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)

An ultrastructural study was performed on rabbit epithelial RK-13 cells and CD4+ human T lymphocyte lines infected with various recombinant vaccinia viruses (RVVs) expressing genes of human immunodeficiency virus (HIV): the mature p17 or p24 gag domain alone, the entire or truncated gag gene, the reverse transcriptase domain, or the gag-pol genes with a frameshift mutation. Cells infected with RVVs that produced the gag polyprotein with a predicted Mr of more than 48K showed budding and release of HIV-like particles into the extracellular space. These particles were not observed in cells expressing a truncated gag gene (p17 and p24 regions). Mature HIV-like particles were observed extracellularly when the entire gag gene and the protease region of the pol gene were expressed. In contrast, in cells infected with RVVs that contained the gag-pol gene with a frameshift mutation, neither recognizable budding structures nor extracellular HIV-like particles could be detected. These results suggest that the gag gene, particularly its 3' terminus, is necessary for the assembly of HIV particles. In addition, the protease region of the pol gene seems to be required for morphological maturation of HIV particles, but complete proteolytic cleavage of the gag protein may prevent bud formation.
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PMID:Role of the gag and pol genes of human immunodeficiency virus in the morphogenesis and maturation of retrovirus-like particles expressed by recombinant vaccinia virus: an ultrastructural study. 191 28

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

Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-Leu*Phe-) and (b) between the p51 and RNase H domain (-Phe*Tyr-) within p66 of RT in the HIV-1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a beta-branched (Ile) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a beta-branched (Val) residue in the P2 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.
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PMID:Mutating P2 and P1 residues at cleavage junctions in the HIV-1 pol polyprotein. Effects on hydrolysis by HIV-1 proteinase. 204 56

The tandemly arrayed miniexon genes of the trypanosomatid Crithidia fasciculata are interrupted at specific sites by multiple copies of an inserted element. The element, termed Crithidia retrotransposable element 1 (CRE1), is flanked by 29-base-pair target site duplications and contains a long 3'-terminal poly(dA) stretch. A single 1,140-codon reading frame is similar in sequence to the integrase and reverse transcriptase regions of retroviral pol polyproteins. Cloned lines derived from a stock of C. fasciculata have unique arrangements of CRE1s. In different cloned lines, CRE1s, in association with miniexon genes, are located on multiple chromosomes. By examining the arrangement of CRE1s in subclones, we estimate that the element rearranges at a rate of ca. 1% per generation. These results indicate that the C. fasciculata miniexon locus is the target for a novel retrotransposon.
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PMID:A rapidly rearranging retrotransposon within the miniexon gene locus of Crithidia fasciculata. 215 19

A synthetic DNA fragment encoding a protease precursor of the human immunodeficiency virus type 2 (HIV2) was cloned and expressed in bacteria and yeast. A recombinant plasmid encoding a hybrid polypeptide consisting of human superoxide dismutase and an HIV2 protease precursor of 113 amino acids was constructed for regulated intracellular expression in bacteria. Induction of this plasmid produced an autoprocessed form of the retroviral enzyme possessing the correct molecular weight. Overexpression and secretion of the protease from yeast was achieved with an expression vector encoding the yeast pheromone alpha-factor signal/leader sequence fused to a protease precursor of 115 amino acids. Amino-terminal sequence analysis confirmed that the viral enzyme exported from yeast was correctly processed from its precursor by cleavage of the predicted Ala-Pro peptide bond located at the NH2 terminus of the protease in the pol open reading frame. No additional amino acid residues were required at the COOH terminus of the protease for this autoproteolytic event. The HIV2 protease expressed in bacteria and yeast was active in an in vitro assay when tested on the HIV1 polyprotein precursor, myristylated Pr53gag. Two synthetic peptides representing junction sequences in the HIV1 gag-pol precursor were used to assay purified HIV2 protease. The enzyme exhibited a kcat/KM of 23.2 min-1 mM-1 on the HIV1 matrix-capsid junction peptide and a kcat/KM of 71.4 min-1 mM-1 on the protease-reverse transcriptase junction peptide. These rates show that the HIV2 enzyme is efficient at hydrolyzing the HIV1 peptide junctions, revealing the analogous nature of the substrate specificities of the two enzymes.
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PMID:Recombinant HIV2 protease processes HIV1 Pr53gag and analogous junction peptides in vitro. 219 46

Hepatitis B virus DNA replicates via its own polymerase that also acts as reverse transcriptase (Summers and Mason, 1982). This enzyme is encoded by a 3.5 Kb mRNA transcript covering the whole genome. Since the same transcript also codes for the core protein, and since the core open reading frame (ORF) is located upstream of the pol ORF, it has been suggested that the polymerase is first produced as a core-pol fusion protein that subsequently undergoes cleavage. This is already known to be the case with retrovirus reverse transcriptase, for which a gag-pol fusion protein is made first and the latter protein is liberated by proteolytic cleavage. We investigated this problem using mutants that were modified at the translation initiation codon for the core and precore ORF. Our findings suggested that polymerase translation occurred from the internal AUG codon independently of core protein synthesis, and that obligatory production of the core-pol fusion protein is accordingly unlikely.
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PMID:Translation of hepatitis B virus DNA polymerase from the internal AUG codon, not from the upstream AUG codon for the core protein. 222 32

We have made a computer-assisted search for homology among polymerases or putative polymerases of various viruses and a transposable element, the Drosophila copia-like element 17.6. The search revealed that the putative polymerase (second open reading frame) of the copia-like element 17.6 bears close resemblance in overall structural organization to the pol gene product of Moloney murine leukaemia virus (M-MuLV): they show significant homology to each other at both the N- and C-terminal portions, suggesting that the 17.6 putative polymerase carries two enzymatic activities, related to reverse transcriptase and DNA endonuclease. The putative polymerase of cauliflower mosaic virus (CaMV) shows striking homology with the putative polymerase of 17.6 over almost its entire length, but it lacks the DNA endonuclease-related sequence. Furthermore, it was shown that the N-terminal ends of the M-MuLV pol product and the CaMV and 17.6 putative polymerases exhibit strong sequence homology with the gag-specific protease (p15) of Rous sarcoma virus (RSV) as well as the amino acid sequence predicted from the gag/pol spacer sequence of human adult T-cell leukaemia virus (HTLV). These p15-related sequences contain a highly conserved stretch of amino acids which show a close similarity with sequences around the active site amino acids Asp-Thr-Gly of the acid protease family, suggesting that they have an activity similar to acid protease. On the basis of the alignment of reverse transcriptase-related sequences, a dendrogram representing phylogenetic relationships among all the viruses compared together with 17.6 was constructed and its evolutionary implication is discussed.
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PMID:Close structural resemblance between putative polymerase of a Drosophila transposable genetic element 17.6 and pol gene product of Moloney murine leukaemia virus. 240 86

We have determined the complete 9202 nucleotide sequence of the visna lentivirus. The deduced genetic organization most closely resembles that of the AIDS retrovirus in that there is a novel central region separating pol and env. Moreover, there is a close phylogenetic relationship between the conserved reverse transcriptase and endonuclease/integrase domains of the visna and AIDS viruses. These findings support the inclusion of the AIDS virus in the retroviral subfamily Lentivirinae.
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PMID:Nucleotide sequence of the visna lentivirus: relationship to the AIDS virus. 241 Jan 40

Expression of a region of the Moloney murine leukemia virus (M-MuLV) pol gene in Escherichia coli resulted in the synthesis of reverse transcriptase activity which could be detected in crude extracts. Construction of deletions at the 3' terminus of this gene resulted in a 4-fold increase in the level of the reverse transcriptase activity in the soluble fraction of crude lysates and yielded the high level production of a stable protein species of Mr = 71,000. Purification of this protein by column chromatography on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, and hydroxylapatite indicated that it was a multifunctional enzyme containing RNase H and reverse transcriptase activity. The Mr = 71,000 species had a sedimentation coefficient of 4.65 S by glycerol gradient centrifugation, indicating that the enzyme was a monomer. Using poly(A)+ mRNAs primed with oligo(dT), the enzyme synthesized double-stranded DNA copies between 1.3 and 9.9 kilobases in length. Synthesis of long cDNA required 8 mM Mg2+, 4 mM Mn2+, 2 mM dNTPs, and saturating levels of enzyme. Actinomycin D efficiently limited the enzyme to the first strand synthesis. Additional characteristics of the fusion protein are described.
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PMID:Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. 241 Apr 13

We have determined the complete nucleotide sequence of the copia element present at the white-apricot allele of the white locus in Drosophila melanogaster. This transposable element is 5,146 nucleotides long and contains a single long open reading frame of 4,227 nucleotides. Analysis of the coding potential of the large open reading frame, which appears to encode a polyprotein, revealed weak homology to a number of retroviral proteins, including a protease, nucleic acid-binding protein, and reverse transcriptase. Better homology existed between another part of the copia open reading frame and a region of the retroviral pol gene recently shown to be distinct from reverse transcriptase and required for the integration of circular DNA forms of the retroviral genome to form proviruses. Comparison of the copia sequence with those of the Saccharomyces cerevisiae transposable element Ty, several vertebrate retroviruses, and the D. melanogaster copia-like element 17.6 showed that Ty was most similar to copia, sharing amino acid sequence homology and organizational features not found in the other genetic elements.
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PMID:Complete nucleotide sequence of the Drosophila transposable element copia: homology between copia and retroviral proteins. 241 Jul 72


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