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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)

Human VA-2 cells infected with baboon type C virus were cloned and fused to Syrian hamster cells, and 33 primary hybrid colonies were obtained. These cells segregated human chromosomes and retained the complete hamster genome. Assays for type C viral p30 antigen and reverse transcriptase were performed in conjunction with analyses of 30 gene-enzyme systems representing 22 different human chromosomes. The results comfirmed that a gene, Bevi, previously assigned to human chromosome 6, dominantly controls baboon type C virus expression in hybrid cells. Representative hybrid colones were studied by nucleic acid hybridization techniques for the presence of integrated proviral DNA using complementary 3H-DNA transcripts of the baboon viral RNA genome. For each of 12 clones examined, there was a concordance between the presence of human chromosome 6, the presence of baboon type C proviral DNA sequences and virus expression. Clones which segregated chromosome 6 as judged by isozyme and karyological analyses lost detectable proviral DNA sequences and failed to produce virus. No syntenic association between the replication of baboon virus and the presence of 21 other human chromosomes was deteced. We conclude that Bevi is a preferred integration site for the baboon type C provirus in the human genome.
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PMID:A gene (Bevi) on human chromosome 6 is an integration site for baboon type C DNA provirus in human cells. 8 Feb 84

Human cells from 13 different individuals were fused to mouse and rat cells producing abundant type C viral particles. The results demonstrate that incorporation of active DNA polymerase (reverse transcriptase) into mature type C particles is suppressed in many of the hybrid clones but not in the parental mouse cell clones. This low particle-associated DNA polymerase phenotype was a heritable trait for over 100 cell generations but reversion to a high particle-associated DNA polymerase phenotype was possible. In contrast, no evidence for suppression of viral p30 antigen was found. These results suggest that human cells contain a factor(s) capable of interfering with the normal maturation of the mouse retrovirus DNA polymerase protein; however, it was not possible to assign this function to any of 20 different human chromosomes tested. It is suggested that these somatic cell hybrids may be useful in examining individual events in retrovirus packaging and release.
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PMID:Regulation of expression of type C virion DNA polymerase (reverse transcriptase) in human x mouse and human x rat hybrid cells. 9

Human immunodeficiency virus 1 (HIV-1) nucleocapsid protein p15 was produced as a fusion protein with glutathione S-transferase (GST) in Escherichia coli. Rapid purification of GST::p15 in an active form by one-step glutathione-agarose chromatography was accomplished in the presence of an antioxidant. Recombinant p15 fused to GST was shown to stimulate the dimerization of viral RNA. HIV-1 reverse transcriptase-catalyzed in vitro synthesis of minus-strand cDNA from synthetic human tRNA(Lys3UUU) and natural bovine tRNA(Lys3SUU) primer molecules was enhanced by GST::p15. GST produced in E.coli revealed no effect with respect to RNA dimerization and cDNA synthesis, demonstrating that both activities reside in the p15 portion of the fusion protein.
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PMID:Recombinant HIV-1 nucleocapsid protein p15 produced as a fusion protein with glutathione S-transferase in Escherichia coli mediates dimerization and enhances reverse transcription of retroviral RNA. 128 Feb 40

High-level expression of a transpositionally competent Ty1 element fused to the inducible GAL1 promoter on a 2 microns plasmid (pGTy1) overcomes transpositional dormancy in Saccharomyces cerevisiae. To investigate the mechanisms controlling the rate of Ty1 retrotransposition, we quantitated transposition and Ty1 gene products in cells induced and uninduced for expression of pGTy1. The increase in Ty1 transposition was 45- to 125-fold greater than the increase in Ty1 RNA effected by pGTy1 induction. Translational efficiency of Ty1 RNA was not altered in transposition-induced cells, since p190TYA1-TYB1 protein synthesis increased in proportion to steady-state Ty1 RNA levels. Therefore, expression of a pGTy1 element increases the efficiency of Ty1 transposition at a posttranslational level. Galactose induction of pGTy1 enhanced TYA1 protein processing and allowed detection of processed TYB1 proteins, which are normally present at very low levels in uninduced cells. When the ability of genomic Ty1 elements to complement defined mutations in HIS3-marked pGTy1 elements was examined, mutations in the protease domain or certain mutations in the integrase domain failed to be complemented, but mutations in the reverse transcriptase domain were partially complemented by genomic Ty1 elements. Therefore, the activity of Ty1 elements in yeast cells may be limited by the availability of Ty1 protease and possibly integrase. These results suggest that Ty1 transposition is regulated at the level of protein processing and that this regulation is overcome by expression of a pGTy1 element.
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PMID:Posttranslational control of Ty1 retrotransposition occurs at the level of protein processing. 131 8

Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.
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PMID:On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli. 138 56

Unlike many other reverse transcriptase genes, the polymerase (P) gene of the hepatitis B viruses is expressed by translational initiation from its own AUG codon rather than by ribosomal frameshifting during translation of the overlapping core gene (C). To explore the mechanism of its translation, we have fused the P gene of duck hepatitis B virus to the bacterial lacZ gene at a point 3' to the C-P overlap; this allows detection of the products of P translation with antisera to the lacZ-encoded protein. The C and P/Z coding regions were cloned downstream of a heterologous promoter and expressed in COS-7 cells. A single, bicistronic mRNA containing both C and P sequences is detected in these cells, and translational initiation occurs efficiently at the internally situated P AUG. Mutations affecting C translation have only minimal effects on P expression, in contrast to what would be expected from a modified scanning model for translation. We conclude that P translation depends on a mechanism other than scanning to allow internal entry of ribosomes to the region of the P initiator.
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PMID:Mechanism of translation of the hepadnaviral polymerase (P) gene. 169 11

Retroviral RNA is copied into DNA by reverse transcriptase when the viral genome enters into its life cycle. In the case of human immunodeficiency virus (HIV), massive amounts of unintegrated viral DNA reportedly appear in the early phase of primary infection. However, the relationship between the accumulation of this DNA and the cytopathic effect (CPE) remains obscure. In an attempt to delineate this association, we examined the appearance of the unintegrated viral DNA by means of two experimental systems: (1) primary infection of highly susceptible MOLT-4#8 cells and (2) induction of CPE by cell-fusion of persistently infected MOLT-4#8 cells. A correlation was observed between the accumulation of unintegrated viral DNA and the appearance of CPE, both when MOLT-4#8 cells were infected with cell-free virus and when persistently infected MOLT-4#8 cells were co-cultured with uninfected cells. Persistently infected cells did not fuse spontaneously in culture, because they lack the CD4-molecule on their surfaces. However, when treated with polyethylene glycol (PEG), the cells fused, exhibited ballooning degeneration, and released fewer viruses. After PEG treatment, unintegrated viral DNA also appeared. Since such DNA is generally not detected in persistently infected cells, it is possible that some cellular mechanism exists to suppress the synthesis of viral DNA and that the fusion induced by PEG treatment cancels the suppression. Treatment of persistently infected cells with Ca2+ ionophore and Ca2+ antagonist also resulted in the accumulation of unintegrated viral DNA and inhibited virus release. These findings suggest that the induction of unintegrated HIV DNA may be an effective strategy for reducing the release of the virus.
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PMID:Unintegrated DNA in cells infected in vitro with human immunodeficiency virus (HIV): a new approach to suppression of virus release. 169 87

Overexpression of the reverse transcriptase was designed in E. coli. For a high level of expression, HIV protein was expressed as a protein fusion with beta-galactosidase. When the proviral DNA fragment covering the 3' half of the gag gene and the entire pol gene was ligated to the 3' end of the lacZ gene to fuse the truncated gag to lacZ in frame, a small quantity of reverse transcriptase was produced, indicating that frameshifting and post-translational processing have occurred. Much more reverse transcriptase was produced when the entire pol region was directly fused to the lacZ gene. From a one liter culture of bacteria, 1 mg of highly purified reverse transcriptase consisting of approximately equimolar amounts of two species (p64 and p51) was obtained. These proteins had identical N-termini consistent with the deduced amino acid sequence and therefore, might be correctly processed from the fusion protein in E. coli by the protease encoded by the pol region. The purified reverse transcriptase was enzymatically as active as the enzyme purified from the virus particles, and immunoreactive to the sera of HIV carriers with high sensitivity and specificity.
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PMID:Overproduction of human immunodeficiency virus type I reverse transcriptase in Escherichia coli and purification of the enzyme. 169 13

The capsid protein and the reverse transcriptase of cauliflower mosaic virus (CaMV) are encoded by two genes (ORF IV and ORF V) that lie in different translation reading frames. A comparison can be drawn between the synthesis of both CaMV proteins and the fusion protein in a yeast retrotransposon, Ty, resulting from a +1 frameshifting event which fuses two out-of-phase ORFs encoding the structural protein and the reverse transcriptase of Ty. For this reason, we constructed a yeast expression vector containing CaMV ORF VII fused to CaMV ORF III by a fragment of 452 bp including the overlapping region of ORF IV and ORF V, ORF VII and ORF III being used as reporter genes. We characterized two proteins (22 and 50 kDa) synthesized from this plasmid in the yeast expression system. We demonstrated that the 50-kDa polypeptide is not synthesized from a +1 frameshifting event but is probably a dimeric form of the 22-kDa protein. From this result we conclude that the CaMV reverse transcriptase is not produced by a mechanism of ribosomal frameshifting.
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PMID:The cauliflower mosaic virus reverse transcriptase is not produced by the mechanism of ribosomal frameshifting in Saccharomyces cerevisiae. 170 75

The retroviruses of the avian sarcoma-leukosis virus group synthesize their viral protease (PR) in two precursor forms--as a carboxy-terminal domain of the Gag precursor and as an embedded domain within the Gag-Pol precursor. We have shown previously that the Gag-derived PR is fully capable of processing the Gag precursor in the absence of the embedded PR (R.P. Bennett, S. Rhee, R.C. Craven, E. Hunter, and J.W. Wills, J. Virol. 65:272-280, 1991). In this study, we examined the question of whether or not the PR domain of Gag-Pol has an essential role in the maturation of the Pol proteins. The Gag-Pol precursor was expressed in the absence of Gag by use of a simian virus 40-based vector in which the gag and pol reading frames were fused. The fusion protein accumulated to high levels in transfected cells without being released into the medium but could be rescued into particles by coexpression of the Gag protein from a second vector. The resulting particles contained mature Gag and Pol proteins and active reverse transcriptase (RT). Using this complementation system, the effects of PR defects in the Gag and/or Gag-Pol proteins on the activation of RT were examined. The results showed that the presence of a functional PR on the Gag precursor, but not on Gag-Pol, was required for full activation of RT. The embedded PR of Gag-Pol was unable to carry out any detectable processing of the Gag precursor and was able to activate RT to only a low level in the absence of a functional Gag PR domain. Finally, some point mutations in the Gag-Pol PR domain inhibited activation of RT in trans by a wild-type PR, suggesting that the correct conformation of the PR domain in Gag-Pol is prerequisite for activation of RT.
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PMID:Role of the avian retroviral protease in the activation of reverse transcriptase during virion assembly. 171 18


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