer, reverse transcriptases with the methionine replaced by valine (YVDD), serine (YSDD), or alanine (YADD) were 70 to 100% as active as the wild type, while those with the glycine substitution (YGDD) were approximately 5 to 10% as active. A proline substitution (YPDD) completely inactivated the enzyme. With a poly(rA).oligo(U) template-primer, only the activity of mutants with YVDD was similar to that of the wild type, while mutants with YADD and YSDD were approximately 5 to 10% as active as the wild-type enzyme. The reverse transcriptases with the YGDD and YPDD mutations demonstrated no activity above background. Proviruses containing the reverse transcriptase with the valine mutation (YVDD) produced viruses with infectivities similar to that of the wild type, as determined by measurement of p24 antigen in culture supernatants and visual inspection of syncytium formation. In contrast, proviruses with reverse transcriptases containing the YADD and YSDD mutations were less infectious than wild-type virus. These results point to the critical role of methionine of the YMDD motif in the activity of HIV-1 reverse transcriptase and subsequent replication potential of the virus.
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PMID:In vitro enzymatic activity of human immunodeficiency virus type 1 reverse transcriptase mutants in the highly conserved YMDD amino acid motif correlates with the infectious potential of the proviral genome. 138 71

Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNA(Lys) partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNA(Lys) we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase alpha, while avian retrovirus RT was neither affected by tRNA(Lys) nor by its specific primer tRNA(Trp). Although the strongest inhibition was observed with tRNA(Lys), other tRNas like tRNA(Phe) and tRNA(Trp) inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNA(Lys) with pancreatic RNase abolished the inhibition; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNA(Lys) isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNA(Lys) was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNA(Lys) the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNA(Lys) primer, are different.
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PMID:Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys). 168 23

The virally encoded protease of human immunodeficiency virus (HIV) is responsible for specific cleavage events leading to the liberation of the enzymes reverse transcriptase, integrase, ribonuclease H, and the core proteins from the gag-pol and gag polyprotein precursors. Utilizing gag polyprotein synthesized in vitro, we have shown that this substrate is sequentially cleaved by purified HIV protease to yield products that on the basis of their sizes and immunoreactivities correspond to p15, p6, p7, p17, and finally mature p24. We have placed unique restriction sites flanking the p17-p24 domain in order to facilitate replacement of cleavage site sequences by utilizing oligonucleotide cassettes. Replacement of the rapidly cleaved methionine-methionine bond at the p24-p15 junction with tyrosine-proline or replacement of the tyrosine-proline bond at the p17-p24 junction with methionine-methionine results in sites that cannot be efficiently cleaved. A basic amino acid at the p17-p24 scissile bond is not tolerated. Replacement of this cleavage site with an inverted repeat amino acid sequence gives intermediate rates of cleavage. In an attempt to convert the p17-p24 domain into a p24-p15 domain, residues flanking the scissile bond were exchanged in an expanding iterative fashion. When four residues flanking the scissile bond had been replaced, the rate of cleavage relative to that of the native p17-p24 sequence was increased fourfold. The cleavage rate of the native p24-p15 sequence is still some 10-fold greater than that of the p17-p24 sequence, suggesting that more-distant residues significantly affect the cleavage rate.
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PMID:Mutagenesis of protease cleavage sites in the human immunodeficiency virus type 1 gag polyprotein. 198 79

To evaluate the relationship between pseudouridine increase in biological fluids and retroviral cell transformation, we have studied the effect of retrovirus infection and/or transformation on the rate of pseudouridine excretion by chick embryo fibroblasts. The results show that: pseudouridine excretion by chick embryo fibroblasts transformed by Rous sarcoma virus is several times higher than that of normal cells; this increased excretion precedes by many hours the appearance of the morphological signs of transformation and it is always present when neosynthesized infectious viral particles are released into the culture medium; and pseudouridine excretion was also increased in cells infected by a mutant of Rous sarcoma virus (RAV-1) which, lacking the src gene, does not transform the cells but replicates normally. To investigate if pseudouridine overproduction is related to an altered turnover rate of specific transfer RNA (tRNA) species which functions as primer of retrovirus reverse transcriptase, the concentration of non-acylated proline-accepting tRNA and non-acylated tryptophan-accepting tRNA, primers of reverse transcriptase of murine leukemia virus and of Rous sarcoma virus, respectively, has been measured, the former in normal and transformed AKR thymus and the latter in normal fibroblasts and in fibroblasts infected by Rous sarcoma virus or by its nontransforming mutant. The results show that in both systems a significant increase of the primer tRNA species occurs in the infected or transformed cells.
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PMID:Pseudouridine excretion and transfer RNA primers for reverse transcriptase in tumors of retroviral origin. 241 41

We have previously described the construction of a mutant of Moloney murine leukemia virus bearing a deletion at the normal site of integration of the viral DNA. We have now recovered a revertant of the virus after abortive infection of mouse cells and have determined the structure of the new virus. The revertant is a recombinant virus containing a 500-base-pair patch of new sequences derived from the mouse genome. The integration site was perfectly restored to the wild-type sequence, although the patch of DNA was overall only 80% homologous to Moloney murine leukemia virus. Surprisingly, the tRNA primer binding site was no longer homologous to the usual proline tRNAs, but was a perfect match for glutamine tRNA. This result suggests that the Moloney murine leukemia virus reverse transcriptase is not specific to one tRNA, but can utilize different tRNAs to prime the synthesis of viral DNA. Comparisons with published reports allowed the identification of sequences that are 94% homologous to the patch sequence, present in one of the endogenous retroviral sequences of the mouse. No replication-competent members of this family, utilizing the glutamine tRNA primer, have been previously isolated.
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PMID:Isolation of a recombinant murine leukemia virus utilizing a new primer tRNA. 241 55

Synthetic oligonucleotides complementary to putative retroviral primer-binding sites were used as hybridization probes to detect novel retroviruslike sequences. An 8.1-kilobase element with structural features of a retroviral provirus was isolated from a human genomic library by this approach. Nucleotide sequence analysis of its 600-base-pair long terminal repeats revealed characteristic motifs known as regulatory signals for RNA polymerase II transcription: CCAAT, TATA, and ATTAAA. In addition, a putative pol gene displays apparent homologies to conserved regions of retroviral reverse transcriptase. The 5' long terminal repeat is flanked at its 3' end by a putative primer-binding site for reverse transcription with homology to tRNA(Pro). This element is therefore termed HuRRS-P (human retrovirus-related sequence-proline). There are 20 to 40 copies of HuRRS-P homologous sequences in DNAs of human and simian origin.
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PMID:Isolation of novel human retrovirus-related sequences by hybridization to synthetic oligonucleotides complementary to the tRNA(Pro) primer-binding site. 243 28

The germline ecotropic murine leukemia (MuLV) proviruses of BALB/c and C57BL/6 (B6) mice were analyzed to determine the molecular basis of low virus expression in these mouse strains and to determine the mechanism of interaction of these two proviruses. Previous work had demonstrated that the BALB/c endogenous ecotropic provirus was infectious but unable to induce XC cell syncytia formation, and that induced (BALB/c X B6) hybrid cells expressed 10- to 50-fold more XC syncytia than induced parental cells. Two independently isolated DNA clones of the B6 endogenous ecotropic provirus were noninfectious following transfection into cells, and cell lines that expressed this viral genome produced noninfectious MuLV. Nucleotide sequencing of the mutant region of the B6 provirus indicated that the defective nature of this provirus resulted from an amino acid substitution of proline for alanine in the central portion of reverse transcriptase. From the analysis of the virus produced by induced hybrid cells, and the patterns of steady-state viral RNA in induced cells, we propose that the enhanced XC cell syncytia formation observed in hybrid cells is due to trans-complementation of viral proteins and not viral recombination or trans-activation of viral genome expression.
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PMID:Mechanism of interaction between endogenous ecotropic murine leukemia viruses in (BALB/c X C57BL/6) hybrid cells. 244 99

Hemoglobin Long Island has two separate amino acid abnormalities of beta-globin structure: an extension of the NH2 terminus by a methionine residue and a histidine-to-proline substitution at the normal second position. The NH2-terminal methionine residue, the translation product of an AUG initiation codon, is present only transiently in nascent proteins. Because of the general biological implications of this abnormality, we investigated the nature of the genetic defect of this mutant. We determined the sequence of the relevant portion of the beta-globin mRNA by means of dideoxynucleotide chain termination of the complementary DNA (cDNA) in which an oligonucleotide complementary to codons 10-17 was used as a primer for reverse transcriptase. A histidine-to-proline substitution was confirmed in the mutant mRNA by identifying an adenine-to-cytosine transversion in the second codon. However, we were unable to find any other abnormality at either the AUG initiation codon or in the 56 bases upstream from the adenine-to-cytosine transversion (encompassing most of the 5' untranslated region of the mutant beta-globin mRNA). Thus, it appears that this single lesion probably interferes with the poorly understood methionine-cleaving mechanism that modulates most of prokaryotic and eukaryotic proteins.
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PMID:Hemoglobin Long Island is caused by a single mutation (adenine to cytosine) resulting in a failure to cleave amino-terminal methionine. 345 55

A human type C retrovirus [human T-cell leukemia (lymphoma) virus; HTLV], recently isolated from young adult patients with cutaneous T-cell lymphoma or leukemia, was not detectably related to other known animal retroviruses in molecular hybridization studies, by comparison of reverse transcriptase and the major core protein p24. The p24 core protein was purified to homogeneity. The amino acid composition, the COOH-terminal amino acid, and the NH(2)-terminal amino acid sequence of the first 25 residues of this major internal structural protein were determined. These results were then compared to the known structure of the internal core protein of other retroviruses. The compositional data reveal that HTLV p24 is chemically distinct from p30-p24 of other animal retroviruses, in agreement with the earlier immunological analyses. However, HTLV p24 shares the common NH(2)-terminal proline and COOH-terminal leucine of all mammalian type C viral p30s. In addition, like bovine leukemia virus (BLV), HTLV lacks the common prolylleucylarginine tripeptide and the larger conserved region found near the NH(2) terminus of the other mammalian type C viral p30s. Alignment of the amino acid sequence of HTLV p24 with previously determined sequences of other retrovirus proteins, including BLV p24, reveals statistically significant sequence homology only to BLV. The results reported here demonstrate that HTLV p24 is related to but chemically distinct from the major core protein of other retroviruses. Similarly, previous results showed that there was no immunological crossreactivity of the p24 protein and reverse transcriptase of HTLV with other retroviruses, including BLV, and no nucleic acid sequence homology. However, the present results, combined with the common size of the p24 and reverse transcriptase, suggest that HTLV may be closer to BLV than any other known retrovirus.
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PMID:Primary structure analysis of the major internal protein p24 of human type C T-cell leukemia virus. 628 Jan 75


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