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)

A new system for recovery of rabies virus from cDNA plasmid, the transcription of which was driven by cellular RNA polymerase II, was developed. The plasmid contains full-length viral cDNA flanked by hammerhead ribozyme and hepatitis delta ribozyme sequences, arranged downstream of the cytomegalovirus (CMV) promotor. Transfection with the full-length cDNA plasmid together with helper plasmids encoding viral N, P, and L proteins without supply of T7 RNA polymerase produced a recombinant rabies virus in several cell lines. The efficiency of recovery between the conventional T7 promotor system and the new CMV promotor system was compared using these plasmid constructs. The newly established system is applicable to various cell lines and allows rapid and efficient generation of recombinant rabies virus.
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PMID:An improved method for recovering rabies virus from cloned cDNA. 1250 38

Expression of rabies virus glycoprotein (G) by G cDNA-transfected mammalian cells resulted in the production of only a fusion-negative form. Low pH-dependent fusion activity, however, was seen when the expression was done under control of the T7 promoter with the help of recombinant vaccinia virus (RVV-T7) that provided T7 RNA polymerase. Fusion-inactive G proteins were transported to the cell surface as being detected by a conformational epitope-specific monoclonal antibody (mAb; #1-46-12). The fusion-inactive G proteins were recognized by most of our 13 conformation-specific mAbs, except for one mAb, #1-30-44, that recognized the low pH-sensitive conformational epitope. When the G gene expression was done with the help of RVV-T7, although most G proteins remained in the epitope-negative form, a small fraction of G gene products were 1-30-44 epitope-positive, and cell fusion activity could be seen when cells were exposed to low pH conditions. From these results, we conclude that acquisition of low pH-dependent fusion activity is closely related to structural maturation of the G protein to form the low pH-sensitive 1-30-44 epitope. Such maturation seems to be dependent on certain rabies virus-induced cellular conditions or functions, which might also be provided in part by the vaccinia virus infection. We further assume that expression of G cDNA alone mostly results in the production of mis-folded and/or differently folded forms of G protein, and only a small fraction is correctly folded even under RVV-T7-mediated expression conditions.
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PMID:Further characterization of the rabies virus glycoproteins produced by virus-infected and G cDNA-transfected cells using a monoclonal antibody, #1-30-44, which recognizes an acid-sensitive epitope. 1282 95

To improve efficiency of recovery of rabies virus from cloned cDNA, we established a BHK cell clone that stably expresses T7 RNA polymerase, which we named BHK/T7-9. We also constructed new helper plasmids for expression of nucleoprotein and RNA polymerase of the RC-HL strain using the pTM1 plasmid vector, which makes the T7 RNA polymerase-transcripts from the plasmid cap-independent for translation. After co-transfection of these helper plasmids and the previously constructed full-length genome plasmid of the RC-HL strain to BHK/T7-9 cells, recombinant rabies virus was efficiently recovered from the cloned cDNA.
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PMID:Improved recovery of rabies virus from cloned cDNA using a vaccinia virus-free reverse genetics system. 1452 22

When the rabies virus G cDNA was expressed with the help of T7 RNA polymerase provided by a recombinant vaccinia virus (RVV-T7), functional G proteins were produced in terms of their ability to induce low pH-dependent syncytium formation and the formation of conformational epitopes, including the acid-sensitive epitope recognized by mAb #1-30-44. Such an ability and the 1-30-44 epitope formation, however, were not associated with the G gene products when G cDNA was expressed without the help of RVV-T7 using a tetracycline-regulated expression vector (pTet-G), although they were normally transported to the surface of established G protein-producing BHK-21 (G-BHK) cells. But, when the G-BHK cells were treated with 2.5 m M sodium butyrate (NaB) after the removal of tetracycline, we could observe not only a much increased frequency of G protein-producing cells, but also the greatly enhanced maturation of the protein. Another short acylate, sodium propionate (NaP), similarly induced increased G protein synthesis at a concentration of 2.5 m M as NaB; however, such proteins were mostly not endowed with the fusion activity nor the 1-30-44 epitope, while NaP at a higher concentration as 5.0 m M did induce similarly the increased production and enhanced maturation of G protein, including the 1-30-44 epitope formation. From these results, we conclude that functional maturation of G protein to acquire fusogenic activity is correlated with 1-30-44 epitope formation, and 2.5 m M NaB not only stimulates G protein production, but also provides such cellular conditions as are required for the structural and functional maturation of the protein.
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PMID:Studies on the conditions required for structural and functional maturation of rabies virus glycoprotein (G) in G cDNA-transfected cells. 1555 43

Rabies virus P protein is a co-factor of the viral RNA polymerase. It has been shown previously that P mRNA directs the synthesis of four N-terminally truncated P products P2, P3, P4, and P5 due to translational initiation by a leaky scanning mechanism at internal Met codons. Whereas P and P2 are located in the cytoplasm, P3, P4, and P5 are found in the nucleus. Here, we have analyzed the molecular basis of the subcellular localization of these proteins. Using deletion mutants fused to GFP protein, we show the presence of a nuclear localization signal (NLS) in the C-terminal part of P (172-297). This domain contains a short lysine-rich stretch ((211)KKYK(214)) located in close proximity with arginine 260 as revealed by the crystal structure of P. We demonstrate the critical role of lysine 214 and arginine 260 in NLS activity. In the presence of Leptomycin B, P is retained in the nucleus indicating that it contains a CRM1-dependent nuclear export signal (NES). The subcellular distribution of P deletion mutants indicates that the domain responsible for export is the amino-terminal part of the protein. The use of fusion proteins that have amino terminal fragments of P fused to beta-galactosidase containing the NLS of SV40 T antigen allows us to identify a NES between residues 49 and 58. The localization of NLS and NES determines the cellular distribution of the P gene products.
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PMID:Nucleocytoplasmic shuttling of the rabies virus P protein requires a nuclear localization signal and a CRM1-dependent nuclear export signal. 1578 Aug 78

The RNA polymerase of rabies virus (RV) is a two-protein complex composed of L (a large catalytic component) and P (a non-catalytic phosphoprotein cofactor) proteins. We generated a gene-deficient RV lacking the entire P gene from HEP-Flury (HEP) strain, one of the most attenuated RV strains, by the method of reverse genetics. This P gene-deficient (def-P) virus could replicate and produce progeny viruses with a slightly retarded rate in the cell lines that constitutively express the P protein. The def-P virus could perform the primary RNA transcription by the virion-associated polymerase even in the infected host without de novo P protein synthesis. However, the def-P virus required the newly synthesized P protein for the secondary RNA transcription and genome RNA replication of virus. No progeny virus was produced in the infected host that did not express P protein. The def-P virus was apathogenic in adult and suckling mice even when inoculated intracranially. On the other hand, inoculation of the def-P virus into mice induced a high titer of virus-neutralizing antibody and protected mice from lethal challenge with the CVS strain. These results demonstrated that the def-P virus could induce strong protective immunity against rabies virus without the production of progeny virus and the severe host damage. The def-P virus would be a potential resource of safe live-attenuated rabies vaccine.
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PMID:Characterization of P gene-deficient rabies virus: propagation, pathogenicity and antigenicity. 1589 3

Rabies virus P protein is a cofactor of RNA polymerase. We investigated other potential roles of P (CVS strain) by searching for cellular partners using two-hybrid screening. We isolated a cDNA encoding the signal transducer and activator of transcription 1 (STAT1) that is a critical component of interferon type I (IFN-alpha/beta) and type II (IFN-gamma) signaling. We confirmed this interaction by glutathione S-transferase-pull-down assay. Deletion mutant analysis indicated that the carboxy-terminal part of P interacted with a region containing the DNA-binding domain and the coiled-coil domain of STAT1. The expression of P protein inhibits IFN-alpha- and IFN-gamma-induced transcriptional responses, thus impairing the IFN-induced antiviral state. Mechanistic studies indicate that P protein does not induce STAT1 degradation and does not interfere with STAT1 phosphorylation but prevents IFN-induced STAT1 nuclear accumulation. These results indicate that rabies P protein overcomes the antiviral response of the infected cells.
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PMID:Rabies virus P protein interacts with STAT1 and inhibits interferon signal transduction pathways. 1625 75

In tropical and subtropical climates, the shipment of animal brains for rabies diagnosis may be a problem because brain specimens sometimes arrive decomposed at the diagnostic laboratory. In this situation, reverse transcription-polymerase chain reaction (RT-PCR) may serve as a potential solution because of its high sensitivity. However, little is known about the stability of rabies viral RNA in decomposed brain tissue. To determine the stability of rabies virus genomic RNA in brain samples, 72 mice were inoculated with the challenge virus strain-11 of rabies virus. After incubation period, mice were euthanized to obtain their brains. These were categorized in 2 different groups. In the first group, 36 brains were kept at room temperature (25-27 degrees C) immediately after euthanasia. In the second group, the other 36 inoculated brains were frozen at -70 degrees C and later maintained at room temperature. In both groups, RT-PCR was performed at days 0, 1, 2, 3, 4, 7, 10, 12, 16, 18, 23, and 26 by using primers previously described in the literature and a primer set specifically designed for a Mexican variant of vampire-bat rabies. Reverse-transcriptase PCR experiments were performed in 3 different inoculated brains, in which the direct fluorescent antibody (DFA) test was previously conducted to detect rabies viral antigen in the brains kept at room temperature and in the frozen brains. The DFA test resulted positive in both groups up to day 7. In brain samples stored at ambient temperature (25-27 degrees C), the intensity of the RT-PCR band started to diminish by day 12; however, rabies virus genome could be successfully amplified by RT-PCR up to 23 days. These results indicate that brain samples kept at ambient temperature (up to 27 degrees C) may reach a reference laboratory in an adequate state for rabies diagnosis by RT-PCR.
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PMID:Use of reverse transcription-polymerase chain reaction to determine the stability of rabies virus genome in brains kept at room temperature. 1656 65

Unlike vesicular stomatitis virus, rabies virus glycoprotein gene has not been successfully relocated closer to promoter-proximal regions by reverse genetics. Here we describe an efficient system for the Evelyn-Rokitnicki-Abelseth (ERA) rabies virus with the glycoprotein gene switched with the matrix protein gene, creating a reshuffled virus ERAgm (gene order N-P-G-M-L). With the aid of an autogene plasmid, the T7 RNA polymerase containing a nuclear location signal from the SV40 large T antigen facilitated virus recovery. The rearranged ERAgm rabies virus replicated as well as the parental ERA (gene order N-P-M-G-L) virus, reaching 10(9) ffu/ml in infected BSR cells. The altered glycoprotein gene position in viral genome presented an alternative way to study the pathogenicity of rabies virus. This also provides a potential novel method for rabies vaccine development.
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PMID:Glycoprotein gene relocation in rabies virus. 1785 Sep 11

Rabies remains endemic within a number of countries in Southeast Europe including Romania, Bulgaria and Turkey. With the probable expansion of the European Union eastwards, it is likely that rabies elimination programs will be increased to reduce the burden of disease in new accession countries. A clear understanding of the epidemiology of the virus in this area of Europe is vital before such programs are introduced. With the exception of Turkey, the red fox (Vulpes vulpes) is the principal disease reservoir in Southeastern Europe. However, cases of rabies in the dog (Canis familiaris) are regularly reported. In contrast to Northern Europe, the raccoon dog (Nyctereutes procyonoides) does not appear to be a vector in the south. This study summarises the current rabies situation in Southeast Europe and demonstrates the phylogenetic relationships between the viruses in a number of the countries within the region. Rabies virus RNA was extracted from original samples and a fragment of the nucleoprotein gene amplified by reverse-transcriptase PCR. Automated sequencing was used to derive nucleoprotein gene sequences and these were used to prepare a molecular phylogeny of rabies viruses in Southeast Europe. In Bulgaria, the dog is the main vector bringing rabies into contact with humans and livestock. However, other species may also act as reservoirs for the disease, complicating the development of elimination strategies. The fox is the principal reservoir species for rabies in Romania although cases in dogs are regularly reported. Despite a gradual decline in dog rabies, urban pockets of the disease remain in many regions of Turkey. Furthermore, there is some evidence that the fox has been a significant vectorfor rabies and may be responsible for increases in rabies in cattle in the Aegean region of the country. Throughout the region there is evidence for cross-border movement of rabies by both wildlife and canine vectors.
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PMID:Epidemiology of rabies in Southeast Europe. 1863 79


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