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)

La Crosse virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology 330:493-500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3' ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 10(8) infectious viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSs-expressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdelNSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutoff and induced little apoptosis. Nevertheless, both viruses grew in Vero cells to similar titers. Our reverse genetics system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and to develop potential vaccine candidates.
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PMID:Efficient cDNA-based rescue of La Crosse bunyaviruses expressing or lacking the nonstructural protein NSs. 1605 34

The 1679-nt single-stranded RNA genome of hepatitis delta virus (HDV) is circular in conformation. It is able to fold into an unbranched rodlike structure via intramolecular base-pairing. This RNA is replicated by host RNA polymerase II (Pol II). Such transcription is unique, because Pol II is known only for its ability to act on DNA templates. The present study addressed the ability of the HDV RNA replication to tolerate insertions of up to 1000 nt of non-HDV sequence either at an end of the rodlike RNA structure or at a site embedded within the rod. The insertions did not interfere with the ability of primary transcripts to be processed in vivo by ribozyme cleavage and ligation. The insertions greatly reduced the ability of genomes to replicate. However, when total RNA from such transfected cells was used to transfect new recipient cells, replicating HDV RNAs could be detected by Northern analyses. The size of the emerged RNAs was consistent with loss of the inserted sequences. RT-PCR, cloning, and sequencing showed that recovery involved removal of inserted sequences with or without small deletions of adjacent RNA sequences. Such restoration of the RNA genome is consistent with a model requiring intramolecular template-switching (RNA recombination) during RNA-directed transcription, in combination with biological selection for maintenance of the rodlike structure of the template.
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PMID:Restoration in vivo of defective hepatitis delta virus RNA genomes. 1661 66

Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that replicates and transcribes in the nucleus of infected cells. Therefore, efficient synthesis of BDV RNA in the nucleus is critical for the development of a reverse genetics system for this virus. Here, we report the development of such a system using the RNA polymerase II (Pol II) promoter. The BDV minigenome cDNA was flanked by hammerhead ribozyme and hepatitis delta ribozyme sequences and inserted downstream of the Pol II promoter. To improve the efficacy of minigenome expression, we estimated the effects of several signal sequences within the minigenome constructs. We found that insertion of the SV40 nuclear import sequence into the Pol II constructs significantly enhances the replication of the minigenome even in cells lacking the SV40 large T antigen. This novel system is theoretically applicable to any mammalian cell line and would be valuable for analyzing host- or cell-type-dependent differences in BDV replication and production. We could demonstrate here the cell-type-dependent inhibitory effect of the viral protein X on BDV polymerase activity. This system may be useful for various research fields not only of BDV but also of other negative-sense RNA viruses.
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PMID:Development of a novel Borna disease virus reverse genetics system using RNA polymerase II promoter and SV40 nuclear import signal. 1669 79

Avian metapneumovirus (AMPV) causes an acute respiratory disease in turkeys and is associated with "swollen head syndrome" in chickens, contributing to significant economic losses for the U.S. poultry industry. With a long-term goal of developing a better vaccine for controlling AMPV in the United States, we established a reverse genetics system to produce infectious AMPV of subgroup C entirely from cDNA. A cDNA clone encoding the entire 14,150-nucleotide genome of AMPV subgroup C strain Colorado (AMPV/CO) was generated by assembling five cDNA fragments between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme of a transcription plasmid, pBR 322. Transfection of this plasmid, along with the expression plasmids encoding the N, P, M2-1, and L proteins of AMPV/CO, into cells stably expressing T7 RNA polymerase resulted in the recovery of infectious AMPV/CO. Characterization of the recombinant AMPV/CO showed that its growth properties in tissue culture were similar to those of the parental virus. The potential of AMPV/CO to serve as a viral vector was also assessed by generating another recombinant virus, rAMPV/CO-GFP, that expressed the enhanced green fluorescent protein (GFP) as a foreign protein. Interestingly, GFP-expressing AMPV and GFP-expressing human metapneumovirus (HMPV) could be recovered using the support plasmids of either virus, denoting that the genome promoters are conserved between the two metapneumoviruses and can be cross-recognized by the polymerase complex proteins of either virus. These results indicate a close functional relationship between AMPV/CO and HMPV.
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PMID:Recovery of avian metapneumovirus subgroup C from cDNA: cross-recognition of avian and human metapneumovirus support proteins. 1673 18

Lacking an RNA-dependent RNA polymerase, hepatitis delta virus (HDV), which contains a circular RNA of 1.7 kilobases, is nonetheless able to replicate its RNA by use of cellular transcription machineries. Previously, we have shown that the replications of genomic- and antigenomic-strand HDV RNAs have different sensitivities to alpha-amanitin, suggesting that these two strands are synthesized in different transcription machineries in the cells, but the nature of these transcription machineries is not clear. In this study, we performed metabolic labeling and immunofluorescence staining of newly synthesized HDV RNA with bromouridine after HDV RNA transfection into hepatocytes and confirmed that HDV RNA synthesis had both alpha-amanitin-sensitive and -resistant components. The antigenomic RNA labeling was alpha-amanitin resistant and localized to the nucleolus. The genomic RNA labeling was alpha-amanitin sensitive and more diffusely localized in the nucleoplasm. Most of the genomic RNA labeling appeared to colocalize with the PML nuclear bodies. Furthermore, promyelocytic leukemia protein, RNA polymerase II (Pol II), and the Pol I-associated transcription factor SL1 could be precipitated together with hepatitis delta antigen, suggesting the association of HDV replication complex with the Pol I and Pol II transcription machineries. This conclusion was further confirmed by an in vitro replication assay. These findings provide additional evidence that HDV RNA synthesis occurs in the Pol I and Pol II transcription machineries, thus extending the capability of the cellular DNA-dependent RNA polymerases to utilizing RNA as templates.
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PMID:RNA-templated replication of hepatitis delta virus: genomic and antigenomic RNAs associate with different nuclear bodies. 1677 35

It was reported previously that hepatitis delta virus (HDV), the only animal virus in which replication is performed by cellular RNA polymerase(s), undergoes RNA recombination. However, the previous RNA transfection system was somewhat limited in terms of practical application. Cultured cells were transfected with plasmids expressing replication-competent genotypes I and IIb HDV genomic RNAs to develop a better system for studying the fundamental aspects of HDV RNA recombination and HDV-related RNA species were examined using restriction fragment length polymorphisms and sequence analysis of cloned RT-PCR products. This novel experimental system generated efficiently recombinants between the two parental HDV sequences, but not between replication-defective HDV constructs. The genome organization of the HDV recombinants produced in this system resembled that observed previously in cultured cells co-transfected with genome I and IIb RNAs. These data indicate that replication-dependent HDV RNA recombination can be catalyzed by host RNA polymerases in cultured cells co-transfected with two cloned HDV sequences. This new DNA-based system is simpler than the previous RNA-based method of study, and generates a higher recombination frequency, facilitating study of HDV RNA recombination.
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PMID:Detection of hepatitis delta virus recombinants in cultured cells co-transfected with cloned genotypes I and IIb DNA sequences. 1686 Aug 82

While this volume covers many different aspects of hepatitis delta virus (HDV) replication, the focus in this chapter is on studies of the structure and replication of the HDV RNA genome. An evaluation of such studies is not only an integral part of our understanding of HDV infections but it also sheds new light on some important aspects of cell biology, such as the fidelity of RNA transcription by a host RNA polymerase and on various forms of post-transcriptional RNA processing. Representations of the replication of the RNA genome are frequently simplified to a form of rolling-circle model, analogous to what have been described for plant viroids. One theme of this review is that such models, even after some revision, deceptively simplify the complexity of HDV replication and can fail to make clear major questions yet to be solved.
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PMID:Structure and replication of hepatitis delta virus RNA. 1690 18

HDV replicates its circular RNA genome using a double rolling-circle mechanism and transcribes a hepatitis delta antigen-encodeing mRNA from the same RNA template during its life cycle. Both processes are carried out by RNA-dependent RNA synthesis despite the fact that HDV does not encode an RNA-dependent RNA polymerase (RdRP). Cellular RNA polymerase II has long been implicated in these processes. Recent findings, however, have shown that the syntheses of genomic and antigenomic RNA strands have different metabolic requirements, including sensitives to alpha-amanitin and the site of synthesis. Evidence is summarized here for the involvement of other cellular polymerases, probably pol I, in the synthesis of antigenomic RNA strand. The ability of mammalian cells to replicate HDV RNA implies that RNA-dependent RNA synthesis was preserved throughout evolution.
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PMID:HDV RNA replication: ancient relic or primer? 1690 19

The hepatitis delta virus (HDV) is an RNA virus that depends on DNA-dependent RNA polymerase (RNAP) for its transcription and replication. While it is generally accepted that RNAP II is involved in HDV replication, its interaction with HDV RNA requires confirmation. A monoclonal antibody specific to the carboxy terminal domain of the largest subunit of RNAP II was used to establish the association of RNAP II with both polarities of HDV RNA in HeLa cells. Co-immunoprecipitations using HeLa nuclear extract revealed that RNAP II interacts with HDV-derived RNAs at sites located within the terminal stem-loop domains of both polarities of HDV RNA. Analysis of these regions revealed a strong selection to maintain a rod-like conformation and demonstrated several conserved features. These results provide the first direct evidence of an association between human RNAP II and HDV RNA and suggest two transcription start sites on both polarities of HDV RNA.
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PMID:The human RNA polymerase II interacts with the terminal stem-loop regions of the hepatitis delta virus RNA genome. 1695 88

The objective of the present study was to detect hepatitis E virus (HEV) in different samples from naturally infected pigs and to characterise genetically the detected strains. Serum, bile, liver, lymph nodes and faeces of 69 animals from 1 week to 4 months of age with different pathological conditions were collected. Reverse transcriptase-polymerase chain reaction (RT-PCR) to detect HEV and histopathology of tissues was conducted. Positive RT-PCR samples were sequenced and phylogenetically analysed. HEV was detected in at least one sample in 26 out of 69 animals (37.7%). Bile was the most frequently positive sample, followed by mesenteric lymph nodes, liver, faeces and serum. HEV was detected in pigs of 1 (n = 7), 2 (n = 8) and 3 (n = 11) months of age. A total of 22 of 69 (31.9%) pigs had mild to moderate hepatitis and 15 of them were HEV RT-PCR positive in at least one of the tested samples. The highest sensitivity of viral detection was achieved using samples that cannot be obtained from live pigs, such as liver, mesenteric lymph node and bile. Phylogenetic analyses confirmed that all Spanish swine HEV strains detected belonged to genotype III. Therefore, genotype III strains are present in a relative high proportion of pigs between 1 and 3 months of age. Through this study, it cannot be ruled out if concomitant infections may influence the distribution of HEV in infected pigs.
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PMID:Detection of hepatitis E virus in liver, mesenteric lymph node, serum, bile and faeces of naturally infected pigs affected by different pathological conditions. 1699 12


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