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)

Bovine leukemia virus protease was purified to homogeneity and assayed by using murine leukemia virus Pr65gag, a polyprotein precursor of the viral core structural proteins, as the substrate. A chemical analysis of the protease, including an amino acid composition and NH2- and COOH-terminal amino acid sequence analysis, revealed that it has an Mr of 14,000 and is encoded by a segment of the viral RNA located between the gag gene and the putative reverse transcriptase gene. As expected from the nucleotide sequence data (Rice et al., Virology 142:357-377, 1985), the reading frame for the protease is different from both the gag and reverse transcriptase reading frames. The 5' end of the protease open reading frame extends 38 codons upstream from the codon for the NH2-terminal residue of the mature viral protease and overlaps the gag open reading frame by 7 codons. The 3' end of the protease open reading frame extends 26 codons beyond the codon for the COOH-terminal residue of the mature protease and overlaps 8 codons of the reverse transcriptase open reading frame. Several lines of evidence, such as protein mapping of the gag polyprotein precursor, the characteristic structure of the mRNA, and promotion of the synthesis of a gag polyprotein precursor by lysine tRNA in vitro, suggest that the protease could be translated by frameshift suppression of the gag termination codon. In vitro synthesized bovine leukemia virus gag-related polyproteins were cleaved by the protease into fragments which were the same size as the known components of bovine leukemia virus, suggesting that the specificity of cleavage catalyzed in vitro by the purified protease is the same as the specificity of cleavage found in the virus.
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PMID:Bovine leukemia virus protease: purification, chemical analysis, and in vitro processing of gag precursor polyproteins. 300 29

By using a DNA fragment primarily encoding the reverse transcriptase (pol) region of the Syrian hamster intracisternal A particle (IAP; type A retrovirus) gene as a probe, human endogenous retrovirus genes, tentatively termed HERV-K genes, were cloned from a fetal human liver gene library. Typical HERV-K genes were 9.1 or 9.4 kilobases in length, having long terminal repeats (LTRs) of ca. 970 base pairs. Many structural features commonly observed on the retrovirus LTRs, such as the TATAA box, polyadenylation signal, and terminal inverted repeats, were present on each LTR, and a lysine (K) tRNA having a CUU anticodon was identified as a presumed primer tRNA. The HERV-K LTR, however, had little sequence homology to either the IAP LTR or other typical oncovirus LTRs. By filter hybridization, the number of HERV-K genes was estimated to be ca. 50 copies per haploid human genome. The cloned mouse mammary tumor virus (type B) gene was found to hybridize with both the HERV-K and IAP genes to essentially the same extent.
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PMID:Molecular cloning and long terminal repeat sequences of human endogenous retrovirus genes related to types A and B retrovirus genes. 300 97

Pancreatic poly(A) RNA isolated from the channel catfish (Ictalurus punctatus) was enriched for sequences corresponding to somatostatin mRNA on isokinetic sucrose gradients. Double-stranded cDNA was synthesized and inserted into the Pst I site pBR322 via the poly(dG) . poly(dC) tailing method. Escherichia coli was transformed with this DNA, and colonies containing somatostatin cDNA sequences were identified by hybridization using a primer-extended somatostatin cDNA. The somatostatin cDNA was obtained by extending a 5'-labeled undecanucleotide primer complementary to somatostatin mRNA with reverse transcriptase using catfish poly(A) RNA as a template. The synthetic primer d(T-T-C-C-A-G-A-A-G-A-A) was deduced from the amino acid sequence Phe-Phe-Trp-Lys present in somatostatin-14. Twenty positive colonies were obtained upon screening 2000 transformants. The restriction maps of the plasmid DNA obtained from the positive colonies were examined. Nineteen of these plasmids contained sequences corresponding to somatostatin-14, while one contained a sequence corresponding to somatostatin-22. The nucleotide sequence of pancreatic somatostatin-14 is reported here. The cDNA contains 350 nucleotides in the 3' noncoding region, 345 nucleotides in the coding region, and 104 nucleotides in the 5'-untranslated region. The mRNA codes for a precursor to somatostatin which is 114 amino acids in length. The preprosomatostatin has a sequence of hydrophobic amino acids at the NH2 terminus, followed by a connecting peptide of approximately 75 amino acids. The sequence Arg-Lys precedes somatostatin-14. Analysis of genomic DNA from the channel catfish reveals that somatostatin-14 and somatostatin-22 are present on different restriction fragments.
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PMID:The structure of cloned DNA complementary to catfish pancreatic somatostatin-14 messenger RNA. 617 39

For diagnosis of HIV-1 infection, attempts were made to detect anti-HIV-1 IgG in urine by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant reverse transcriptase (RT) and p17 as antigens. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate and recombinant protein-enzyme conjugate. The enzymes used as labels were horseradish peroxidase for RT and Escherichia coli beta-D-galactosidase for p17. The complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with epsilon N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Finally, bound enzyme activity was assayed by fluorometry. Urine samples were collected from 100 seronegative subjects and 70 seropositive subjects. The sensitivity and specificity were both 100% with unconcentrated urine samples. The positivity was confirmed by preincubation of urine samples with excess of the antigens. The positivity and negativity with one of the two antigens could be confirmed with the other antigen. The positivity with low signals could be confirmed by concentration of urine samples. Detection of anti-HIV-1 IgG in urine by the immune complex transfer enzyme immunoassay using different antigens would make diagnosis of HIV-1 infection possible.
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PMID:Detection of antibody IgG to HIV-1 in urine by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens for diagnosis of HIV-1 infection. 750 5

A contribution of the 51-kDa subunit of human immunodeficiency virus type-1 reverse transcriptase to activities of the parental heterodimer (p66/p51) was assessed in "selectively deleted" heterodimers whose p51 component contained C-terminal truncations of 13, 19, or 25 residues. Analyses included (i) efficiency of reconstitution into heterodimer, (ii) retention of polymerase and ribonuclease H (RNase H) function, and (iii) interaction with the HIV replication primer, tRNA(Lys,3). Our data suggest that these features of heterodimer reverse transcriptase can be modulated by the extent of the C-terminal p51 deletion. Severely impaired tRNA binding in a selectively deleted heterodimer whose 51-kDa subunit lacks 13 residues, despite retention of enzymatic functions, strengthens arguments for p51 involvement in tRNA binding.
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PMID:Modulation of HIV-1 reverse transcriptase function in "selectively deleted" p66/p51 heterodimers. 750 7

We have selected and plaque purified zidovudine (3'-azido-3'-deoxythymidine [AZT])-resistant mutants from an infectious molecular clone of feline immunodeficiency virus (FIV). The patterns of cross-resistance and drug susceptibilities of these mutants were similar to those of the AZT-resistant FIV that we previously selected in vitro from a wild-type FIV population and to those of the most common AZT-resistant clinical isolates of human immunodeficiency virus type 1. Two AZT-resistant mutants of FIV, one selected from a normal population and one selected from the molecular clone, each reverted rapidly to an AZT-sensitive phenotype when passaged in the absence of drug. Sequence analysis of the reverse transcriptase (RT)-encoding region from the plaque-purified AZT-resistant FIV revealed a single base change at position 2939, resulting in a Glu-to-Lys substitution at amino acid 202 of the RT. Similar analyses of plaque-purified revertants showed that the phenotypic reversion was not the result of a genotypic reversion at this position and that no additional mutations existed within the RT-encoding region of the revertants. Moreover, RTs purified from the mutant and revertant were both resistant to the 5'-triphosphate of AZT. These results indicate the complexity of AZT resistance and suggest the presence of additional factors, outside the RT-encoding region, which may contribute to AZT resistance.
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PMID:Rapid phenotypic reversion of zidovudine-resistant feline immunodeficiency virus without loss of drug-resistant reverse transcriptase. 750 82

It has not been unambiguously demonstrated whether the priming reaction of human immunodeficiency virus, type 1 (HIV-1) cDNA synthesis initiates with either the 2'-OH or 3'-OH group of the 3'-terminal adenosine residue of tRNA(Lys-3). In this report, we synthesized tRNA(Lys-3) of which the 3'-terminal adenosine residue lacks either a 2'-OH or 3'-OH. These tRNA molecules were used for the HIV-1 cDNA-priming reaction in a cell-free system consisting of a 141-base RNA template and purified HIV-1 reverse transcriptase. It was found that under the conditions used, the tRNA containing the 2'-deoxyadenosine was able to initiate the cDNA synthesis, while the tRNA with the 3'-deoxyadenosine was not. The results show that retroviral reverse transcriptase specifically primes cDNA synthesis from the 3'-OH group. This is in contrast to bacterial reverse transcriptase, which initiates cDNA synthesis from the 2'-OH group of an internal guanosine residue of a template RNA.
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PMID:Specificity of priming reaction of HIV-1 reverse transcriptase, 2'-OH or 3'-OH. 750 35

Thiazolo-iso-indolinone derivatives with high specificity toward the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) were identified. The most potent compound, BM +51.0836, inhibited HIV-1 RT at a 50% inhibitory concentration of 90 nM in vitro. In cell culture assays, similar 50% inhibitory concentrations were obtained with high specificity for HIV-1. These substances were equally active against a zidovudine-resistant isolate. No antiviral effect was observed with an HIV-2 isolate. HIV-1 isolates resistant to the thiazolo-iso-indolinones were generated in cell culture, and the nucleotide sequences of the respective RT genes were analyzed subsequently. Comparison of the deduced amino acid sequences with the wild-type sequence showed an amino acid change at position 181 (Tyr to Cys). Substitutions of amino acid Lys-101 and Lys-103 as well as Tyr-181 and/or Tyr-188 by site-directed mutagenesis led to resistance against the thiazolo-iso-indolinones. A chimeric HIV-2 RT, substituted with amino acids at positions 179 to 190 from HIV-1, acquired only partial susceptibility to BM +51.0836.
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PMID:Viral resistance to the thiazolo-iso-indolinones, a new class of nonnucleoside inhibitors of human immunodeficiency virus type 1 reverse transcriptase. 750 44

High level resistance to 3'-azido-3'-deoxythymidine (AZT, zidovudine or Retrovir) is conferred by the presence of four or five mutations (Met-41-->Leu; Asp-67-->Asn; Lys-70-->Arg; Thr-215-->Tyr or Phe; Lys-219-->Gln) in the human immunodeficiency virus (HIV) reverse transcriptase. The order of appearance of these five mutations in asymptomatic patients during therapy has been studied. This has enabled us to propose a model for the acquisition of zidovudine resistance mutations during the treatment of high-risk asymptomatic HIV-infected individuals. A consistent acquisition pattern of mutations at codons 41, 70 and 215 was observed in 17 individuals. Complex mixtures of HIV species containing different combinations of single and linked double resistance mutations were present early in zidovudine therapy in isolates from two patients studied in detail. From these mixtures the linked Leu-41/Tyr-215 genotype outgrew all others initially. The development of each new virus population is likely to be mediated primarily by the increase in the level of drug resistance rather than changes in the growth kinetics of the virus. This leads us to conclude that one major driving force in the outgrowth of different mutant viruses is the selective advantage conferred by higher levels of drug resistance.
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PMID:Zidovudine treatment results in the selection of human immunodeficiency virus type 1 variants whose genotypes confer increasing levels of drug resistance. 750 70

COS-7 cells transfected with human immunodeficiency virus type 1 (HIV-1) proviral DNA produce virus in which three tRNA species are most abundant in the viral tRNA population. These tRNAs have been identified through RNA sequencing techniques as tRNA(3Lys) the primer tRNA in HIV-1, and members of the tRNA(1,2Lys) isoacceptor family. These RNAs represent 60% of the low-molecular-weight RNA isolated from virus particles, while they represent only 6% of the low-molecular-weight RNA isolated from the COS cell cytoplasm. Thus, tRNA(Lys) is selectively incorporated into HIV-1 particles. We have measured the ratio of tRNA(3Lys) molecules to copies of genomic RNA in viral RNA samples and have calculated that HIV-1 contains approximately eight molecules of tRNA(3Lys) per two copies of genomic RNA. We have also obtained evidence that the Pr160gag-pol precursor is involved in primer tRNA(3Lys) incorporation into virus. First, selective tRNA(Lys) incorporation and wild-type amounts of tRNA(3Lys) were maintained in a protease-negative virus unable to process Pr55gag and Pr160gag-pol precursors, indicating that precursor processing was not required for primer tRNA incorporation. Second, viral particles containing only unprocessed Pr55gag protein did not selectively incorporate tRNA(Lys), while virions containing both unprocessed Pr55gag and Pr160gag-pol proteins demonstrated select tRNA(3Lys) packaging. Third, studies with a proviral mutant containing a deletion of most of the reverse transcriptase sequences and approximately one-third of the integrase sequence in the Pr160gag-pol precursor resulted in the loss of selective tRNA incorporation and an eightfold decrease in the amount of tRNA(3Lys) per two copies of genomic RNA. We have also confirmed herein finding of a previous study which indicated that the primer binding site is not required for the selective incorporation of tRNA(Lys).
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PMID:Role of Pr160gag-pol in mediating the selective incorporation of tRNA(Lys) into human immunodeficiency virus type 1 particles. 751 Nov 67


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