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)

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 extend our previous studies of the function of the Cys-His box of Rous sarcoma virus NC protein, we have constructed a series of point mutations of the conserved or nonconserved amino acids of the proximal Cys-His box and a one-amino-acid deletion. All mutants were characterized for production of viral proteins and particles, for packaging and maturation of viral RNA, for reverse transcriptase activity, and for infectivity. Our results indicated the following. (i) Mutations affecting the strictly conserved amino acids cysteine 21, cysteine 24, and histidine 29 were lethal; only the mutant His-29----Pro was still able to package viral RNA, most of it in an immature form. (ii) Mutation of the highly conserved glycine 28 to valine reduced viral RNA packaging by 90% and infectivity 30-fold, whereas mutant Gly-28----Ala was fully infectious. This suggests a steric hindrance limit at this position. (iii) Shortening the distance between cysteine 24 and histidine 29 by deleting one amino acid abolished the maturation of viral RNA and yielded noninfectious particles. (iv) Substitution of tyrosine 22 by serine lowered viral RNA packaging efficiency and yielded particles that were 400-fold less infectious; double mutant Tyr-22Thr-23----SerSer had the same infectivity as Tyr-22----Ser, whereas mutant Thr-23----Ser was fully infectious. (v) Changing glutamine 33 to a charged glutamate residue did not affect virus infectivity. Similarities and differences between our avian mutants and those in murine retroviruses are discussed.
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PMID:Point mutations in the proximal Cys-His box of Rous sarcoma virus nucleocapsid protein. 216 81

The NH2-terminal amino acid sequence of Moloney murine leukemia virus reverse transcriptase was determined to be Thr-Leu-Asn-Ile-Glu-Asp-Glu-Tyr-Arg-Leu-His-Glu-. The comparison of the amino acid analysis data obtained after carboxypeptidase Y digestion with the published nucleotide sequence (T. M. Shinnick, R. A. Lerner, and J. G. Sutcliffe, Nature (London) 293, 543-548, 1981) led to the conclusion that the COOH-terminus is Leu coded by CTC in nucleotide positions 4608-4610, and the tentative COOH-terminal sequence is Pro-Asp-Thr-Ser-Thr-Leu-Leu-OH. In light of these and previously reported results the complexity and map order of the pol gene are discussed.
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PMID:Amino- and carboxyl-terminal sequence of Moloney murine leukemia virus reverse transcriptase. 241 14

The human neuropeptide Y (NPY) gene was isolated from a human genomic DNA library. The transcription unit spans approximately 8 kilobase pairs and is interrupted by three intervening sequences. The first exon contains only nontranslated DNA. The site where transcription initiates was determined by primer extension analysis using a primer derived from a human cDNA, pheochromocytoma RNA and avian myeloblastosis virus reverse transcriptase. A TATA-like sequence and a CAAT-like sequence occur 25 and 70 base pairs 5' to the transcription start site, respectively. The second exon begins with the initiator Met for preproNPY and extends to the Arg (residue 63) which precedes the Tyr-amide of mature NPY. The third exon contains the coding region for 27 amino acids, and the fourth exon codes for the terminal heptapeptide and the 3' nontranslated DNA. Transcriptional control elements were investigated by fusing 581 base pairs of the 5' sequences of the NPY gene to the promoterless structural gene for chloramphenicol acetyltransferase. NPY promoter activity was assayed by transfection of these hybrid constructions into CA-77 and PC12 cells followed by the determination of chloramphenicol acetyltransferase activity in cellular extracts. DNA sequences located within 530 bases of the start of transcription are sufficient for transient expression in the two neuronally derived cell lines examined.
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PMID:Characterization, sequence, and expression of the cloned human neuropeptide Y gene. 242 15

Human immunodeficiency virus (HIV) isolates with reduced sensitivity to zidovudine (3'-azido-3'-deoxythymidine, AZT) from individuals with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex were studied to determine the genetic basis of their resistance. Most were sequential isolates obtained at the initiation of and during therapy. Comparative nucleotide sequence analysis of the reverse transcriptase (RT) coding region from five pairs of sensitive and resistant isolates identified three predicted amino acid substitutions common to all the resistant strains (Asp67----Asn, Lys70----Arg, Thr215----Phe or Tyr) plus a fourth in three isolates (Lys219----Gln). Partially resistant isolates had combinations of these four changes. An infectious molecular clone constructed with these four mutations in RT yielded highly resistant HIV after transfection of T cells. The reproducible nature of these mutations should make it possible to develop rapid assays to predict zidovudine resistance by performing polymerase chain reaction amplification of nucleic acid from peripheral blood lymphocytes, thereby circumventing current lengthy HIV isolation and sensitivity testing.
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PMID:Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). 247 83

The production of Moloney murine leukaemia virus from chronically infected cells was inhibited after starvation of glutamine. While the rate of synthesis of the precursor of the core proteins, Pr65gag, was not affected in the starved cells, its proteolytic processing was blocked. Pulse-chase experiments indicated that glutamine was required during the synthesis of Pr65gag to facilitate its subsequent processing. In addition, the synthesis of Pr200gag-pol, the precursor of the protease, reverse transcriptase and endonuclease, was inhibited in the glutamine-starved cells. Starvation for other essential amino acids such as tyrosine and isoleucine affected neither the synthesis nor the processing of the virus proteins. These results suggest that the readthrough mechanism which enables synthesis of the Pr200gag-pol polyprotein is modulated in the chronically infected cells by glutamine levels. Since the viral protease is part of the pol gene, its synthesis may be inhibited in the glutamine-starved cells and Pr65gag is therefore not processed.
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PMID:Glutamine starvation of murine leukaemia virus-infected cells inhibits the readthrough of the gag-pol genes and proteolytic processing of the gag polyprotein. 348 14

We have used an oligodeoxynucleotide of defined sequence to detect and quantitate proenkephalin mRNA in the poly(A)-containing fraction of RNA from bovine adrenal medullas. The decahexamer 5'-d(G-G-T-A-G-T-C-C-A-T-C-C-A-C-C-A)-3' was synthesized to be complementary to the codons specifying the amino acid sequence NH2-Trp-Trp-Met-Asp-Tyr-Gln-COOH. This stretch of amino acids occurs in peptide I, one of the intermediates in the biosynthetic pathway of the enkephalins in bovine adrenal medulla. This pathway starts with a precursor (proenkephalin) of about 45 kilodaltons [Stern, A. S., Jones, B. N., Shively, J. E., Stein, S. & Udenfriend, S. (1981) Proc. Natl. Acad. Sci. USA 78, 1962-1966]. The decahexamer hybridized to adrenal poly(A)+RNA and was extended into cDNA with reverse transcriptase (RNA-dependent DNA nucleotidyltransferase). Five main discrete products ranging in size from 115 to 168 nucleotides were observed. The sequences of these extensions were found to be identical over the approximately 70 nucleotides sequenced from their 5' termini and corresponded exactly to the sequence expected from the amino acid sequence of peptide I. These cDNAs and the decahexamer itself hybridized to an adrenal medullary poly(A)+RNA species of about 1500 nucleotides, sufficient in size to code for the proposed proenkephalin. At saturation, approximately 2 fmol of the decahexamer were bound per microgram of mRNA; thus, the proenkephalin mRNA represents about 0.1% of the total poly(A)+RNA population in the tissue.
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PMID:Detection and partial characterization of proenkephalin mRNA. 694 86

[Met]enkephalin and [Leu]enkephalin are derived from a protein in bovine adrenal medulla that contains multiple copies of [Met]enkephalin [Kilpatrick, D. L., Taniguchi, T., Jones, B. N., Stern, A. S., Shively, J. E., Hullihan, J., Kimura, S., Stein, S. & Udenfriend, S. (1981) Proc. Natl. Acad. Sci. USA 78, 3265--3268.] Here we characterize pro-enkephalin mRNA from bovine and human tissue by use of an oligodeoxynucleotide pentadecamer probe complementary to codons for Tyr-Gly-Gly-Phe-Met ([Met]enkephalin). This probe hybridizes specifically to a species of poly(A)-RNA from adrenal medulla and human pheochromocytoma, (1400--1450 bases), and also to [Met]enkephalin-containing pro-opiomelanocortin mRNAs from bovine pituitary (1200 bases) and from mouse pituitary tumor cell (1100 bases). A cloned cDNA probe (144 bases) complementary to the region of pro-opiomelanocortin mRNA that codes for lipotropin does not hybridize to the RNA from bovine adrenal medulla, demonstrating that the latter RNA is not pro-opiomelanocortin mRNA. The pentadecamer probe was extended to make cDNA with reverse transcriptase after hybridizing it to adrenal poly(A)-RNA. The sequence of an extended cDNA, 62 bases in length, was found to correspond exactly to that expected from the amino acid sequence of peptide E (a bovine adrenal peptide containing [Met]- and [Leu]enkephalin sequences). This cDNA also forms a specific hybrid with the RNA from bovine adrenal and human pheochromocytoma, confirming that these species of RNA are pro-enkephalin mRNA.
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PMID:Partial characterization of the mRNA that codes for enkephalins in bovine adrenal medulla and human pheochromocytoma. 695 89

The major cause of viral resistance to the potent human immunodeficiency virus type 1 reverse transcriptase (RT) inhibitor nevirapine is the mutation substituting cysteine for tyrosine-181 in RT (Y181C RT). An evaluation, against Y181C RT, of previously described analogs of nevirapine revealed that the 2-chlorodipyridodiazepinone 16 is an effective inhibitor of this mutant enzyme. The detailed examination of the structure-activity relationship of 2-substituted dipyridodiazepinones presented below shows that combined activity against the wild-type and Y181C enzymes is achieved with aryl substituents at the 2-position of the tricyclic ring system. In addition, the substitution pattern at C-4, N-5, and N-11 of the dipyridodiazepinone ring system optimum for inhibition of both wild-type and Y181C RT is no longer the 4-methyl-11-cyclopropyl substitution preferred against the wild-type enzyme but rather the 5-methyl-11-ethyl (or 11-cyclopropyl) pattern. The more potent 2-substituted dipyridodiazepinones were evaluated against mutant RT enzymes (L100I RT, K103N RT, P236L RT, and E138K RT) that confer resistance to other non-nucleoside RT inhibitors, and compounds 42, 62, and 67, with pyrrolyl, aminophenyl, and aminopyridyl substituents, respectively, at the 2-position, were found to be effective inhibitors of these mutant enzymes also.
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PMID:Novel non-nucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. 4. 2-Substituted dipyridodiazepinones as potent inhibitors of both wild-type and cysteine-181 HIV-1 reverse transcriptase enzymes. 749 Jul 32

All known DNA polymerases require primers for the initiation of DNA synthesis. While cellular polymerases and reverse transcriptases use free hydroxyl groups of RNA or DNA, the DNA polymerases of certain animal viruses and bacteriophages depend upon hydroxyl groups of amino acid residues within proteins as primers for DNA synthesis. Recently, the reverse transcriptase of a hepadnavirus has been shown to prime RNA-directed DNA synthesis from an internal site of the polypeptide (G.H. Wang and C. Seeger, Cell 71:663-670, 1992). In this report we demonstrate that a tyrosine residue of the polymerase polypeptide is the site of a phosphodiester linkage with the first nucleotide of minus-strand DNA. This tyrosine residue is located within an amino-terminal domain of the polymerase polypeptide and is indispensable for the priming of reverse transcription. Our results demonstrate that the hepatitis B virus reverse transcriptase can initiate DNA synthesis without the requirement for tRNA as a primer.
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PMID:Reverse transcription in hepatitis B viruses is primed by a tyrosine residue of the polymerase. 750 42


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