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)

Rabies virus M protein was expressed in Escherichia coli in the form of a fusion protein with maltose binding protein (MBP) and purified by amylose affinity column chromatography after extraction. In order to investigate the possible regulatory role of M protein in viral transcription, an assay system for rabies virion-associated transcriptase activity was established by using the ribonucleoprotein (RNP) cores prepared from purified virions. Analysis of the products of the transcription assay system showed that the products are sensitive to RNase and are positive-strand RNA. Addition of the fusion protein to the system after cleavage with a proteinase Factor Xa (FXa), which cleaves the fusion protein into the M protein and MBP, resulted in an efficient and dose-dependent inhibition of the transcription. Furthermore, addition to the system of anti-M protein monoclonal antibody significantly restored the transcription. Control experiments with the same transcription assaying system using rabies virus nucleoprotein expressed as a fusion protein with MBP and cleaved with FXa did not result in an inhibition of the transcription. These results suggest that the M protein of rabies virus has the property to down-regulate virion-associated transcription.
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PMID:Rabies virus M protein expressed in Escherichia coli and its regulatory role in virion-associated transcriptase activity. 864 3

A reverse genetics approach was applied to generate a chimeric nonsegmented negative strand RNA virus, rabies virus (RV) of the Rhabdoviridae family, that expresses a foreign protein. DNA constructs containing the entire open reading frame of the bacterial chloramphenicol acetyltransferase (CAT) gene and an upstream RV cistron border sequence were inserted either into the nontranslated pseudogene region of a full-length cDNA copy of the RV genome or exchanged with the pseudogene region. After intracellular T7 RNA polymerase-driven expression of full-length antigenome RNA transcripts and RV nucleoprotein, phosphoprotein and polymerase from transfected plasmids, RVs transcribing novel monocistronic mRNAs and expressing CAT at high levels, were recovered. The chimeric viruses possessed the growth characteristics of standard RV and were genetically stable upon serial cell culture passages. CAT activity was still observed in cell cultures infected with viruses passaged for more than 25 times. Based on the unprecedented stability of the chimeric RNA genomes, which is most likely due to the structure of the rhabdoviral ribonucleoprotein complex, we predict the successful future use of recombinant rhabdovirus vectors for displaying foreign antigens or delivering therapeutic genes.
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PMID:Highly stable expression of a foreign gene from rabies virus vectors. 869 89

The RNA polymerase of rabies virus consists of two subunits, the large (L) protein and the phosphoprotein (P), with 2,127 and 297 amino acids, respectively. When these proteins were coexpressed via the vaccinia virus-T7 RNA polymerase recombinant in mammalian cells, they formed a complex as detected by coimmunoprecipitation. Analysis of P and L deletion mutants was performed to identify the regions of both proteins involved in complex formation. The interaction of P with L was not disrupted by large deletions removing the carboxy-terminal half of the P protein. On the contrary, P proteins containing a deletion in the amino terminus were defective in complex formation with L. Moreover, fusion proteins containing the 19 or the 52 first residues of P in frame with green fluorescent protein (GFP) still bound to L. These results indicate that the major L binding site resides within the 19 first residues of the P protein. We also mapped the region of L involved in the interaction with P. Mutant L proteins consisting of the carboxy-terminal 1,656, 956, 690, and 566 amino acids all bound to the P protein, whereas deletion of 789 residues within the terminal region eliminated binding to P protein. This result demonstrates that the carboxy-terminal domain of L is required for the interaction with P.
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PMID:Mapping the interacting domains between the rabies virus polymerase and phosphoprotein. 949 45

Rabies virus nucleoprotein (N) encapsidates negative-strand genomic RNA in vivo, and this RNA-N complex, together with the nominal viral phosphoprotein (P) and RNA polymerase (L), forms the active cytoplasmic ribonucleoprotein (RNP) complex in virus-infected cells and the RNP core in virus particles. The RNP complex is capable of initiating viral RNA transcription and replication in vivo and in vitro. To obtain insight into the events leading to the formation of the RNA-N complex, we have investigated the interaction between rabies virus N and the positive-strand leader RNA transcript. Binding studies revealed that recombinant N binds preferentially to rabies virus leader RNA and that N binding to leader RNA was 5 to 10 times stronger than to nonleader RNA. Encapsidation of leader RNA by N could be competetively inhibited by unlabeled leader RNA but not by nonleader RNA. Furthermore, N protein encapsidation of nonleader RNA but not the leader RNA was inhibited when P was simultaneously added into the encapsidation reaction, indicating that P helps confer the specificity of leader RNA encapsidation by N. The initiation signal for leader RNA encapsidation by N has been mapped to nucleotides 20-30 of the RNA sequence which is A rich. Studies with N-deletion mutants indicate that the intact N is required to encapsidate RNA, since deletion of amino acid residues from either the N- or the C-terminus of N abolishes the ability of N to encapsidate leader RNA.
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PMID:The specificity of rabies virus RNA encapsidation by nucleoprotein. 950 Oct 55

In rabies virus, the ribonucleoprotein complex (RNP), the RNA genome (-) and the antigenome (+) are specifically coated by the viral nucleoprotein (N protein), forming the template for transcription and replication bythe viral RNA polymerase. This specific encapsidation starts at the 5' ends of the RNAs. To investigate domains of the N protein that govern binding specificity, we tested in vitro the ability of both full-length and truncated forms of the N protein to interact with a synthetic RNA probe corresponding to the 5' end of the antigenome. UV-LASER cross-linking, which covalently links RNA and proteins in intimate contact, showed that the entire N protein (450 aa) and the NH2-terminal 376 aa (t42) contained all of the determinants for specific interaction. It was demonstrated by affinity chromatography that a peptide near the COOH terminus of t42 (position 298352), which is located in the most conserved region of Rhabdoviridae N proteins, bound directly to the viral RNA. However, no significant sequence similarity was detected between this peptide and known RNA binding proteins in the databases. This suggests both that N proteins may possess a new type of RNA binding motif and that protein folding contributes to the architecture of the RNA binding site.
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PMID:Identification of a region of the rabies virus N protein involved in direct binding to the viral RNA. 960 15

To investigate the RNA polymerase of rabies virus, we cloned a cDNA of the catalytic subunit (called L protein because of its large molecular size) of the HEP-Flury strain, an avirulent strain obtained by high frequencies of serial embryonated hen egg passages. Nucleotide sequencing showed that the cDNA encodes a long polypeptide of 2,127 amino acids (Mr. 242,938). A comparison of the deduced amino acid sequence with that of other strains (PV and SAD B19) indicated that the sequence was highly conserved, except for several amino acid substitutions which were accumulated in some limited regions. A fragment of the cDNA was used for expression in Escherichia coli (E. coli) to prepare the L antigen for raising the antibodies in rabbits. Immunoprecipitation studies with the rabbit antiserum showed that the polypeptides produced in the L cDNA-transfected COS-7 cells displayed almost the same electrophoretic mobility as that of authentic L protein. Immunofluorescence studies indicated that both L and P (another subunit of RNA polymerase) proteins displayed colocalized distribution with the nucleocapsid antigen (N) in the cytoplasmic inclusion bodies, where envelope proteins (G and M) were absent. On the other hand, expression of the L protein alone did not cause inclusion body-like granular distribution, suggesting that the inclusion body-like accumulation depends on certain interaction(s) with other viral gene products, probably with the ribonucleoproteins comprising the inclusion bodies.
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PMID:Studies on rabies virus RNA polymerase: 1. cDNA cloning of the catalytic subunit (L protein) of avirulent HEP-flury strain and its expression in animal cells. 971 1

We investigated the relationship between the two forms of rabies virus P protein, a non-catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37- and 40-kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [3H]leucine and [32P]orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37-kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37- and 40-kDa products in P cDNA-transfected animal cells, while only a 37-kDa product was produced in Escherichia coli. Incubation of 37-kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40-kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37-kDa polypeptide, which depends on certain heparin-sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus-induced factor(s).
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PMID:Studies on the rabies virus RNA polymerase: 2. Possible relationships between the two forms of the non-catalytic subunit (P protein). 988 49

Typical defective interfering (DI) RNAs are more successful in the competition for viral polymerase than the parental (helper) virus, which is mostly due to an altered DI promoter composition. Rabies virus (RV) internal deletion RNAs which possess the authentic RV terminal promoters, and which therefore are transcriptionally active and can be used as vectors for foreign gene expression, are poorly propagated in RV-infected cells and do not interfere with RV replication. To allow DI-like amplification and high-level gene expression from such mini-RNA vectors, we have used an engineered 3' copy-back (ambisense) helper RV in which the strong replication promoter of the antigenome was replaced with the 50-fold-weaker genome promoter. In cells coinfected with ambisense helper virus and mini-RNAs encoding chloramphenicol acetyltransferase (CAT) and luciferase, mini-RNAs were amplified to high levels. This was correlated with interference with helper virus replication, finally resulting in a clear predominance of mini-RNAs over helper virus. However, efficient successive passaging of mini-RNAs and high-level reporter gene activity could be achieved without adding exogenous helper virus, revealing a rather moderate degree of interference not precluding substantial HV propagation. Compared to infections with recombinant RV vectors expressing CAT, the availability of abundant mini-RNA templates led to increased levels of CAT mRNA such that CAT activities were augmented up to 250-fold, while virus gene transcription was kept to a minimum. We have also exploited the finding that internal deletion model RNAs behave like DI RNAs and are selectively amplified in the presence of ambisense helper virus to demonstrate for the first time RV-supported rescue of cDNA after transfection of mini-RNA cDNAs in ambisense RV-infected cells expressing T7 RNA polymerase.
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PMID:Virus promoters determine interference by defective RNAs: selective amplification of mini-RNA vectors and rescue from cDNA by a 3' copy-back ambisense rabies virus. 1019 76

The sequence of the RNA genome of bovine ephemeral fever virus (BEFV) was determined from the start of the L (polymerase) gene to the end of the untranslated 5' trailer sequence, completing the sequence of the 14900 nucleotide (nt) genome. The 6470 nt L gene encodes a single long ORF of 2144 amino acids with a deduced molecular weight of 249766 Da. The 70 nt BEFV 5' trailer region displays partial terminal complementarity with the 3' leader sequence and contains a 26 nt direct repeat of the U-rich domain of the 3' leader region. The 47 nt 5' trailer region of Adelaide River virus (ARV) displays terminal sequence similarity to the BEFV trailer and partial terminal complementarity with the ARV 3' leader sequence, but does not contain the direct repeat sequence. The BEFV L protein contains all characteristic sequence motifs of amino acid blocks I-VI, conserved among RNA polymerase proteins of single-stranded (-) RNA viruses, separated by regions of lower homology. Phylogenetic analysis using the complete BEFV L protein sequence indicated a closer relationship to vesicular stomatitis virus than to rabies virus. Sequence comparison of two conserved central domains encompassing blocks II and III and block VI of the BEFV and ARV L proteins indicated they are closely related. An extended phylogenetic analysis using the block III sequence, confirmed the relationship of these ephemeroviruses to vesiculo- and lyssaviruses and to other single-stranded (-) RNA viruses.
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PMID:RNA polymerase (L) gene and genome terminal sequences of ephemeroviruses bovine ephemeral fever virus and Adelaide River virus indicate a close relationship to vesiculoviruses. 1107 28

In order to identify the viral gene related to the pathogenicity of rabies virus, we tried to establish a reverse genetics system of the attenuated RC-HL strain, which causes nonlethal infection in adult mice after intracerebral inoculation. A full-length genome plasmid encoding the complete antigenomic cDNA of the RC-HL strain and helper plasmids containing cDNAs of the complete open reading frame of the N, P, and L genes, respectively, were constructed. After transfection of these plasmids into BHK-21 cells infected with the T7 RNA polymerase-expressing vaccinia virus, infectious rabies virus with almost the same biological properties as those of the wild-type RC-HL strain was rescued. Using this reverse genetics system of the RC-HL strain, we generated a chimeric virus with the open reading frame of the glycoprotein gene from the parent Nishigahara strain, which kills adult mice after intracerebral inoculation, in the background of the RC-HL genome. Since the chimeric virus killed adult mice following intracerebral inoculation, it became evident that the open reading frame of the glycoprotein gene is related to the pathogenicity of the Nishigahara strain for adult mice.
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PMID:Rescue of rabies virus from cloned cDNA and identification of the pathogenicity-related gene: glycoprotein gene is associated with virulence for adult mice. 1153 76


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