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 normal reverse transcription of retroviral RNA is a complex process which depends on the orchestration of several steps throughout the virus life cycle. During the assembly of retroviruses, reverse transcriptase (RT) is directed into the virion as a component of the Gag-Pol polyprotein. In the maturation of the Gag-Pol polyprotein of human immunodeficiency virus type 1 (HIV-1), cleavage by the viral protease occurs during viral budding. After infection, reverse transcription of viral RNA into double-stranded DNA is completed in the cytoplasm of the infected cell. In this study, the processing and reverse transcription of HIV-1 have been examined by separate expression of mature HIV-1 RT and proviral molecules bearing RT mutations. The effects of RT expression in trans during virion release and after viral entry were investigated. Constitutive expression of HIV-1 RT was established in CD4- and non-CD4-expressing cells via the coexpression of its individual subunits, and three HIV-1 RT mutant constructs were generated. The results indicate that a bona fide RT trans complementation does not occur during virion release or after infection. However, after infection of an RT-expressing cell with a high titer RT-defective virus, intracellular reverse transcription can be detected.
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PMID:Expression of human immunodeficiency virus type 1 reverse transcriptase in trans during virion release and after infection. 864 23

We have found that isoguanine (iG) can pair with thymine (iG.T) and the non-natural base, 5-methylisocytosine (iG.iCM) during template directed synthesis catalyzed by AMV reverse transcriptase. The ratio of these pairings is about 1:10, irrespectively which of the templates, poly(C,iG) or poly(I,iG) is used. This ratio corresponds to the ratio of 2-OH and 2-keto tautomers in monomer in aqueous solution and apparently it is not influenced by the template context. Our results indicate also that formation of the reverse transcriptase catalyzed base pairs between iG and A, G or C can occur only at a low frequency, comparable to the frequency, of mismatches of.(ABSTRACT TRUNCATED)
Acta Biochim Pol 1996
PMID:Miscoding properties of isoguanine (2-oxoadenine) studied in an AMV reverse transcriptase in vitro system. 879 Jul 29

The Gag-Pol polyprotein of human immunodeficiency virus type 1 is not required for efficient viral particle assembly or release. However, in this report we demonstrate that the synthesis of a truncated Gag-Pol precursor due to a premature termination codon in pol can reduce the ability of a full-length provirus to direct the formation of viral particles. Marked effects on particle production were seen when premature termination codons were introduced into the integrase (IN)-coding region. By contrast, a mutant which lacked both IN and reverse transcriptase (RT) formed particles with normal efficiency. Particle production by IN mutants was restored to wild-type levels when a second premature termination codon was introduced at the 5' end of the RT-coding sequence. Particle formation was similarly restored by a second site mutation in the viral protease (PR) gene which prevented proteolytic processing of the Gag polyprotein. Finally particle formation was restored in the presence of A77003, a specific inhibitor of human immunodeficiency virus type 1 PR. These results suggest that the effects of a lack of IN sequences on particle formation require the synthesis of a Gag-Pol precursor which contains RT sequences and are due to inappropriate PR activity.
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PMID:Lack of integrase can markedly affect human immunodeficiency virus type 1 particle production in the presence of an active viral protease. 879 22

Simian and human foamy viruses (SFV and HFV) encode a transcriptional transactivator, Tas, which governs the levels of viral transcripts initiated by both the promoter in the long terminal repeat (LTR) and the internal promoter (IP) located within the env gene of these viruses. Tas-responsive target elements,(TRE) LTR in the LTR and (TRE) IP in the env gene, are located 5' of the TATA box in both viral promoters and function as orientation- and position-independent enhancers. We have identified a strong Tas-responsive element, designated TRE (GP), near the 3' end of the gag gene and preceding the pol gene of SFV-1. In transient-expression assays with plasmids containing reporter genes, a 59-bp DNA fragment containing TRE (GP) (nucleotides 2224 to 2282) functioned as an enhancer element, dependent on Tas, in several cell types and in the context of a heterologous basal promoter. DNase footprinting revealed that the fusion protein glutathione S-transferase-Tas, purified from genetically engineered bacteria, interacts with about 40 hp (nucleotides 2237 to 2279) in the TRE (GP). A low degree of sequence homology was noted between TRE (GP) and TRE (IP). In virus-infected cells, novel transcripts with 5' ends immediately upstream from the reverse transcriptase translation frame (nucleotides 2611 to 5778) were identified. Upstream of the start site for these transcripts is a TATA box (nucleotides 2575 to 2579), which was required for transcription in transient-expression assays. Although a spliced mRNA initiated in the viral LTR is implicated in the synthesis of the HFV Pol polyprotein which encodes protease, reverse transcriptase, and integrase, it is possible that SFV-1 contains a promoter within the pol gene for initiating a reverse transcriptase transcript. Taken together, these studies define a novel Tas-responsive enhancer element, which binds the viral transactivator, and a potential promoter within the pol gene.
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PMID:The simian foamy virus type 1 transcriptional transactivator (Tas) binds and activates an enhancer element in the gag gene. 879 26

In human foamy virus (HFV) the reverse transcriptase is expressed independently of the Gag protein as a 127-kDa Pol precursor molecule. Evaluating the mechanism of Pol expression we identified a spliced mRNA which uses the main 5' splice donor and a splice acceptor site located in the gag gene. The significance of this spliced transcript for HFV Pol expression was studied by constructing a virus with a mutated splice acceptor site. This virus was unable to express detectable Pol proteins after transient transfection. Replication of the mutant was studied by a sensitive assay based on HFV transactivator-stimulated expression of an integrated lacZ gene under control of the HFV long terminal repeat. Whereas in the first 2 weeks after transfection the mutant replicated 3 to 5 order of magnitude less well than wild-type virus, extracellular titers obtained thereafter were similar to those of wild-type virus. This increase in replication competence was accompanied by a reversion of the mutated splice acceptor site. The results underlined the importance of the spliced pol transcript for HFV replication and pointed to a second mechanism of Pol expression. Indicator gene assays suggest that this other mechanism is likely to be a transactivator-dependent cryptic promoter in the gag gene which gives rise to Pol-encoding transcripts.
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PMID:Expression of human foamy virus reverse transcriptase involves a spliced pol mRNA. 886 27

We have determined the crystal structure of dUTP pyrophosphatase (dUTPase) from feline immunodeficiency virus (FIV) at 1.9 A resolution. The structure has been solved by the multiple isomorphous replacement (MIR) method using a P6(3) crystal form. The results show that the enzyme is a trimer of 14.3 kDa subunits with marked structural similarity to E. coli dUTPase. In both enzymes the C-terminal strand of an anti-parallel beta-barrel participates in the beta-sheet of an adjacent subunit to form an interdigitated, biologically functional trimer. In the P6(3) crystal form one trimer packs on the 6(3) screw-axis and another on the threefold axis so that there are two independent monomers per asymmetric unit. A Mg2+ ion is coordinated by three asparate residues on the threefold axis of each trimer. Alignment of 17 viral, prokaryotic, and eukaryotic dUTPase sequences reveals five conserved motifs. Four of these map onto the interface between pairs of subunits, defining a putative active site region; the fifth resides in the C-terminal 16 residues, which is disordered in the crystals. Conserved motifs from all three subunits are required to create a given active site. With respect to viral protein expression, it is particularly interesting that the gene for dUTPase (DU) resides in the middle of the Pol gene, the enzyme cassette of the retroviral genome. Other enzymes encoded in the Pol polyprotein, including protease (PR), reverse transcriptase (RT), and most likely integrase (IN), are dimeric enzymes, which implies that the stoichiometry of expression of active trimeric dUTPase is distinct from the other Pol-encoded enzymes. Additionally, due to structural constraints, it is unlikely that dUTPase can attain an active form prior to cleavage from the polyprotein.
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PMID:Crystal structure of dUTP pyrophosphatase from feline immunodeficiency virus. 897 51

Our group demonstrated recently that the 3' ends of several families of tRNA-derived SINEs (short interspersed repetitive elements) originated from the 3' ends of LINEs (long interspersed repetitive elements) [Ohshima et al. (1996) Mol. Cell. Biol. 16:3756-3764]. Two fully characterized examples of such organization were provided by the tortoise Pol III/SINE and the salmonid HpaI family of SINEs, and two probable examples were provided by the tobacco TS family of SINEs and the salmon SmaI family of SINEs. This organization of SINEs can explain their potential to retropose in the genome since it appears reasonable that the sites for recognition of LINEs by reverse transcriptase should be located within the 3'-end sequences of LINEs. We now add another example to this category of SINEs. In the bovine genome, there are Bov-tA SINEs, which belong to the superfamily of tRNA-derived families of SINEs, and Bov-B LINEs, which were recently demonstrated to belong to a LINE family. Moreover, Bov-tA and Bov-B share the same 3'-end tail. We propose a possible scenario whereby the composite structure of the bovine Bov-tA family of SINEs might have been generated from the Bov-B family of LINEs during evolution.
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PMID:The 3' ends of tRNA-derived SINEs originated from the 3' ends of LINEs: a new example from the bovine genome. 907 Oct 12

Characterization of human foamy virus (HFV) gag-encoded precursors and the search for a Gag-Pol polyprotein and mature proteins derived from proteolytic processing were carried out in HFV-infected cells and with purified preassembled cores and extracellular virus by Western blotting and radioimmunoprecipitation using antisera against synthetic peptides corresponding to putative Gag and protease proteins. Precursor proteins, Pr78gag/74gag and Pr135pol, were found in the nucleus of epithelial and fibroblast cells 3-4 days after HFV infection. Kinetic analysis of HFV Pr78gag and Pr74gag indicated that Pr78gag is a precursor to Pr74gag. South-Western blot analysis indicated that Pr78gag and Pr74gag have properties associated with nucleic acid binding protein although they lack the typical zinc-finger motifs found in retroviral nucleocapsid proteins. Western blot analyses of preassembled HFV cores isolated from the cytoplasm of infected cells and purified by sucrose gradient centrifugation demonstrated the presence of Pr78gag/74gag and Pr135pol, but no proteolytically processed Gag proteins were observed. The majority of extracellular HFV particles were found to have pentagon-shaped cores, as observed intracellularly, and are believed to be the immature extracellular form of the virus. The highest concentration of extracellular particles, estimated by EM, Western blot, and reverse transcriptase assays were found in sucrose gradient fractions having a density of 1.21-1.24 g/cm3. Western blot analysis revealed that Pr78gag/74gag and Pr135pol were the major viral proteins associated with these extracellular particles, as only small amounts of putative proteolytically cleaved capsid (p32) were observed. Our results support the notion that Pol is translated independent of Gag in HFV-infected cells.
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PMID:Protein composition and morphology of human foamy virus intracellular cores and extracellular particles. 912 38

Retroviral reverse transcription starts with the extension of a cellular tRNA primer bound near the 5' end of the viral genomic RNA at a site called the primer binding site (PBS). Formation of the HIV-1 initiation complex between tRNA3(Lys), viral RNA and reverse transcriptase probably occurs during encapsidation of these components. tRNA3(Lys) is thought to be selectively packaged by interaction with the reverse transcriptase domain of the Pr160Gag-Pol precursor protein, then annealed to the PBS of viral RNA with the help of the nucleocapsid protein. tRNA3(Lys) and HIV-1 viral RNA form a highly-structured complex, with extended interactions between the two molecules. Two different modes of reverse transcription have been distinguished: initiation, a tRNA3(Lys)-specific and distributive mode of polymerization corresponding to the addition of the first five nucleotides, followed by elongation, a non-specific and processive mode of DNA synthesis. These two modes are reminiscent of the initiation and elongation processes previously observed with DNA-dependent RNA polymerases.
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PMID:Structural and functional evidence that initiation and elongation of HIV-1 reverse transcription are distinct processes. 915 Aug 89

These studies were performed to characterize retroviruses found in cell lines spontaneously developed from peripheral blood mononuclear cells (PBMNC) from 6 multiple sclerosis patients, a patient with progressive myelopathy and a healthy control. The cell lines are B-lymphoblastoid and produce Epstein-Barr virus (EBV) particles or express EBV proteins. The B-lymphoblastoid cell lines are also characterized by production of low, fluctuating amounts of retrovirus. The low productivity complicates purification and characterization, but implementation of product-enhanced reverse transcriptase (PERT) assays has provided a highly useful tool for monitoring retrovirus production. By electron microscopy, the retroviral particles appear type-C-like. Functional assays indicate the presence of Pol, Gag and Env. Indirect ELISA demonstrates a significant relation between disease activity and reactivity towards retroviral peptides. Molecular characterization is primarily based on RT-PCR, cloning, sequencing and Northern- or Southern analyses. Molecular characterization is continuing.
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PMID:Characterization of retroviruses from patients with multiple sclerosis. 917 40


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