Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Double-stranded RNA viruses have an RNA-dependent RNA polymerase activity associated with the viral particles which is indispensable for their replication cycle. Using the yeast L-A double-stranded RNA virus we have investigated the mechanism by which the virus encapsidates its genomic RNA and RNA polymerase. The L-A gag gene encodes the principal viral coat protein and the overlapping pol gene is expressed as a gag-pol fusion protein which is formed by a -1 ribosomal frameshift. Here we show that Gag alone is sufficient for virus particle formation, but that it fails to package the viral single-stranded RNA genome. Encapsidation of the viral RNA requires only a part of the Pol region (the N-terminal quarter), which is presumably distinct from the RNA polymerase domain. Given that the Pol region has single-stranded RNA-binding activity, these results are consistent with our L-A virus encapsidation model: the Pol region of the fusion protein binds specifically to the viral genome (+) strand, and the N-terminal gag-encoded region primes polymerization of Gag to form the capsid, thus ensuring the packaging of both the viral genome and the RNA polymerase.
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PMID:Pol of gag-pol fusion protein required for encapsidation of viral RNA of yeast L-A virus. 143 38

The plus strand of the L-A double-stranded RNA virus of Saccharomyces cerevisiae has two large open reading frames, ORF1, which encodes the major coat protein, and ORF2, which encodes a single-stranded RNA-binding protein having a sequence diagnostic of viral RNA-dependent RNA polymerases. ORF2 is expressed only as a Gag-Pol-type fusion protein with ORF1. We have constructed a plasmid which expresses these proteins from the yeast PGK1 promoter. We show that this plasmid can support the replication of the killer toxin-encoding M1 satellite virus in the absence of an L-A double-stranded RNA helper virus itself. This requires ORF2 expression, providing a potential in vivo assay for the RNA polymerase and single-stranded RNA-binding activities of the fusion protein determined by ORF2. ORF1 expression, like a host ski- mutation, can suppress the usual requirement of M1 for the MAK11, MAK18, and MAK27 genes and allow a defective L-A (L-A-E) to support M1 replication. These results suggest that expression of ORF1 from the vector makes the cell a ski- phenocopy. Indeed, expression of ORF1 in a wild-type killer makes it a superkiller, suggesting that a target of the SKI antiviral system may be the major coat protein.
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PMID:Expression of yeast L-A double-stranded RNA virus proteins produces derepressed replication: a ski- phenocopy. 198 95

We have used the vaccinia virus-T7 RNA polymerase-based expression system for studies on the activity of proteases from various retroviruses on homologous and heterologous Gag polyproteins in eukaryotic cells. Proteases from human immunodeficiency virus (HIV) types 1 and 2, equine infectious anaemia virus, human T cell leukaemia virus type 1 and human spumavirus were produced and were shown to cleave their cognate Gag substrates produced in trans. Analysis of cross reactivity revealed that lentivirus proteases cleaved only lentivirus Gag proteins and oncovirus proteases acted primarily on oncovirus Gag proteins. The HIV-2 protease cleaved the HIV-1 Gag precursor almost as efficiently as HIV-1 protease. Expression of the 5' end of the human spumavirus pol gene revealed that it encodes a functional protease that acts specifically on the human spumavirus Gag polyprotein. This assay will allow further investigation on the activity and specificity of retrovirus proteases in eukaryotic cells.
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PMID:Analysis of cross reactivity of retrovirus proteases using a vaccinia virus-T7 RNA polymerase-based expression system. 756 54

The Pol region of the Gag-Pol fusion protein of the L-A double-stranded (ds) RNA virus of Saccharomyces cerevisiae has (i) a domain essential for packaging viral positive strands, (ii) consensus amino acid sequence patterns typical of RNA-dependent RNA polymerases, and (iii) two single-stranded RNA binding domains. We describe here a third single-stranded RNA binding domain (Pol residues 374 to 432), which is unique in being cryptic. Its activity is revealed only after deletion of an inhibitory region C terminal to the binding domain itself. This cryptic RNA binding domain is necessary for propagation of M1 satellite dsRNA, but it is not necessary for viral particle assembly or for packaging of viral positive-strand single-stranded RNA. The cryptic RNA binding domain includes a sequence pattern common among positive-strand single-stranded RNA and dsRNA viral RNA-dependent RNA polymerases, suggesting that it has a role in RNA polymerase activity.
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PMID:A cryptic RNA-binding domain in the Pol region of the L-A double-stranded RNA virus Gag-Pol fusion protein. 805 76

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.
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PMID:Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein. 808 57

Packaging cell lines are important tools for transferring genes into eukaryotic cells. Human immunodeficiency virus type 1 (HIV-1)-based packaging cell lines are difficult to obtain, in part owing to the problem that some HIV-1 proteins are cytotoxic in a variety of cells. To overcome this, we have developed an HIV-1-based packaging cell line which has an inducible expression system. The tetracycline-inducible expression system was utilized to control the expression of the Rev regulatory protein, which in turn controls the expression of the late proteins including Gag, Pol, and Env. Western blotting (immunoblotting) demonstrated that the expression of p24gag and gp120env from the packaging cells peaked on days 6 and 7 postinduction. Reverse transcriptase activity could be detected by day 4 after induction and also peaked on days 6 and 7. Defective vector virus could be propagated, yielding titers as high as 7 x 10(3) CFU/ml, while replication-competent virus was not detectable at any time. Thus, the cell line should enable the transfer of specific genes into CD4+ cells and should be a useful tool for studying the biology of HIV-1. We have also established an inducible HIV-1 Env-expressing cell line which could be used to propagate HIV-1 vectors that require only Env in trans. The env-minus vector virus titer produced from the Env-expressing cells reached 2 x 10(4) CFU/ml. The inducible HIV-1 Env-expressing cell line should be a useful tool for the study of HIV-1 Env as well.
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PMID:Inducible human immunodeficiency virus type 1 packaging cell lines. 867 79

We report the generation of recombinant vaccinia viruses (VVs) expressing the gag, pol, bel-1, and bet open reading frames of human foamy virus (HFV), and the establishment of a transient, VV-T7 RNA polymerase-directed expression system for the HFV env gene. The correct expression of the HFV proteins was demonstrated by radioimmunoprecipitation using monospecific rabbit antisera, by analysis of the subcellular distribution (for VVgag, VVpol, VVbel-1, and VVbet), and by the ability to induce syncytium formation (for the env expression system). The HFV pol gene was successfully expressed using its own ATG start codon. Foamy viruses are regarded as retroviruses with intracytoplasmatic capsid assembly. However, when VVgag and VVpol were used to study the HFV Gag-Pol protein interaction and particle formation, no HFV capsid structures were observed in singly or doubly infected cells. In addition, no cleavage of the Pr74gag precursor molecule by the pol-encoded protease was detected in doubly infected cells. Our results indicate that foamy virus particle assembly is fundamentally different from that of other retroviruses.
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PMID:Characterization of human foamy virus proteins expressed by recombinant vaccinia viruses. 910 Sep 94

A human cell line constitutively expressing the HIV-1 gag and pol genes products was established. The cell line was established by stably transfecting 293 cells with a plasmid construct that expresses the HIV Gag and Pol and can confer the transfectants resistant to mycophenolic acid. Particles generated from transient expression of the plasmid construct were noninfectious when pseudotyped with HIV envelope or with amphotropic murine leukemia virus envelope proteins. However, virus-like Gag particles produced by the stable cell line were appropriately processed, exhibited a wild-type retrovirus particle density, and possessed significant reverse-transcriptase (RT) activities. Continuous passage of the cell line either in the presence or absence of mycophenolic acid had no major effects on the Gag processing efficiency, particle assembly, or RT activity release. It was also demonstrated that the proteolytic processing of the virus-like particles released from the cell line was inhibited by an HIV protease inhibitor, saquinavir. The establishment of a stable cell line producing noninfectious but proteolytically processed HIV Gag particles offers a safe, convenient tool for biochemical and immunological analysis of virus-like particle assembly and is very useful for the development of anti-HIV protease drugs.
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PMID:A human cell line constitutively expressing HIV-1 Gag and Gag-Pol gene products. 989 Apr 17

Tat-mediated trans-activation of the HIV-1 long terminal repeat (LTR) occurs through the phosphorylation of the carboxy-terminal domain of the RNA polymerase II. The kinase complex, pTEFb, composed of cyclin T1 (CycT1) and CDK9, mediates this process. The trans-activation response (TAR) RNA-binding protein 2 (TRBP2) increases HIV-1 LTR expression through TAR and protein kinase R (PKR) binding, but not through interactions with the Tat-CycT1-CDK9 complex. TRBP2 and the Tat-CycT1-CDK9 complex have overlapping binding sites on TAR RNA. TRBP2 and CycT1 increased Tat trans-activation in NIH 3T3 cells with additive effects. Upon transfection of HIV-1 pLAI, pNL4-3, pMAL, and pAD molecular clones, reverse transcriptase (RT) activity and p24 concentration were decreased 200- to 900-fold in NIH 3T3 cells compared with HeLa cells in both cells and supernatants. In murine cells, cotransfection of the HIV clones with CycT1 or TRBP2 increased modestly the expression of RT activity in cell extracts. The analysis of Gag expression in murine cells transfected with CycT1 compared with human cells showed a 20-fold decrease in expression and a strong processing defect. The expression of both CycT1 and TRBP2 had a more than additive activity on RT function in cell extracts and on viral particle production in supernatant of murine cells. These results suggest an activity of CycT1 and TRBP2 at different steps in HIV-1 expression and indicate the requirement for another posttranscriptional factor in murine cells for full HIV replication.
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PMID:Additive activity between the trans-activation response RNA-binding protein, TRBP2, and cyclin T1 on HIV type 1 expression and viral production in murine cells. 1458 7

The major human tRNALys isoacceptors, tRNALys1,2 and tRNALys3, are selectively packaged into HIV-1 during assembly, where tRNALys3 acts as the primer for initiating reverse transcription. In this report, we shall review the evidence that supports a model for the formation of a tRNALys packaging complex, whose components include the precursor proteins Gag and Gag-Pol, viral genomic RNA, tRNALys, and lysyl-tRNA synthetase (LysRS). In the model proposed, the tRNALys packaging complex is formed when a Gag/Gag-Pol/viral RNA complex interacts with a tRNALys/LysRS complex, with Gag interacting with LysRS, and Gag-Pol interacting with tRNALys. The incorporation of Gag-Pol into HIV-1 requires its interaction with Gag multimers whose polymerization is promoted by RNA. Reverse transcriptase sequences within Gag-Pol also bind to tRNALys, and this binding is required for tRNALys packaging into viruses. LysRS, the enzyme that aminoacylates tRNALys, is also incorporated into HIV-1, and this protein is a strong candidate for being the signal that specifically targets tRNALys for viral incorporation. Newly-synthesized LysRS is a main source of viral LysRS, and its incorporation into viruses occurs via its interaction with Gag and independently of tRNALys packaging. While tRNALys incorporation into viruses depends upon its interaction with LysRS, tRNALys aminoacylation is not a requirement for viral packaging.
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PMID:The tRNALys packaging complex in HIV-1. 1518 44


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