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)

In HeLa cells, RNA polymerase I (Pol I)-mediated transcription is severely inhibited soon after infection with poliovirus. We have developed a gel retardation assay to analyze DNA-protein complexes formed at the Pol I promoter. We show here that two complexes (A and C) formed by nuclear extracts from uninfected cells disappear after infection of cells with poliovirus. In contrast, a new, rapidly migrating complex (D) is formed in virus-infected cell extract. This change in the mobility of gel-retarded complexes correlates well with the kinetics of inhibition of rRNA transcription in virus-infected cells. Incubation of nuclear extracts from mock-infected cells with bacterially expressed, purified poliovirus protease 3C results in the disappearance of complexes A and C with concomitant generation of complex D. A partially purified transcription factor fraction derived from uninfected cells that contains complex A is able to restore Pol I transcription when added to virus-infected cell extracts, suggesting that this complex plays an important role in Pol I transcription. These results suggest that poliovirus proteinase 3C may have an important role in the shutoff of Pol I transcription in cells infected with poliovirus.
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PMID:Infection of HeLa cells with poliovirus results in modification of a complex that binds to the rRNA promoter. 131 18

A mutagenic oligonucleotide cassette was used to introduce single and tandem amino acid substitutions into the proteinase 3C coding region of an infectious poliovirus type 1 cDNA. The sites targeted for mutagenesis, residues 60, 61, and 66, are located within a putative helical loop structure which may be involved in substrate recognition by the enzyme. Fourteen viable 3C proteinase mutants were isolated. A Lys----Arg substitution at position 60 resulted in cold sensitivity for growth at 33 degrees. Replacement of Lys 60 with Ile, either singly or in combination with substitutions at position 61, resulted in viruses that produced three- to fivefold more 3D RNA polymerase than wild-type poliovirus. 3C-mediated processing of the remaining sites within the polyprotein was not noticeably affected. The overproduction of 3D is a consequence of more efficient processing of the carboxy-terminal Gln-Gly amino acid pair of 3C. Together with a previous report in which substitution of Val 54 with an Ala residue results in a poliovirus that produces decreased levels of 3D, these observations provide evidence that the putative loop region (residues 51-66) may be a functional domain involved in recognition of the carboxy-terminal Gln-Gly cleavage site of 3C.
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PMID:A genetic locus in mutant poliovirus genomes involved in overproduction of RNA polymerase and 3C proteinase. 215 85

All picornaviral genes are expressed as a single, large polyprotein, which is proteolytically processed into the system produces functional proteins, including viral protease 3C, which plays a major role in processing the precursor proteins. To study the function of the two putative proteases 3C and leader (L) in processing, we constructed several cDNA plasmids encoding various regions of the FMDV type A12 genome. These plasmids, containing FMDV cDNA segments under the control of the T7 promoter, were transcribed in vitro by using T7 RNA polymerase and then translated in rabbit reticulocyte lysates. The expressed FMDV gene products were identified by immunoprecipitation with specific antisera and analyzed by gel electrophoresis. The results demonstrate the following: (i) the leader protein, L, is processed from the structural protein precursor, P1, in the absence of any P2 or P3 region proteins; (ii) protein 2A remains associated with the structural protein precursor, P1, rather than the precursor, P2; (iii) the processing of the P1-2A/P2 junction is not catalyzed by 3C or L; (iv) the proteolytic processing of polyproteins from the structural P1 region (except VP4/VP2) and the nonstructural P2 and P3 region is catalyzed by 3C.
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PMID:Proteolytic processing of foot-and-mouth disease virus polyproteins expressed in a cell-free system from clone-derived transcripts. 304 Oct 41

Comparisons of the RNA polymerase and capsid sequences of small round structured viruses (SRSVs) have recently shown these are genetically diverse viruses which fall into two distinct groups. The genomes of two group I viruses, Southampton and Norwalk viruses have been characterized; however, similar data for the genetic group II SRSVs have not been available until now. We report here the complete genome sequence of a recent group II SRSV, Lordsdale virus. The Lordsdale virus genome is 7555 nt in length and has a similar organization to the group I SRSVs. The large ORF in the 5' half of the genome (5100 nt) is shorter than the group I SRSV ORF1 (5367 nt), but has the characteristic 2C helicase, 3C protease and 3D RNA polymerase enzyme motifs. ORF2, encoding the structural protein is of a similar size to the group I viruses but the small 3'-terminal ORF is significantly larger in group II. A highly conserved sequence of 28 nt was identified at the start of Lordsdale virus ORF1 and repeated at the start of ORF2. These conserved motifs are typical of the animal caliciviruses. Comparison of the 150 N-terminal amino acids in the ORF1 protein revealed little identity between the two SRSV genetic groups, reflecting the shorter ORF1 in the group II virus. Recombinant baculoviruses containing ORF2 and ORF3 sequences were constructed and used to express large quantities of the group II Lordsdale virus structural protein. The capsid protein formed virus-like particles by self assembly which resembled 'empty' SRSVs.
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PMID:Human enteric Caliciviridae: the complete genome sequence and expression of virus-like particles from a genetic group II small round structured virus. 756 76

The RNA polymerase gene of human coronavirus (HCV) 229E encodes a large polyprotein that contains domains with motifs characteristic of both papain-like cysteine proteinases and proteinases with homology to the 3C proteinase of picornaviruses. In this study, we have, first, expressed the putative HCV 229E 3C-like proteinase domain as part of a beta-galactosidase fusion protein in Escherichia coli and have shown that the expressed protein has proteolytic activity. The substitution of one amino acid within the predicted proteinase domain (His-3006-->Asp-3006) abolishes, or at least significantly reduces, this activity. Amino-terminal sequence analysis of a purified, 34-kDa cleavage product shows that the bacterial fusion protein is cleaved at the dipeptide Gln-2965-Ala-2966, which is the predicted amino-terminal end of the putative 3C-like proteinase domain. Second, we have confirmed the proteolytic activity of a bacterially expressed polypeptide with the amino acid sequence of the predicted HCV 229E 3C-like proteinase by trans cleavage of an in vitro translated polypeptide encoded within open reading frame 1b of the RNA polymerase gene. Finally, using fusion protein-specific antiserum, we have identified a 34-kDa, 3C-like proteinase polypeptide in HCV 229E-infected MRC-5 cells. This polypeptide can be detected as early as 3 to 5 h postinfection but is present in the infected cell in very low amounts. These data contribute to the characterization of the 3C-like proteinase activity of HCV 229E.
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PMID:Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity. 776 94

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

Host cell RNA polymerase II (Pol II)-mediated transcription is inhibited by poliovirus infection. This inhibition is correlated to a specific decrease in the activity of a chromatographic fraction which contains the transcription factor TFIID. To investigate the mechanism by which poliovirus infection results in a decrease of TFIID activity, we have analyzed a component of TFIID, the TATA-binding protein (TBP). Using Western immunoblot analysis, we show that TBP is cleaved in poliovirus-infected cells at the same time postinfection as when Pol II transcription is inhibited. Further, we show that one of the cleaved forms of TBP can be reproduced in vitro by incubating TBP with cloned, purified poliovirus encoded protease 3C. Protease 3C is a poliovirus-encoded protease that specifically cleaves glutamine-glycine bonds in the viral polyprotein. The cleavage of TBP by protease 3C occurs directly. Finally, incubation of an uninfected cell-derived TBP-containing fraction (TFIID) with protease 3C results in significant inhibition of Pol II-mediated transcription in vitro. These results demonstrate that a cellular transcription factor can be directly cleaved both in vitro and in vivo by a viral protease and suggest a role of the poliovirus proteinase 3C in host cell Pol II-mediated transcription shutoff.
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PMID:Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro. 838 Aug 94

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

The RNA genome of hepatitis A virus (HAV) encodes a giant polyprotein that is putatively cleaved proteolytically into four structural and seven non-structural proteins. So far, most of the proposed non-structural proteins and their respective cleavage sites have not been identified. A vaccinia virus recombinant (vRGORF) containing the complete HAV ORF under the control of the bacteriophage T7 promoter was used to express HAV in recombinant animal cells (BT7-H) that constitutively expressed T7 DNA-dependent RNA polymerase. A HAV-specific 27.5 kDa expression product was identified as peptide 2B. The 27.5 kDa 2B antigen was also found in HAV-infected MRC-5 cells. The N-terminal amino acid residues of the new peptide 2B are Ala-Lys-Ile-Ser-Leu-Phe and polyprotein cleavage between 2A and 2B occurred at amino acids 836-837 (Gln-Ala). Furthermore, heterologous expression in the same system of regions P1-P2 and of the protease 3C (3Cpro) gene, showed that P1-P2 polyprotein is not cleaved autocatalytically but by 3Cpro. Hence, 3Cpro is effective in cleaving the polyprotein 2A-2B junction.
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PMID:Identification of hepatitis A virus non-structural protein 2B and its release by the major virus protease 3C. 862 28

Transcription factor IIIC (TFIIIC) is a general RNA polymerase III transcription factor that binds the B-box internal promotor element of tRNA genes and the complex of TFIIIA with a 5S rRNA gene. TFIIIC then directs the binding of TFIIIB to DNA upstream of the transcription start site. TFIIIB in turn directs RNA polymerase III binding and initiation. Human TFIIIC contains five different subunits. The 243-kDa alpha subunit can be specifically cross-linked to B-box DNA, but its sequence does not reveal a known DNA binding domain. During poliovirus infection, TFIIIC is cleaved and inactivated by the poliovirus-encoded 3C protease (3Cpro). Here we analyzed the cleavage of TFIIIC subunits by 3Cpro in vitro and during poliovirus infection of HeLa cells. Analyses of the DNA binding activities of the resulting subcomplexes indicated that an N-terminal 83-kDa domain of the alpha subunit associates with the beta subunit to generate the TFIIIC DNA binding domain. Cleavage with 3Cpro also generated an approximately 125-kDa C-terminal fragment of the alpha subunit which remained associated with the gamma and epsilon subunits.
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PMID:DNA binding domain and subunit interactions of transcription factor IIIC revealed by dissection with poliovirus 3C protease. 875 15


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