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)

Immunoglobulin G directed against the DNA polymerase of Rauscher murine leukemia virus (R-MuLV) could bind to 125I-labeled DNA polymerase of spleen necrosis virus (SNV), a member of the reticuloendotheliosis virus (REV) species. Competition radioimmunoassays showed the specificity of this cross-reaction. The antigenic determinants common to SNV and R-MuLV DNA polymerases were shared completely by the DNA polymerases of Gross MuLV, Moloney MuLV, RD 114 virus, REV-T, and duck infectious anemia virus. Baboon endogenous virus and chicken syncytial virus competed partially for antibodies directed against the common antigenic determinants of SNV and R-MuLV DNA polymerases. DNA polymerases of avian leukosis viruses, pheasant viruses, and mammalian type B and D retroviruses and particles with RNA-dependent DNA polymerase activity from the allantoic fluid of normal chicken eggs and from the medium of a goose cell culture did not compete for the antibodies directed against the common antigenic determinants of SNV and R-MuLV DNA polymerases. We also present data about a factor in normal mammalian immunoglobulin G that specifically inhibits the DNA polymerases of REV and mammalian type C retrovirus DNA polymerases.
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PMID:Specific antigenic relationships between the RNA-dependent DNA polymerases of avian reticuloendotheliosis viruses and mammalian type C retroviruses. 615 4

The three enzyme forms (alpha, alpha beta-, and beta-form) of the RNA-dependent DNA polymerase (the reverse transcriptase) from three strains of avian sarcoma virus (ASV B77, ASV tsLA334, and ASV QV2) and one exogenous (avian myeloblastosis virus (AMV)) and one endogenous avian leukosis virus (Rous-associated virus type-0 (RAV-0) were compared with each other in subunit structure and catalytic properties. Despite the gross similarity in the subunit molecular weight (Mr(alpha) = 65,000 and Mr(beta) = 92,000), minor differences were found in the molecular size of the subunit as determined by SDS-gel electrophoresis, the order being ASV tsLA334 less than or equal to ASV QV2 less than ASV B77 less than or equal to RAV-0 = AMV. The structural differences were supported by analysis of peptide fragments after treatment with S. aureus V8 protease. Although the general catalytic properties of the purified enzymes from the five virus strains were similar, the selectivety of template-primer differed in the RAV-0 enzymes. The template-primer selectivity of the reverse transcriptases from all five virus strains tested was also found to be greatly influenced by the reaction temperature for DNA synthesis, resulting in a temperature-dependent increase of poly(dG) synthesis over [(A)m] . [(dT)12-18]-dependent poly(dT) synthesis. The RAV-0 enzymes required a lower temperature for DNA synthesis, particularly for [(C)n] . [(dG)12-18]-dependent poly(dG) synthesis.
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PMID:Reverse transcriptase associated with avian sarcoma-leukosis viruses. II. Comparison of subunit structure and catalytic properties. 617 24

OK10, a defective leukemia virus, is produced as a defective particle by so-called nonproducer transformed quail fibroblasts. OK10 defective viral particles contain an 8-kilobases (kb)-long genomic RNA, lack any detectable reverse transcriptase activity, and are not infectious. We studied the genetic content of OK10 RNA extracted from both virions and infected cells. As shown by RNA-cDNA hybridizations in stringent conditions, about 77% (6.4 kb) of the OK10 8.0kb RNA was related to avian leukosis viruses in the three structural genes gag, pol, and env, as well as in the c region. The remainder of the OK10 genome-encoding capacity (</=1.6 kb) was homologous to the MC29-specific transforming sequence myc(m) and therefore has been named myc(o). EcoRI restriction analysis of the OK10 integrated proviral DNA with different probes indicated the presence of only one provirus in the OK10 QB5 clone, which agreed with the gene order: 5'-gag-Deltapol-myc(o)-Deltaenv-c- 3'. Heteroduplex molecules formed between the viral OK10 8.0-kb RNA and the 6.8-kb SacI DNA fragment of the Prague A strain of Rous sarcoma virus confirmed that structure and indicated that the myc(o) sequence formed a continuous RNA stretch of 1.4 to 1.6 kb long between Deltapol and Deltaenv. We also examined the myc(o)-containing mRNA's transcribed in OK10-transformed cells. OK10-transformed quail fibroblasts (OK10 QB5) transcribed two mRNA species of 8.0 and 3.6 kb containing the myc(o) sequence. The genetic content of the 3.6-kb species made it a possible maturation product of the genome size 8-kb species by splicing out the gag and pol sequences. In OK10-transformed bone marrow cells (OK10 BM), a stable bone marrow-derived cell line producing OK10, the myc(o) sequence was found in four RNA species of 11.0, 8.0, 7.0, and 3.6 kb. Again, the genetic content of these mRNA's indicated that (i) the 3.6-kb species could be spliced out of the 8.0-kb-genome size mRNA and (ii) the 11.0-kb-long mRNA could represent a read-through of the OK10 provirus, the corresponding maturation product being, then, a 7.0-kb mRNA. The 7.0- and 3.6- kb mRNA's both contained the myc(o) sequence, but no sequences related to the gag or pol gene. In conclusion, whereas the myc sequences have been generally thought to be expressed through a gag-onc fusion protein, as for MC29 and CMII viruses, our experiments indicate that they could also be expressed as a non-gag-related product made from a subgenomic mRNA in the OK10-transformed cells.
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PMID:Subgenomic mRNA in OK10 defective leukemia virus-transformed cells. 628 57

In avian leukosis virus, processing by the viral protease (PR) appears to activate reverse transcriptase (RT), since PR-defective virions have extremely feeble reverse transcriptase activity. We showed previously that when such detergent-treated virions are digested in vitro with PR, the Gag precursor is completely and properly matured, but the Gag-Pol precursor is not. In particular, the junction between Gag and Pol, i.e., between the PR and RT domains in Gag-Pol, remains refractory to cleavage, and reverse transcriptase is hardly activated. We have now investigated processing between Gag and Pol in greater detail, both in vitro and in vivo. In vivo, three mutations designed to destroy or alter the cleavage site at the N-terminus of RT failed to abrogate processing, suggesting that nearby cryptic cleavage sites can be used by PR, and thus that in virions this portion of Gag-Pol is in an extended conformation. By contrast, resistance to cleavage was observed in vitro in a series of N- and C-terminally truncated Gag-Pol substrates, produced by in vitro translation or in the baculovirus-insect cell system. This resistance was maintained even in short polypeptides, implying that the inability to be processed in vitro is a consequence of local conformation. In the previously described Gag mutant cs22, which is unable to undergo full activation of PR, we found that in vivo in quail cells the only cleavages made in the Gag-Pol polypeptide are at the NC-PR and the PR-RT junctions, suggesting that in wild-type avian leukosis virus, processing of Gag-Pol begins by cleavage immediately upstream and downstream of the PR domain. Taken together, these results suggest a model in which in immature virions the segment of polypeptide between PR and RT is held in an extended but inherently unstable conformation, and that in vivo the first cleavage in Gag-Pol must occur in this region. In the absence of virion structure this segment of polypeptide collapses into its most stable conformation, preventing cleavage. Based on amino acid sequence, we predict that this portion of Gag-Pol adopts a coiled coil conformation reminiscent of a leucine zipper.
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PMID:Proteolytic cleavage at the Gag-Pol junction in avian leukosis virus: differences in vitro and in vivo. 752 75

A current model for the generation of transforming retroviruses proposes that read-through RNAs, containing both viral and cellular sequences, are copackaged with viral genomic RNA. It is, however, possible that a cellular mRNA is occasionally encapsidated into a retroviral particle, even though viral packaging sequences are absent. We have generated recombinant proviruses following copackaging of an avian leukosis viral genomic RNA and a neo-containing RNA completely devoid of retroviral sequences. In these studies, we used the packaging cell line SE21Q1b, which has the unique ability to randomly package cellular mRNA into retroviral particles. We describe 10 recombinants obtained following copackaging of nonhomologous RNAs. Our data show that recombination is not occurring at the DNA level in the parental SE21Q1b cells but is occurring at the RNA level, during reverse transcription. These data further suggest that reverse transcriptase can preferentially jump between templates at short stretches of homology in otherwise unrelated RNAs. We conclude that retroviral sequences are not required for packaged mRNA to be reverse transcribed and to be included in integrated proviruses.
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PMID:A model system for nonhomologous recombination between retroviral and cellular RNA. 768 1

Protease (PR)-defective avian leukosis virus particles display 300-fold-reduced levels of reverse transcriptase (RT) activity relative to wild-type particles. This observation suggests that during virion assembly RT is activated by proteolytic maturation of the Gag-Pol polyprotein precursor. To study the relationship between proteolytic cleavage and RT activation, we subjected PR-defective virion cores to digestion with purified viral PR and analyzed the structure of the major polypeptides produced as well as RT activity. Under conditions in which Gag precursors were fully matured, the RT domain was only incompletely released from the Gag-Pol precursor, remaining tethered to the upstream Gag domains PR or NC-PR. In the same reaction, RT activity was stimulated only three-fold, or 100-fold less than expected for a fully active RT. The poor activation suggested that the NC or PR domains could repress RT activity. To test this idea, we constructed recombinant baculoviruses expressing 19 different fusion proteins with upstream Gag or downstream Pol sequences attached to RT. Each protein was partially purified and assayed for its inherent RT activity. The results are consistent with the idea that Gag sequences can inhibit RT activity but indicate that the size of the Pol domain as well as the status of the PR domain (wild-type or mutant) also can profoundly influence activity. Several of the constructed Gag-Pol fusion proteins contained a wild-type PR domain. Some of these underwent intracellular PR-mediated processing, while others did not. All proteins in which the PR domain was preceded by upstream Gag sequences showed specific proteolysis. By contrast, all proteins initiated with a methionine placed one residue upstream of the natural N terminus of PR failed to show specific proteolysis. Amino-terminal sequencing of one such protein yielded the correct amino acid sequence and showed that the initiating methionine was not removed. One interpretation of these findings is that activation of PR requires the generation of the precise N terminus of the mature PR.
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PMID:Reverse transcriptase and protease activities of avian leukosis virus Gag-Pol fusion proteins expressed in insect cells. 769 75

A reverse transcriptase polymerase chain reaction (RT-PCR) for avian leukosis virus (ALV) was developed for the detection of contamination of vaccines produced in embryonated eggs and cell cultures derived from chicken. ALV is highly pathogenic and induces a wide spectrum of disease in infected animals. ALV can be divided into five subgroups (A-E). The envelope glycoprotein (env gp85) is the main antigen determinant and responsible for subgroup classification. Viral RNA of all subgroups (A-E) was isolated and amplified using three sets of primers. Subsequently, restriction endonuclease analysis confirmed the product identity and discriminated between subgroups. In specific pathogen free (SPF) eggs experimentally inoculated with ALV, viral RNA was found in allantoic fluids, as well as in vaccines spiked with different subgroups of ALV. No adventitious virus was detected in commercially available preparations. This system provides a rapid and specific in vitro method for the detection of ALV RNA as an extraneous agent and may be applied for quality control of avian vaccines.
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PMID:Use of reverse transcriptase polymerase chain reaction for detection of vaccine contamination by avian leukosis virus. 922 Mar 92

In an early step in the retroviral infectious process, reverse transcriptase copies the genomic RNA of the virus into complementary minus-strand DNA. The primer for this synthetic event is a molecule of cellular tRNA, which is annealed by its 3' 18 nucleotides to a region of the genomic RNA termed the primer-binding site (PBS); the sequence of the PBS and hence the identity of the tRNA depend upon the retrovirus species. In addition to the primer tRNA, retrovirus particles contain a substantial number of other tRNA molecules. The latter tRNA population is enriched for the tRNA species which serves as primer for the virus. While there is considerable evidence that the enrichment for the primer species can be attributed to the pol gene product, nothing is known regarding mechanisms of annealing the primer to the PBS. We have analyzed pol- mutants of avian leukosis virus (ALV) and murine leukemia virus (MuLV) for the presence of primer at the PBS in virion genomic RNA. Remarkably, the results were different for the two viruses: the PBS was substantially occupied by primer in MuLV but not in ALV. Previous data indicates that the Pol-dependent enrichment of the primer within the virion is much greater in ALV than in MuLV. We therefore propose that the absence of primer at the PBS in pol- ALV is due to the deficiency of the primer species within the particle. The results suggest that, at least in MuLV, the tRNA is unwound by either the Gag protein or a cellular protein for annealing to the PBS. Further, the C-terminal 17 amino acids of Gag are unnecessary for this function in MuLV.
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PMID:Placement of tRNA primer on the primer-binding site requires pol gene expression in avian but not murine retroviruses. 926 22

A reverse transcriptase polymerase chain (RT-PCR) assay was developed to detect avian leukosis retrovirus (ALV) in egg albumen. Eggs of Single Comb White Leghorns were from a commercial breeder (stock F) and from a pathogen-free flock (stock N). RT-PCR was undertaken on isolated RNA from 20 unfertilized egg samples using seven sets of primers that correspond to the ALV gp85 envelope glycoprotein which determines the ALV subgroup classification. An ELISA assay for ALV gs antigen of egg albumen was positive for all stock F birds tested and negative for all stock N birds. Virus isolation was undertaken by inoculating egg albumen, feather pulp, or blood from five stock F chickens onto cultures of chicken embryo fibroblasts (C/E). IFA analysis of the inoculated C/E cultures indicated that all stock F birds tested contained infectious ALV. For the virus-positive stock F chickens, RT-PCR analyses using primers designed to detect all ALV subgroups detected ALV in 15/15 (100%) egg albumen samples, while primers designed to detect subgroup A ALV were positive for 12/15 (80%) egg albumen samples. RT-PCR products were not detected from five egg albumen samples from five stock N chickens by any primer sets. Direct sequencing using primers specific for subgroup A ALV verified the viral subgroup in the RT-PCR amplification products. The combined use of RT-PCR and direct sequencing of the RT-PCR product provides a new approach for identifying ALV-infected poultry.
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PMID:Detection of avian leukosis virus in albumen of chicken eggs using reverse transcription polymerase chain reaction. 1020 92

Retroviral particles contain two positive-strand genomic RNAs linked together by noncovalent bonds that can be dissociated under mild conditions. We studied genomic RNAs of wild-type and mutant avian leukosis viruses (ALVs) in an attempt to (i) better understand the site(s) of RNA dimerization, (ii) examine whether the primer binding site (PBS) and tRNA primer are involved in dimerization, and (iii) determine the structure of genomic RNA in protease-deficient (PR(-)) mutants. We showed that extensively nicked wild-type ALV genomic RNAs melt cooperatively. This implies a complex secondary and/or tertiary structure for these RNAs that extends well beyond the 5' dimerization site. To investigate the role of the PBS-tRNA complex in dimerization, we analyzed genomic RNAs from mutant viruses in which the tRNA(Trp) PBS had been replaced with sequences homologous to the 3' end of six other chicken tRNAs. We found the genomic RNAs of these viruses are dimers that dissociate at the same temperature as wild-type viral RNA, which suggests that the identity of the PBS and the tRNA primer do not affect dimer stability. We studied two ALV PR(-) mutants: one containing a large (>1.9-kb) inversion spanning the 3' end of gag and much of pol, rendering it deficient in PR, reverse transcriptase, and integrase, and another with a point mutation in PR. In both of these mutant viruses, the genomic RNA appears to be either primarily or exclusively monomeric. These data suggest that ALV can package its RNA as monomers that subsequently dimerize.
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PMID:Studies of the genomic RNA of leukosis viruses: implications for RNA dimerization. 1043 3


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