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)

Recently we described rescue of defective Kunjin virus (KUN) RNAs with small deletions in the methyltransferase and RNA polymerase motifs of the ns5 gene, using BHK cells stably expressing KUN replicon RNA (repBHK cells) as helper (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). We have now extended our previous observations and report successful trans-complementation of defective KUN RNAs with most of the ns5 gene deleted or substituted with a heterologous (dengue virus) ns5 sequence. Replication of full-length KUN RNAs with 3'-terminal deletions of 136 (5%), 933 (34%), and 1526 (56%) nucleotides in the ns5 gene was complemented efficiently in transfected repBHK cells. RNA with a larger deletion of 2,042 nucleotides (75%) was complemented less efficiently, and RNA with an even larger deletion of 2,279 nucleotides (84%) was not complemented at all. Chimeric KUN genomic RNA containing 87% of the KUN ns5 gene replaced by the corresponding sequence of the dengue virus type 2 ns5 gene was unable to replicate in normal BHK cells but was complemented in repBHK cells. These results demonstrate for the first time complementation of flavivirus RNAs with large deletions (as much as 75%) in the RNA polymerase gene and establish that translation of most of the N-terminal half of NS5 is essential for complementation in trans. A model of formation of the flavivirus replication complex implicating a possible role in RNA replication of conserved coding sequences in the N-terminal half of NS5 is proposed based on the complementation and earlier results with KUN and on reported data with other flaviviruses.
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PMID:trans-Complementation analysis of the flavivirus Kunjin ns5 gene reveals an essential role for translation of its N-terminal half in RNA replication. 1051 33

Successful trans-complementation of the defective Kunjin virus (KUN) RNA FLdGDD with a deletion of the RNA polymerase motif GDD in the NS5 gene by using a BHK cell line, repBHK, that continuously produced a functionally active KUN replication complex (RC) from replicon RNA was recently reported (A. A. Khromykh, M. T. Kenney, and E. G. Westaway, J. Virol. 72:7270-7279, 1998). In order to identify whether this complementation of FLdGDD RNA was provided by the wild-type NS5 protein alone or with the help of other nonstructural (NS) proteins also expressed in repBHK cells, we generated BHK cell lines stably producing the individual NS5 protein (SRns5BHK) or the NS1-NS5 polyprotein (SRns1-5BHK) by using a heterologous expression vector based on a modified noncytopathic Sindbis replicon. Western blot analysis with anti-NS5 antibodies showed that the level of production of NS5 was significantly higher in SRns5BHK cells than in SRns1-5BHK cells. Despite the higher level of expressed NS5, trans-complementation of FLdGDD RNA was much less efficient in SRns5BHK cells than in SRns1-5BHK cells and produced at least 100-fold less of the secreted complemented virus. In contrast, efficient complementation of KUN RNA with lethal cysteine-to-alanine mutations in the NS1 gene was achieved both in BHK cells producing the individual KUN NS1 protein from the Sindbis replicon vector and in repBHK cells, with both cell lines expressing similar amounts of NS1 protein. These results clearly demonstrate that flavivirus NS5 coexpressed with other components of the viral replicase possesses much higher functional (trans-complementing) activity than individually expressed NS5 and that efficient trans-complementation of mutated flavivirus NS1 and NS5 proteins occurs by different mechanisms. The results are interpreted and discussed in relation to our proposed model of formation of the flavivirus RC largely based on previous ultrastructural and biochemical analyses of KUN replication.
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PMID:Efficient trans-complementation of the flavivirus kunjin NS5 protein but not of the NS1 protein requires its coexpression with other components of the viral replicase. 1055 44

Sequence motifs within the non-structural protein NS5 or NS5B of the members of the family Flaviviridae suggest that this protein is the RNA-dependent RNA polymerase. This protein has now been expressed in various in vitro systems and used in polymerase assays. To understand the role of the RNA polymerase in RNA replication, this review will examine not only the polymerase protein but also the other proteins in the RNA replication complex. To date, several groups have investigated the interaction of these proteins both in vitro and in vivo and also the interaction of these proteins with the RNA signals at the 3' terminus of the RNA. Infectious clones and replicons containing the non-structural proteins have now been generated and these will be useful tools in understanding the processes of initiation and elongation of both positive and negative RNA synthesis.
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PMID:Flaviviridae polymerase and RNA replication. 1060 40

The hepatitis C virus is a single-stranded RNA virus with a genome approximately 9,000 nucleotides in length. The genome consists of a single, large open reading frame (ORF) and 5' and 3' untranslated regions. The highly conserved 5' untranslated region is 341 nucleotides in length with a complex secondary structure and may function as an internal ribosomal entry site (IRES). The 3' untranslated region is approximately 500 nucleotides in length and contains a hypervariable region, followed by a poly(U) sequence and a highly conserved 98-nucleotide element with a stable secondary structure. The ORF codes form a single polyprotein that is processed into as many as 10 polypeptides, including a capsid protein (core), two envelope proteins (E1 and E2), and nonstructural proteins (NS2, NS3, NS4, and NS5). Potentially suitable antiviral targets include the IRES, protease, helicase, and RNA polymerase. In vitro studies show that antisense oligonucleotides can inhibit the production of structural HCV proteins and may be therapeutically useful if the problems of stability and delivery can be solved. The binding of HCV envelope proteins to CD81, a potential receptor for viral entry into hepatocytes, has recently been described and also raises the possibility of agents to block the binding to CD81 or the entry of the virus into cells.
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PMID:Hepatitis C--virology and future antiviral targets. 1065 56

Extracellular ATP can function as a glial trophic factor as well as a neuronal transmitter. In astrocytes, mitogenic signalling by ATP is mediated by metabotropic P(2Y) receptors that are linked to the extracellular signal regulated protein kinase (Erk) cascade, but the types of P(2Y) receptors expressed in astrocytes have not been defined and it is not known whether all P(2Y) receptor subtypes are coupled to Erk by identical or distinct signalling pathways. We found that the P(2Y) receptor agonists ATP, ADP, UTP and 2-methylthioATP (2MeSATP) activated Erk and its upstream activator MAP/Erk kinase (Mek). cRaf-1, the first kinase in the Erk cascade, was activated by 2MeSATP, ADP and UTP but, surprisingly, cRaf-1 was not stimulated by ATP. Furthermore, ATP did not activate B-Raf, the major isoform of Raf in the brain, nor other Mek activators such as Mek kinase 1 (MekK1) and MekK2/3. Reverse transcriptase-polymerase chain reaction (RT - PCR) studies using primer pairs for cloned rat P(2Y) receptors revealed that rat cortical astrocytes express P(2Y(1)), a receptor subtype stimulated by ATP and ADP and their 2MeS analogues, as well as P(2Y(2)) and P(2Y(4)), subtypes in rats for which ATP and UTP are equipotent. Transcripts for P(2Y(6)), a pyrimidine-preferring receptor, were not detected. ATP did not increase cyclic AMP levels, suggesting that P(2Y(11)), an ATP-preferring receptor, is not expressed or is not linked to adenylyl cyclase in rat cortical astrocytes. These signal transduction and RT - PCR experiments reveal differences in the activation of cRaf-1 by P(2Y) receptor agonists that are inconsistent with properties of the P(2Y(1)), P(2Y(2)) and P(2Y(4)) receptors shown to be expressed in astrocytes, i.e. ATP=UTP; ATP=2MeSATP, ADP. This suggests that the properties of the native P(2Y) receptors coupled to the Erk cascade differ from the recombinant P(2Y) receptors or that astrocytes express novel purine-preferring and pyrimidine-preferring receptors coupled to the ERK cascade.
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PMID:P(2Y) purinoceptor subtypes recruit different mek activators in astrocytes. 1069 92

Most of the seven flavivirus nonstructural proteins (NS1 to NS5) encoded in the distal two-thirds of the RNA positive-sense genome are believed to be essential components of RNA replication complexes. To explore the functional relationships of these components in RNA replication, we used trans-complementation analysis of full-length infectious RNAs of Kunjin (KUN) virus with a range of lethal in-frame deletions in the nonstructural coding region, using as helper a repBHK cell line stably producing functional replication complexes from KUN replicon RNA. Recently we showed that replication of KUN RNAs with large carboxy-terminal deletions including the entire RNA polymerase region in the NS5 gene, representing 34 to 75% of the NS5 coding content, could be complemented after transfection into repBHK cells. In this study we have demonstrated that KUN RNAs with deletions of 84 to 97% of the NS1 gene, or of 13 to 63% of the NS3 gene including the entire helicase region, were also complemented in repBHK cells with variable efficiencies. In contrast, KUN RNAs with deletions in any of the other four nonstructural genes NS2A, NS2B, NS4A, and NS4B were not complemented. We have also demonstrated successful trans complementation of KUN RNAs containing either combined double deletions in the NS1 and NS5 genes or triple deletions in the NS1, NS3, and NS5 genes comprising as much as 38% of the entire nonstructural coding content. Based on these and our previous complementation results, we have generated a map of cis- and trans-acting elements in RNA replication for the nonstructural coding region of the flavivirus genome. These results are discussed in the context of our model on formation and composition of the flavivirus replication complex, and we suggest molecular mechanisms by which functions of some defective components of the replication complex can be complemented by their wild-type counterparts expressed from another (helper) RNA molecule.
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PMID:cis- and trans-acting elements in flavivirus RNA replication. 1070 42

This study of the yellow fever French neurotropic vaccine strain from the Institut Pasteur (FNV-IP) demonstrates that this viral genome is not as stable as that of the 17D-204 vaccine virus. FNV-IP was plaque-purified three times and then passaged eight times in Vero cells. Viral populations from the second and eighth passage post purification were sequenced and compared to the published sequences of FNV-IP. The passage-2 viral population had 31 nucleotide and nine amino acid changes compared to the parental virus while the passage-8 virus had six additional nucleotide changes encoding a single amino acid substitution. The plaque-purified virus also had two sequence deletions in the 3'-noncoding region. The plaque purification resulted in selection of a passage-2 virus that had a mouse LD(50) of 20 pfu/ml, 67-fold greater than parental FNV-IP which had an LD(50) of 0.3 pfu/ml. Subsequent passage in Vero cells resulted in a passage-8 virus which had increased neurovirulence with an LD(50) of 3.2 pfu/ml. The only amino acid difference between the passage-2 and passage-8 viruses was at amino acid 638 of NS5 which lies within domain V of the RNA-dependent-RNA polymerase. Overall, these data indicate that FNV-IP virus has an inherently less stable genome than 17D vaccine virus and a variable viral population.
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PMID:The French neurotropic vaccine strain of yellow fever virus accumulates mutations slowly during passage in cell culture. 1098 83

In order to study whether flavivirus RNA packaging is dependent on RNA replication, we generated two DNA-based Kunjin virus constructs, pKUN1 and pKUN1dGDD, allowing continuous production of replicating (wild-type) and nonreplicating (with a deletion of the NS5 gene RNA-polymerase motif GDD) full-length Kunjin virus RNAs, respectively, via nuclear transcription by cellular RNA polymerase II. As expected, transfection of pKUN1 plasmid DNA into BHK cells resulted in the recovery of secreted infectious Kunjin virions. Transfection of pKUN1dGDD DNA into BHK cells, however, did not result in the recovery of any secreted virus particles containing encapsidated dGDD RNA, despite an apparent accumulation of this RNA in cells demonstrated by Northern blot analysis and its efficient translation demonstrated by detection of correctly processed labeled structural proteins (at least prM and E) both in cells and in the culture fluid using coimmunoprecipitation analysis with anti-E antibodies. In contrast, when dGDD RNA was produced even in much smaller amounts in pKUN1dGDD DNA-transfected repBHK cells (where it was replicated via complementation), it was packaged into secreted virus particles. Thus, packaging of defective Kunjin virus RNA could occur only when it was replicated. Our results with genome-length Kunjin virus RNA and the results with poliovirus replicon RNA (C. I. Nugent et al., J. Virol. 73:427-435, 1999), both demonstrating the necessity for the RNA to be replicated before it can be packaged, strongly suggest the existence of a common mechanism for minimizing amplification and transmission of defective RNAs among the quasispecies in positive-strand RNA viruses. This mechanism may thus help alleviate the high-copy error rate of RNA-dependent RNA polymerases.
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PMID:Coupling between replication and packaging of flavivirus RNA: evidence derived from the use of DNA-based full-length cDNA clones of Kunjin virus. 1131 33

The hepatitis C virus (HCV) is a small enveloped RNA virus belonging to the family flaviviridae and genus hepacivirus. The HCV RNA genome is 9,600 nucleotides in length and encodes a single polyprotein that is post-translationally cleaved into 10 polypeptides including t3 structural (C, E1, and E2) and multiple nonstructural proteins ([NS] NS2 to NS5). The NS proteins include enzymes necessary for protein processing (proteases) and viral replication (RNA polymerase). The virus replicates at a high rate in the liver and has marked sequence heterogeneity. There are 6 genotypes and more than 90 subtypes of HCV, the most common in the United States being 1a and 1b (approximately 75%), 2a and 2b (approximately 15%), and 3 (approximately 7%). Acute hepatitis C is marked by appearance of HCV RNA in serum within 1 to 2 weeks of exposure followed by serum alanine aminotransferase (ALT) elevations, and then symptoms and jaundice. Antibody to HCV (anti-HCV) tends to arise late. In acute resolving hepatitis, HCV RNA is cleared and serum ALT levels fall to normal. However, 55% to 85% of patients do not clear virus, but develop chronic hepatitis C. Chronic hepatitis C is often asymptomatic, but is usually associated with persistent or fluctuating elevations in ALT levels. The chronic sequelae of hepatitis C include progressive hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. Extra-hepatic manifestations include sicca syndrome, cryoglobulinemia, glomerulonephritis, and porphyria cutanea tarda. Knowledge of the course and outcome of hepatitis C is important in developing approaches to management and therapy.
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PMID:Course and outcome of hepatitis C. 1240 73

We have established a T7-based model system for hepatitis C virus (HCV) 1a strain, which involves the use of a replication-defective adenovirus that carries the gene for T7 RNA polymerase and a transcription plasmid containing full-length HCV cDNA clone. To facilitate high-level expression of HCV, sub-confluent Huh7 cells were first infected with adenovirus containing the gene for the T7 RNA polymerase and then transfected with the transcription plasmid. As a negative control, part of NS5B gene of this clone was deleted which abolishes the HCV RNA-dependent RNA polymerase and prevents replication of viral RNA. This model produces high levels of structural (core, E1, E2) and nonstructural proteins (NS5), which were detected by Western blot analysis and immunofluorescence assay. Negative-strand HCV RNA was detected only in the wild-type clone in the presence of actinomycin D, and no RNA was detected with the NS5B deleted mutant control. As a practical validation of this model, we showed that IFN alpha-2b selectively inhibits negative-strand RNA synthesis by blocking at the level of protein translation. The inhibitory effect of IFN alpha-2b is not due reduction of transcription by T7 polymerase or due to intracellular degradation of HCV RNA. This in vitro model provides an efficient and reliable means of assaying negative-strand RNA, protein processing, and testing the antiviral properties of interferon.
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PMID:Interferon alpha-2b inhibits negative-strand RNA and protein expression from full-length HCV1a infectious clone. 1512 7


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