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 mechanism of inhibition of Moloney leukemia virus by N-methylisatin-beta-4',4'-diethylthiosemicarbazone was studied. Experiments that used [3H]leucine for short-pulse labeling in the presence of the drug resulted in a 71% inhibition in the synthesis of Pr-80, the polypeptide precursor of the gag viral proteins. The radioactive pulse products of the polypeptide precursors after a further 2-h chase period showed a normal cleavage of the precursors, with the formation of a reduced amount of final mature viral structural proteins. The experimental evidence indicated that at the inhibitory concentration of 17 microM N-methylisatin-beta-4',4'-diethylthiosemicarbazone, the amount of intracellular viral RNA was not affected, whereas the activities of reverse transcriptase and the other viral protein syntheses were suppressed.
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PMID:Inhibition of the synthesis of Moloney leukemia virus structural proteins by N-methylisatin-beta-4',4'-diethylthiosemicarbazone. 608 72

A native ribonucleoprotein (RNP) complex of avian myeloblastosis virus was prepared under conditions that gave optimal cDNA synthesis. The complex was an autonomous transcriptional unit capable of synthesizing DNA complementary to the RNA virus genome in the absence of exogenous reverse transcriptase (RNA-dependent DNA nucleotidyltransferase), genomic RNA, and primer. The RNA of the RNP complex cannot be translated in an in vitro cell-free translational system. The RNP contains intact viral RNA, the two subunits of the reverse transcriptase (beta and alpha), the p32 polypeptide resulting from the cleavage of the beta subunit into the alpha subunit, and p12. The principal polypeptide constituent of the RNP complex is the highly basic protein p12, which occurs at a molar ratio of 40:1 in relation to the beta subunit of the polymerase. When examined by the electron microscope, the RNP complex appears similar to the beaded structure of chromatin fiber. A significant portion of these molecules are circular, with headlike structures attached. The circular nature of the proviral DNA and the ability of the RNP complex to generate large intact cDNA copies from the natural primer end suggest that the 5' and 3' ends of the viral RNA are in proximity when in the RNP complex.
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PMID:Native ribonucleoprotein is an efficient transcriptional complex of avian myeloblastosis virus. 615 28

A proteolytic activity is associated with structural protein p15 in avian RNA tumor viruses. Its effect on the known intracellular viral polyprotein precursors obtained by immunoprecipitation was investigated. Cleavage of Pr76gag resulted in the sequential appearance of p15, p27, and p19. The intracellular precursor Pr180gag-pol was also cleaved by p15, whereas the intracellular glycoprotein precursors of avian RNA tumor viruses, Pr92env, remained unaffected by p15 under all conditions tested. The specificities of the antibodies used to precipitate the precursors influenced the pattern of intermediates and cleavage products obtained by p15 treatment. If virus harvested from the the Prague strain of Rous sarcoma virus, subgroup C-transformed cells at 15-min intervals was incubated at 37 degrees C for further maturation, RNA-dependent DNA polymerase activity showed an optimum of DNA synthesis with 70S viral RNA or synthetic template-primers after short incubation periods. The presence of additional p15 during incubation resulted in a shift of the enzyme activity peak toward earlier time points. Virus harvested at 3-h intervals contained significant amounts of Pr180gag-pol and Pr76gag. The addition of p15 resulted in the cleavage of Pr180gag-pol and Pr76gag, but only a few distinct low-molecular-weight polypeptides appeared. Treatment of purified RNA-dependent DNA polymerase with p15 in vitro resulted in a disappearance of the beta subunit and an enrichment of the alpha subunit. In addition, a polypeptide of 32 x 10(3) molecular weight was generated. The cleavage pattern observed differed from the one obtained by trypsin treatment.
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PMID:Effect of p15-associated protease from an avian RNA tumor virus on avian virus-specific polyprotein precursors. 615 35

Feline leukemia virus DNA polymerase was purified by ion-exchange and nucleic acid affinity chromatographies. The enzyme consists of a single polypeptide chain of mol. wt. approx. 72,000 as determined by both glycerol density gradient centrifugation and SDS-polyacrylamide gel electrophoresis. The preferred divalent cation for DNA synthesis is Mn2+ on a variety of template-primers, and its optimum concentration appears to be significantly lower than reported results of other mammalian type-C viral enzymes. The purified enzyme also contained RNAase H activity. Both DNA polymerase and RNAase H activities appear to reside on the same molecule as demonstrated by the copurification of both activities through various purification steps. The divalent cation requirement for maximum activity of RNAase H is also similar to that of the DNA polymerase. RNAase H without detectable polymerase activity was generated by a limited chymotrypsin digestion of the purified reverse transcriptase. This RNAase H activity was inhibited equally effectively as RNAase H in the intact reverse transcriptase by antisera prepared against reverse transcriptase of feline leukemia virus. These results indicate that the RNAase H catalytic activity of reverse transcriptase is distinct from the polymerase portion of the molecule. Since the RNAase H activity appears to be more stable, the measurement of RNAase H activity with a proper antibody might be useful for assaying tumor cells for the presence of the viral enzyme.
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PMID:Biochemical and immunological properties of the DNA polymerase and RNAase H activities of purified feline leukemia virus reverse transcriptase. 615 69

We have studied the biosynthesis of avian retrovirus proteins related to reverse transcriptase in permissive avian embryonic cells. Analysis of immune precipitates from avian sarcoma virus (ASV)-infected cells demonstrated the presence of the 180,000-dalton gag-pol "read-through" protein (Pr180gag-pol) and a 130,000-dalton polypeptide (Pr130gag-pol). Pr130gag-pol was found, in serological and peptide mapping studies, to consist primarily of sequences related to reverse transcriptase and the gag-encoded protein p15. Pr180gag-pol was found to be phosphorylated, whereas Pr130gag-pol was not. In addition, only Pr180gag-pol but not Pr130gag-pol was susceptible to cleavage with the virion protease p15. Although the structure of Pr130gag-pol would suggest that it is generated by removal of a portion of the gag region from Pr180gag-pol, an analysis of labeling kinetics has failed to demonstrate unequivocally whether Pr130gag-pol is a cleavage product of Pr180gag-pol or a primary translation product. We were repeatedly unable to detect either Pr180gag-pol or Pr130gag-pol in virus particles released from the cell, whereas both beta and alpha subunits were readily observed. Several presumed intermediates between Pr130gag-pol and the beta subunit of reverse transcriptase were also observed in virions. These studies indicate cleavage of polyemrase precursors at the time of virus budding. On the basis of these data, we present a processing scheme for the generation of reverse transcriptase subunits. We have also examined reverse transcriptase biosynthesis in cells producing two mutants that fail to package the enzyme. Previous work showed that integrated proviruses of both mutants are missing DNA sequences in pol: one mutant, PH9 (Mason et al., J. Virol. 30:132-140, 1979), contains a deletion near the 3' end of pol, whereas the other, SE52d (linial et al., Virology 87:130-141, 1978), may have inserted a host cell sequence near the 5' end of pol. Neither mutant synthesized Pr180gag-pol or Pr130gag-pol, but instead produced novel proteins comprised of sequences shared with gag proteins plus a region antigenically related to reverse transcriptase. Both proteins were defective as precursors to reverse transcriptase. Whereas Pr180gag-pol and Pr130gag-pol were precipitated by an antiserum raised against p32 (a virion protein derived from the portion of the beta subunit removed during processing of beta to alpha [Schiff and Grandgenett, J. Virol. 28:279-291, 1978]), the novel protein synthesized by PH9 ws not precipitated. This suggets that the alpha subunit is generated by a COOH-terminal cleavage of the beta subunit.
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PMID:Synthesis and processing of polymerase proteins of wild-type and mutant avian retroviruses. 616 Feb 63

Visna virus is a retrovirus which replicates in fibroblast-like cells of the sheep choroid plexus through a lytic cycle. Visna virions contain three major low-molecular-weight proteins (p30, p16, and p14) which, together with the genomic RNA and several molecules of reverse transcriptase, constitute the core structure of the virions. The core is surrounded by an envelope containing a major glycoprotein (gp135). By analogy with the oncoviruses, these three groups of structural proteins (i.e., the internal proteins, the envelope glycoprotein, and the reverse transcriptase) are probably encoded by the gag, env, and pol genes, respectively. To elucidate the genetic organization of the visna virus genome and its expression, we studied the synthesis of viral proteins in infected sheep choroid plexus cells. Intracellular viral proteins were detected by immunoprecipitation of pulse-labeled cell extracts with monospecific sera raised against p30, p16, and gp135 and resolution of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoprecipitation with anti-p30 and anti-p16 sera allowed the characterization of the 55,000-dalton polypeptide precursor to internal virion proteins p30, p16, and p14 (Pr55(gag)). Tryptic peptide mapping confirmed the precursor-product relationship between Pr55(gag) and the three internal proteins. In addition, a gag-related polypeptide of 150,000 daltons was also detected. This polypeptide, which was less abundant than Pr55(gag), is a likely precursor to the viral reverse transcriptase (Pr150(gag-pol)). Pr55(gag) and Pr150(gag-pol) are not glycosylated. The precursor related to major envelope protein gp135 is a glycosylated polypeptide with an average molecular weight of 150,000 (gPr150(env)). Pulse-chase experiments indicated that gPr150(env) matures into glycoprotein gp135 intracellularly; however, gp135 was never preponderant in cell extracts. The non-glycosylated from of gPr150(env), which accumulated in the presence of 2-deoxy-d-glucose, appeared as a polypeptide of about 100,000 daltons. These results indicated that visna virus codes for the largest non-glycosylated env-related precursor among all of the retroviruses and therefore probably contains the largest env gene.
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PMID:Precursor polypeptides to structural proteins of visna virus. 617 45

We studied intracellular avian gag proteins (internal structural proteins of virions) in several mammalian cell lines transformed by Rous sarcoma virus. All lines examined contain gag antigens as determined by radioimmune assay. We used the techniques of protein blotting from polyacrylamide gels, which detects nanogram quantities of viral protein, to investigate the size of intracellular viral polypeptides. All of the lines that contained enough viral protein to be amenable to this type of analysis synthesized Pr76, the avian sarcoma virus gag precursor polypeptide, but failed to process it into mature virion proteins. In some cell lines, the recovery of Pr76 was greatly enhanced by the addition of a mixture of protease inhibitors, including the sulfhydryl-blocking reagent N-ethylmaleimide, to the lysis buffer. At least several of the mammalian cells also synthesized a viral polypeptide the size of Pr180, the precursor to reverse transcriptase. Since Rous sarcoma virus does not replicate or replicates extremely poorly in mammalian cells, the lack of processing suggests that cleavage and virion assembly are invariably associated.
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PMID:Avian sarcoma virus gag precursor polypeptide is not processed in mammalian cells. 618 52

A rare cDNA coding for most of the alpha subunit of the Torpedo nicotinic acetylcholine receptor has been cloned into bacteria. The use of a mismatched oligonucleotide primer of reverse transcriptase facilitated the design of an efficient, specific probe for recombinant bacteria. DNA sequence analysis has enabled the elucidation of a large part of the polypeptide primary sequence which is discussed in relation to its acetylcholine binding activity and the location of receptor within the plasma membrane. When used as a radioactive probe, the cloned cDNA binds specifically to a single Torpedo mRNA species of about 2350 nucleotides in length but fails to show significant cross-hybridisation with alpha subunit mRNA extracted from cat muscle.
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PMID:The molecular cloning and characterisation of cDNA coding for the alpha subunit of the acetylcholine receptor. 618 41

We have previously shown that the inhibition of methylation reactions by the treatment of B77 avian sarcoma virus-infected cells with medium containing cycloleucine results in an inhibition in the intracellular accumulation of the spliced subgenomic mRNA for the virion envelope protein precursor, whereas the genome-size RNA accumulates in larger than normal amounts (C. M. Stoltzfus and R. W. Dane, J. Virol. 42:918-931, 1982). To measure the production of virus particles, we have now determined the reverse transcriptase activity in the culture fluid from infected cells treated with various concentrations of cycloleucine. The activity was somewhat greater in the fluid from the cycloleucine-treated cells than it was in the fluid from the control cells, suggesting an enhancement of particle production in the presence of cycloleucine. In contrast, the production of infectious virions, as determined by the focus assay, decreased when the cycloleucine concentration of the medium increased. We determined the polypeptide compositions of purified particles produced from infected cells treated with or without cycloleucine and labeled with [(3)H]leucine. The relative amounts of radioactivity associated with p19 and p27 were approximately the same in all of the preparations. In contrast, significant decreases were observed in the relative amounts of [(3)H]leucine radioactivity associated with the virion glycoproteins gp85 and gp37. The extent of the decrease in the ratio of gp85 to p27 was a function of the cycloleucine concentration and correlated well with the decrease in the infectivity of the virus particles. Therefore, it is probable that the observed reduction of specific infectivity results from the reduced amounts of envelope glycoproteins in the particles budding from cycloleucine-treated cells.
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PMID:Particles of reduced infectivity and deficient in envelope glycoproteins are produced in cycloleucine-treated B77 avian sarcoma virus-infected chicken embryo fibroblasts. 618 42

Spumavirinae or foamy viruses have been shown to have a characteristic RNA-dependent DNA polymerase activity. We demonstrate here the existence of an RNase H activity that copurifies with the 81-kilodalton monomeric polypeptide, which carries the RNA-dependent DNA polymerase activity of simian foamy virus type 1. RNase H degrades RNA hybrid substrates; however, it does not solubilize single-stranded RNAs. Inactivation assays with heat, high levels of bivalent cations, ethidium bromide, and sodium fluoride suggest that the RNase H catalytic site could be topologically independent from the DNA polymerase catalytic site.
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PMID:Characterization of RNase H activity associated with reverse transcriptase in simian foamy virus type 1. 619 Oct 42


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