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)

Infectious bovine respiratory syncytial virus (BRSV) was produced by intracellular co-expression of five plasmid borne cDNAs, each under the control of a T7 RNA polymerase promoter. These separately encoded a full-length, genetically-marked copy of BRSV antigenome along with either BRSV or human respiratory syncytial virus (HRSV) support plasmids, which express N, P, L and M2-1 proteins. HEp2 cells were used in transfection and recombinant vaccinia virus (MVA-T7) provided T7 RNA polymerase to drive the transcription. The recovery of recombinant BRSV (rBRSV) was confirmed by immunological staining of plaques, restriction enzyme digestion and nucleotide sequencing of PCR fragments carrying the genetic markers from the rescued virus. The rBRSV was indistinguishable from its parental wild-type virus in its growth characteristics in cell culture. The present work has completed the entire genome sequence of BRSV strain A51908 (15,140 nt) and has also identified changes in sequence and growth characteristics in cell culture from the original BRSV strain A51908 laboratory isolate.
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PMID:Rescue of bovine respiratory syncytial virus from cloned cDNA: entire genome sequence of BRSV strain A51908. 1172 68

The RNA genome of the vaccine strain 2024 of feline calicivirus was cloned as cDNA and analyzed by nucleotide sequencing. A full-length DNA copy of the viral genome was established and proved to be a source of infectious cRNA after in vitro transcription and RNA transfection. Virus could also be recovered when the DNA construct was introduced into cells containing phage T7 RNA polymerase that was provided by vaccinia virus MVA-T7. After insertion of the sequence encoding the green fluorescent protein into the structural protein-encoding region of the infectious cDNA clone, a defective replicon was recovered that was able to replicate autonomously and was packaged into virus particles when the structural proteins were provided in trans.
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PMID:Feline calicivirus: recovery of wild-type and recombinant viruses after transfection of cRNA or cDNA constructs. 1202 75

The replication-deficient vaccinia virus (VV) MVA-T7 produces large amounts of T7 RNA polymerase and permits efficient protein expression from cDNA of T7-promoted genes. Yet, unlike recombinant VV vTF7-3, (VV) MVA-T7 produces no cytopathic effect in primate cells, thus allowing the study of processes with slow kinetics. We have applied MVA-T7 to aid genome expression of HAV, a representative of the Picornaviridae family that is well known for its inefficient replication in mammalian cell cultures. After cDNA transfection and MVA-T7 infection, empty capsids and mature HAV particles were formed with different kinetics and were characterized by their morphology, protein content, and infectivity. The data suggests that HAV genome replication is initiated from RNA, which was transcribed in vivo by the MVA-T7-encoded T7 RNA polymerase. HAV genome replication was also demonstrated in a recombination assay. After co-expression of two subgenomic HAV cDNAs, both by themselves unable to complete the viral life cycle, infectious HAV was rescued, indicating that replication-dependent genetic recombination has occurred. We propose that the high-level genome expression mediated in vivo by the VV-encoded T7 RNA polymerase augments the amount of viral RNA, such that replication of viruses poorly replicating in cell cytoplasm is detectable.
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PMID:A vaccinia virus MVA-T7-mediated recovery of infectious hepatitis A virus from full-size cDNA or from two cDNAs, both by themselves unable to complete the virus life cycle. 1236 52

Determining the subcellular localization of the L1 ORF2 protein (ORF2p) has been impossible to date because of technical limitations in detecting either endogenous or overexpressed forms of the protein. Here we report visualization of the full-length ORF2p in cultured human cells following expression in a modified vaccinia virus/T7 RNA polymerase (MVA/T7RP) system. The MVA/T7RP system was used to ascertain subcellular localization of L1 ORF1p and ORF2p both as fusions with green fluorescent protein and by immunocytochemistry. Full-length ORF2p was predominantly cytoplasmic, while carboxy-terminal-deleted ORF2p localized additionally to the nucleolus. We mapped a functional nucleolar localization signal in ORF2p. ORF1p appeared in the cytoplasm with a speckled pattern and colocalized with ORF2p in nucleoli in a subset of cells. These findings help explain the presence of chimeras between L1s and small RNA gene sequences recently discovered in the human genome.
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PMID:A potential role for the nucleolus in L1 retrotransposition. 1502 73

Human noroviruses, the most common cause of nonbacterial gastroenteritis, are characterized by high infectivity rate, low infectious dose, and unusually high stability outside the host. However, human norovirus research is hindered by the lack of a cell culture system and a small animal model of infection. Norwalk virus (NV) is the prototype strain of human noroviruses. We report here replication of NV viral RNA and its packaging into virus particles in mammalian cells by intracellular expression of native forms of NV viral RNA devoid of extraneous nucleotide sequences derived from the expression vector by the use of replication-deficient vaccinia virus MVA encoding the bacteriophage T7 RNA polymerase (MVA/T7). Expressed genomic RNA was found to replicate; NV subgenomic RNA was transcribed from genomic RNA by use of NV nonstructural proteins expressed from genomic RNA and was subsequently translated into NV capsid protein VP1. Viral genomic RNA was packaged into virus particles generated in mammalian cells. The cesium chloride (CsCl) density gradient profile of virus particles containing genomic RNA was similar to that of NV purified from stool. These observations indicate that the NV cDNA constructed here is a biologically infectious clone, and that mammalian cells have the ability to replicate NV genomic RNA. This work establishes a mammalian cell-based system for analysis of human norovirus replication and, thus, makes it feasible to investigate antiviral agents in mammalian cells.
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PMID:Replication and packaging of Norwalk virus RNA in cultured mammalian cells. 1600 73


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