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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An in situ hybridization technique has been optimised for use on paraffin-embedded sections of tissues collected from cattle infected experimentally with foot-and-mouth disease virus type O1BFS. Tissue was collected 5 days after infection by direct contact. In situ hybridization was carried out using an RNA probe corresponding to a region of the 3D gene which codes for the RNA polymerase, and labelled with digoxigenin. Consistent, reproducible signal was detected within the epithelial layers of the palatine tonsil, soft palate and pharyngeal tissue studied. This is the first time that a digoxigenin-based system has been used successfully for FMD virus RNA detection with bovine tissue samples.
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PMID:Optimization of an in situ hybridization technique for the detection of foot-and-mouth disease virus in bovine tissues using the digoxigenin system. 773 Apr 40

The foot-and-mouth disease virus (FMDV) Lb gene was cloned into bacterial expression vectors under the control of a T7 RNA polymerase promoter. The Lb protein was expressed in both an in vitro transcription-translation system and in Escherichia coli. In vitro expression of a construct containing the Lb gene fused to a portion of the VP4 and 3D genes demonstrated cis cleavage activity that could be blocked by the thiol protease inhibitor E-64. Lb expressed in E. coli was purified from the soluble fraction by metal chelation chromatography. Purified Lb had trans cleavage activity at the L/P1 junction and cleaved the p220 component of the cap-binding protein complex.
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PMID:Expression in Escherichia coli and purification of biologically active L proteinase of foot-and-mouth disease virus. 778 15

We have constructed a clone encoding the foot-and-mouth disease virus (FMDV) 3C protease gene (p3C) using the polymerase chain reaction. The construct was engineered to contain initiation and termination codons and cloned into a plasmid under the control of the bacteriophage T7 promoter. The p3C gene was expressed both in an in vitro transcription-translation system and in vivo in an Escherichia coli system containing an inducible T7 RNA polymerase gene. In both systems the expressed products were of the appropriate molecular weight and immunologically reactive with bovine convalescent serum. E. coli-expressed 3C protein was mainly found in the insoluble fraction of cell lysates. The E. coli-expressed protease was assayed in an in vitro system with radiolabeled P1 capsid precursor protein and P2 precursor protein as substrates. E. coli-expressed 3C completely processed the P1 and P2 precursors into mature capsid and nonstructural proteins, respectively. The kinetics of processing of P1 by E. coli-expressed 3C revealed the following order of cleavage: VP3-VP1, VP0-VP3, VP1-2A.
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PMID:Characterization of the foot-and-mouth disease virus 3C protease expressed in Escherichia coli. 821 67

An antibody against the Escherichia coli-expressed RNA polymerase of foot-and-mouth disease virus (FMDV) reacts with the virus in ELISA and radioimmunoprecipitation experiments and with a protein of the disrupted virus particle in an immunoblot analysis. Treatment of the virus with trypsin, which cleaves capsid protein VP1 and a 56-kDa polypeptide present in trace amount in the particles, reduces the level of the reaction in ELISA and radioimmunoprecipitation and eliminates the immunoblot reaction. Electron microscopy showed that only approximately 20% of the virus particles reacted with the anti-polymerase antibody, whereas most reacted with an antibody against the immunodominant G-H loop of the virus. In the presence of ammonium ions, the expressed polymerase degrades the RNA of the virus into molecules sedimenting at approximately 12 S, indicating that it can act as a hydrolytic as well as a polymerizing enzyme. Moreover, the RNA in trypsin-treated virus particles is degraded when incubated at 37 degrees C, suggesting that the cleaved 56-kDa protein still possesses hydrolytic activity. In addition, the anti-polymerase antibody, which inhibits the polymerase activity of the E. coli-expressed protein, also partially inhibits the hydrolytic activity of the previously described endonuclease of the virus particle, suggesting that this enzyme is identical with the polymerase or forms part of it.
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PMID:Foot-and-mouth disease virus particles contain replicase protein 3D. 829 May 91

Equine rhinovirus 1 (ERhV1) is a respiratory pathogen of horses which has an uncertain taxonomic status. We have determined the nucleotide sequence of the ERhV1 genome except for a small region at the 5' end. The predicted polyprotein was encoded by 6741 nucleotides and possessed a typical picornavirus proteolytic cleavage pattern, including a leader polypeptide. The genomic structure and predicted amino acid sequence of ERhV1 were more similar to those of foot-and-mouth disease viruses (FMDVs), the only members of the aphthovirus genus, than to those of other picornaviruses. Features which were most similar to FMDV included a 16-amino acid 2A protein which was 87.5% identical in sequence of FMDV 2A, a leader (L) protein similar in size to FMDV Lab and the possibility of a truncated L protein similar in size to FMDV Lb, and a 3C protease which recognizes different cleavage sites. However, unlike FMDV, ERhV1 had only one copy of the 3B (VPg) polypeptide. The phylogenetic relationships of the ERhV1 sequence and nucleotide sequences of representative species of the five genera of the family Picornaviridae were examined. Nucleotide sequences coding for the complete polyprotein, the RNA polymerase, and VP1 were analyzed separately. The phylogenetic trees confirmed that ERhV1 was more closely related to FMDV than to other picornaviruses and suggested that ERhV1 may be a member, albeit very distant, of the aphthovirus genus.
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PMID:Equine rhinovirus 1 is more closely related to foot-and-mouth disease virus than to other picornaviruses. 857 74

cDNA cassettes encoding the foot-and-mouth disease virus (FMDV) structural protein precursor (P1-2A) together with the 3C protease, which cleave this molecule to 1AB, 1C and 1D, were constructed. These cassettes were introduced into vaccinia virus (VV) transfer vectors. Attempts to isolate recombinant VVs constitutively expressing these cassettes were unsuccessful. However, when the P1-2A-3C cassette was placed under the control of the bacteriophage T7 promoter, stable VV/FMDV recombinants were isolated. Co-infection with recombinant VV vTF7-3 (which expresses T7 RNA polymerase) led to the production of correctly processed FMDV capsid proteins. Analysis by sucrose gradient centrifugation showed that material which co-sedimented with natural empty capsid particles (70S) was formed. Electron microscopy revealed empty capsid-like particles with diameters of about 30 nm. Studies using monoclonal antibodies specific for conformational epitopes indicated that the antigenicity of the synthetic particles was similar to whole virions and natural empty capsid particles. Surprisingly, merely the modification of a single amino acid residue within the myristoylation consensus sequence at the N terminus of P1-2A allowed the isolation of a recombinant VV which constitutively expressed the correctly processed proteins. However, the capsid proteins expressed from this mutant cassette failed to assemble into 70S empty particles.
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PMID:Assembly of foot-and-mouth disease virus empty capsids synthesized by a vaccinia virus expression system. 884 14

Mutant forms of the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) have been produced and shown to be severely defective in directing internal initiation of protein synthesis within cells using the vaccinia/T7 RNA polymerase system. Mutants in different regions of the IRES were complemented in trans by coexpression of the intact EMCV IRES but not by coexpression of the related IRES elements from Theiler's murine encephalomyelitis virus (another cardiovirus) or from foot-and-mouth disease virus. Distinct, truncated regions of the EMCV IRES, insufficient to direct internal initiation, were also shown to complement defective EMCV IRES elements. It was necessary for the complementing molecule, whether truncated or full length, to be expressed in the positive sense orientation. RT-PCR analysis provided no support for the idea that any recombination event was responsible for the complementation. The data suggest that multiple activities are performed by distinct functional entities within the IRES in the process of internal initiation of protein synthesis. At least some of these different functions may be achieved by different molecules acting in trans.
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PMID:Complementation of defective picornavirus internal ribosome entry site (IRES) elements by the coexpression of fragments of the IRES. 900 58

Nonstructural proteins 2C, 3CD, 3C, and 3D, and the cellular protein actin, are present in highly purified preparations of foot-and-mouth disease virus (FMDV) and poliovirus. They remain bound in variable amounts to the RNAs when the RNAs are extracted from the viruses with phenol or phenol-sodium dodecyl sulfate (SDS) and, for FMDV, when the RNA is released from the particles by a lowering of the pH below 7. RNA prepared by these methods is rapidly degraded at 37 degrees C, particularly in the presence of NH4+ ions, but hydrolysis can be prevented by antibody against Escherichia coli-expressed 3D, indicating that it is the RNA polymerase that has nuclease activity. In contrast, virion RNA from which the nonstructural proteins and actin have been removed by extraction with guanidine thiocyanate-phenol-chloroform or proteinase K-phenol is stable at 37 degrees C, although its specific infectivity is lower than that of the RNA extracted with phenol or phenol-SDS. The possible implications of the close association of replication complex proteins with the RNA in virus particles are discussed.
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PMID:Foot-and-mouth disease virus and poliovirus particles contain proteins of the replication complex. 931 48

Removal of virus infectivity from blood and biopharmaceutical products prepared from blood is an issue of considerable importance. For biopharmaceutical products, removal can usually be achieved by a series of fractionation steps or by inactivation with a suitable reagent. Irrespective of the methods that are chosen it is vital that the biological activity of the product is not impaired. For blood and unfractionated plasma or serum, the problem is even more challenging. Selective inactivation of the genome is the key step in the preparation of killed virus vaccines. Viruses belonging to all the recognised families can be inactivated by imines. In this paper it is shown that the biological properties of several proteins, including the cell growth-promoting factors in calf serum, are not impaired using conditions which ensure the inactivation of > 10(15) infectious units of poliovirus and foot-and-mouth disease virus (FMDV). Also shown is that both viruses can be inactivated by imines at 4 degrees C, thus providing a method for removing infectivity from protein preparations which are unstable at higher temperatures. The RNA extracted from FMDV inactivated at 4 degrees C was not degraded and contained no hidden breaks but nevertheless was non-infectious. However, it could be amplified by PCR using primers corresponding to the gene coding for a portion of the viral RNA polymerase, but not from that coding for VP1, one of the structural proteins, showing that alteration of a base or bases had occurred in that region. Surprisingly, it could be translated in the rabbit reticulocyte system although some of the products were different from those obtained with unmodified RNA.
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PMID:A universal virus inactivant for decontaminating blood and biopharmaceutical products. 963 48

Removal of virus infectivity from blood and biopharmaceutical products prepared from blood is an issue of considerable importance. Irrespective of the methods that are chosen it is vital that the biological activity of the product is not impaired. For blood and unfractionated plasma or serum, the problem is even more challenging. Selective inactivation of the genome is the key step in the preparation of killed virus vaccines. Imines have been used for more than 30 years for the preparation of inactivated foot-and-mouth disease virus vaccines without any evidence of survival of virus infectivity. Moreover, the immunogenicity of the virus is unimpaired. Viruses belonging to all the recognised families can be inactivated by imines. The biological properties of several proteins, including the cell growth-promoting factors in calf serum, are not impaired using conditions which ensure the inactivation of > 10(15) infectious units of poliovirus and foot-and-mouth disease virus (FMDV). Moreover, both viruses can be inactivated by imines at 4 degrees C, thus providing a method for removing infectivity from protein preparations which are unstable at higher temperatures. The mechanism by which FMDV is inactivated has been studied. We found that the RNA extracted from the virus after inactivation at 4 degrees C was not degraded and contained no hidden breaks but nevertheless was non-infectious. However, it could be amplified by PCR using primers corresponding to the gene coding for a portion of the viral RNA polymerase, but not from that coding for VP1, one of the structural proteins, showing that alteration of a base or bases had occurred in that region.
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PMID:A universal virus inactivant for decontaminating blood and biopharmaceutical products. 1040 83


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