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)

Rats inoculated intraperitoneally with 100 LD50 of Venezuelan Equine Encephalomyelitis virus (VEE), showed a significant decrease of DNA-dependent RNA polymerase type I activity of brain nuclei of 29.7% and 59.3% at 24 and 48 hours after infection, respectively, while the animals had no clinical symptoms of illness. No alterations were observed in the nuclei of mononuclear cells at any time. VEE virus titer was higher in the serum than in the brain. The results suggest that viral infection produced a modification in the activity of this enzyme only in brain, even with a low amount of virus, and had no effect on enzyme activity of mononuclear cells.
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PMID:RNA-polymerase, type I: activity in rat brain cell nuclei and peripheral blood mononuclear cells after Venezuelan equine encephalomyelitis virus infection. 205 41

The 26S mRNA and most of the nsP4 encoding regions of the eastern equine encephalomyelitis (EEE) viral genome have been cloned. Excluding the poly(A) tail, the 26S mRNA region was determined to be 4139 nucleotides long and to share the same general organization as that of other alphaviruses. A highly conserved region of 19 nucleotides, the putative transcriptase recognition site for 26S mRNA synthesis, was present at the 26S/42S junction region of the 42S genomic RNA. Translation of the 26S mRNA began at the first AUG (positions 59 to 61) initiation codon and continued with an open reading frame that coded for a polyprotein of 1258 amino acids ending at a UAA ochre termination codon (positions 3776 to 3778). All four putative posttranslational cleavage sites used to generate the capsid, E3, E2, 6K and E1 proteins were conserved. Transmembrane domains present in the EEE virus structural polyprotein have been identified and their functions discussed. Pairwise comparison of the deduced amino acid sequences of the polyproteins of five alphaviruses (EEE, Venezuelan equine encephalitis, Sindbis, Semliki Forest and Ross River viruses) revealed EEE virus to be more closely related to VEE virus than to the other three viruses.
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PMID:Nucleotide sequence of the genome region encoding the 26S mRNA of eastern equine encephalomyelitis virus and the deduced amino acid sequence of the viral structural proteins. 288 48

Lust, George (Fort Detrick, Frederick, Md.). Alterations of protein synthesis in arbovirus-infected L cells. J. Bacteriol. 91:1612-1617. 1966.-Cellular protein synthesis and ribonucleic acid (RNA) synthesis in mouse L cells were markedly depressed 1 hr after infection with Venezuelan equine encephalomyelitis virus. Host RNA and protein synthesis were inhibited more rapidly by the virus infection than by actinomycin D. In cells infected 4 hr, a cytoplasmic RNA polymerase was demonstrated which was absent in uninfected cells. At this time, deoxyribonucleic acid-directed RNA synthesis catalyzed by the nuclear RNA polymerase was inhibited in vitro in enzyme preparations from nuclei of virus-infected cells. For optimal activity, the cytoplasmic RNA polymerase required the four nucleoside triphosphates, Mg(++), and RNA. The enzyme was insensitive to actinomycin D and deoxyribonuclease, indicating that it catalyzed RNA-directed RNA synthesis. Attempts to purify the induced polymerase further were unsuccessful. Fresh preparations had to be used because the enzymatic activity was unstable.
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PMID:Alterations of protein synthesis in arbovirus-infected L cells. 592 79

In the present study the effect of the attenuated strain TC-83 of the Venezuelan Equine Encephalitis virus on the nuclear transcription in brain cells of rats was assessed. The transcription activity of the DNA depending RNA polymerases (types I and II) in the isolated nuclei of brain of infected rats and controls was determinated by incorporation of the (3H) UTP. Simultaneously a viral replication curve in the brain and the serum was carried out by plaque forming method in chicken embryo cell cultures. RNA polymerase I activity was only significantly reduced after 25 hours of infection, respect to control values, while polymerase II activity was progressive and significantly diminished from inicial stages of the viral infection at 10, 15, 20 y 25 hours post-infection compared to control values. The virus was not detected in the brain but after 25 hours post-infection with very low titers (< 0.7 log10 P.F.U./ml.), while the viral presence in the blood was demonstrated after a 10 hour period. Our results demonstrated a marked effect of the attenuated strain on the brain nuclear transcription, although the presence of the virus was not detected in the brain of the infected rats. This finding suggest a mechanism of action which deserves further studies to elucidate the cerebral metabolic response and the pathogenesis of the Venezuelan Equine Encephalitis infection.
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PMID:[Effect of the attenuated strain (TC-83) of Venezuelan equine encephalitis virus on nuclear transcription in rat brain cells]. 892 28

Alphaviruses have several features that make them attractive as gene delivery platforms, and vectors derived principally from Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEE), are currently being developed as prophylactic and therapeutic vaccines for infectious diseases and cancer. Alphavirus vectors, termed "replicons", retain the nonstructural protein genes encoding the viral replicase, that in turn programme high level cytoplasmic amplification of the vector RNA. We have developed plasmid DNA and recombinant vector particle delivery systems derived from the prototype alphavirus, SIN. Each system uses RNA polymerase II-based expression of alphavirus genome components and both vector formats are highly efficacious towards inducing robust antigen-specific immune responses in vaccinated animals. To increase the potency of SIN vector particles, which are not known to be lymphotropic, the tropism was re-directed for efficient infection of dendritic cells, both in vitro and in vivo.
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PMID:Alphavirus DNA and particle replicons for vaccines and gene therapy. 1171 18

Non-viral self-replicating vectors based on defective viral genomes have been developed for a number of different alphaviruses including Semliki Forest virus (SFV), Sindbis virus (SIN) and Venezuelan equine encephalitis virus (VEE). These vectors can be used for gene delivery as naked RNA or DNA. Recombinant alphavirus RNA can be synthesized in vitro from plasmids containing the alphavirus replicon under the control of a prokaryotic promoter such as SP6 or T7. These self-replicating RNAs have been able to induce protective immune responses in vivo, probably due to the high level of expression of the recombinant antigen in the transfected cells. However, alphavirus vectors based on the direct delivery DNA are probably a better choice due to their higher stability and lower production cost. In these vectors, the alphavirus replicon is placed under the control of a RNA polymerase II promoter. These vectors are more efficient than conventional plasmids in inducing both humoral and cellular immune responses in small animals, allowing the use of significant smaller amounts of DNA for immunization. In addition, due to the transient nature of the alphavirus replicons, possible problems associated with DNA integration into host chromosomes are eliminated.
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PMID:Non-viral amplification systems for gene transfer: vectors based on alphaviruses. 1171 47

A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) was cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. A streptavidin-binding peptide gene fused to a 6His tag was attached downstream to the scFv gene. The recombinant fusion protein was expressed in bacteria as inclusion bodies that were subsequently solubilized with 8 M urea and renatured by an arginine system. Purification of the fusion protein was achieved by immobilized metal affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) and Western blotting results revealed that the fusion protein not only retained VEE antigen binding and specificity properties similar to those of its parent native monoclonal antibody (MAb), but also possessed streptavidin-binding activity. This experimental approach can eliminate the need for chemical biotinylation of antibodies and the risk associated of antibody denaturation and can provide a stable and reproducible reagent for rapid and efficient immunoassay of VEE when detected by horseradish peroxidase (HRP)-conjugated streptavidin.
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PMID:Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to Venezuelan equine encephalitis virus. 1257 5

The genes encoding envelope proteins E1 and E2 of western equine encephalitis virus (WEEV) were respectively cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. The recombinant C-terminal 6xHis-tagged WEEV E1 and E2 were expressed in bacteria as inclusion bodies that were subsequently solubilized with 8M urea, purified by immobilized metal ion affinity chromatography and finally refolded using an arginine system. The purified 6xHis-tagged proteins showed 50kDa bands as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, consistent with the expected sizes of WEEV E1 and E2. The potential of the recombinant WEEV E1 and E2 as antigens for serologic tests to detect anti-WEEV antibodies for diagnosis of WEEV infection was assessed by an enzyme-linked immunosorbent assay with anti-WEEV polyclonal antibodies obtained from the mice infected with WEEV. The anti-WEEV antibodies bound the recombinant WEEV E1 and E2 in a dose dependent manner. On the contrary, antibodies against Venezuelan equine encephalitis virus with a genetic background and a disease spectrum very similar to WEEV, did not bind to the recombinant WEEV E1 and E2. Our results suggest that the recombinant WEEV E1 and E2 possess predominant antigenicity of WEEV and have the potential to be used as antigens in immunoassays to detect anti-WEEV antibodies for serological diagnosis of WEEV infection so as to eliminate the need for preparation of cell culture-derived viral antigens, which is time-consuming, expensive, laborious, tedious, and hazardous.
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PMID:Cloning, expression and purification of envelope proteins E1 and E2 of western equine encephalitis virus and potential use of them as antigens in immunoassays. 1805 49

Venezuelan equine encephalitis virus is a member of the alphavirus family and genus togaviridae. VEEV is highly infectious in aerosol form and has been weaponized in the past making it a potential biothreat agent. At present, there are no FDA approved antiviral treatments or vaccines for VEEV. Artificial microRNAs are small molecules which are expressed through endogenous microRNA machinery by RNA polymerase II. These artificial microRNAs effectively inhibit gene expression and are non-toxic to the host cell. VEEV RNA dependent RNA polymerase (RdRp) is central to VEEV replication. Therefore, we hypothesize that targeted inhibition of VEEV RdRp using artificial microRNAs may efficiently inhibit VEEV replication. Five artificial microRNAs were tested in vitro in BHK cells. Three of these artificial miRNAs showed significant inhibition of VEEV replication. Further, these microRNAs were cloned into the expression vector in combination to see the synergistic effect on VEEV replication. Combination of more than one miRNA did not result in significant inhibition of virus replication. In conclusion, we have shown that RNAi through artificial microRNAs effectively inhibits VEEV replication and is significantly less toxic in comparison to siRNAs.
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PMID:Artificial microRNAs can effectively inhibit replication of Venezuelan equine encephalitis virus. 2398 97