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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To study the mechanisms by which the influenza A virus RNA-dependent RNA polymerase switches from transcription to replication we have devised a riboprobe protection technique with which we analyzed the 3' end sequence of (+)-strand RNA products of an in vitro transcription reaction containing purified virion-RNP complexes in the presence and the absence of the putative regulatory proteins NP and NS1. We found that the addition of these proteins did not result in the synthesis of full-length (+)-strand RNA products resulting from read-through of the polyadenylation signal or replication. Because NS1 and NP are both phosphoproteins we searched for protein kinase activity that might play a role in regulating RNA synthesis. We showed that virion RNP complexes phosphorylated NS1 but possessed no autophosphorylating activity. Soluble NP protein derived from RNP complexes did not phosphorylate NS1, but did phosphorylate casein. When NP protein was dephosphorylated, however, it no longer phosphorylated casein. We also showed that NS1 was an ssRNA-binding protein which binds nonspecifically to all ssRNA, and that this activity is not dependent on its state of phosphorylation.
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PMID:Influenza A virus in vitro transcription: roles of NS1 and NP proteins in regulating RNA synthesis. 182 5

To facilitate further studies of flavivirus transcription, cell extraction methods and in vitro reaction conditions which increased West Nile virus (WNV) RNA-dependent RNA polymerase activity were determined. Subcellular fractions from WNV-infected BHK-21/W12 cells were characterized with regard to their protein and RNA content and in vitro polymerase activity. In both a cytoplasmic fraction, designated S1, and a fraction enriched for outer nuclear membranes, designated S2, seven virus-specific proteins, NS5 (96 kilodaltons [kDa]), NS3 (67 kDa), E (48 kDa), NS1 (47 kDa), ns4a (26 kDa), ns2a (17 kDa), and ns2b (14.5 kDa), were detected. The fractions also contained virus-specific RNA and cellular rRNA and mRNA. Polymerase activity in S1 and S2 fractions from WNV-infected cells was concentrated by pelleting and consisted of two types of enzyme activities: the WNV RNA-dependent RNA polymerase and terminal transferases of cellular origin. Enhanced levels of WNV polymerase activity were obtained from these cell fractions by altering several of the in vitro reaction conditions. Although Mg2+ was the divalent cation preferred by WNV polymerase, virus-specific in vitro transcription was detected at reduced levels when Mn2+ (0.05 or 0.5 mM) was present as the sole divalent cation. Product analysis revealed that the viral polymerase incorporated radiolabeled ribonucleotides into three distinct RNA species. Free single-stranded genome-sized RNA which was LiCl insoluble and RNase sensitive was found by fingerprint analysis to have an oligonucleotide pattern similar to that of WNV genomic RNA. RNA molecules which comigrated as a broad band near the top of the gel were separable into LiCl-insoluble, partially RNase-sensitive replicative-intermediate RNA and LiCl-soluble, RNase-resistant replicative-form RNA. The cellular transferases added UMP or AMP residues to the 3'-termini of cellular mRNA, tRNA, and 18S and 28S rRNA. Although a cellular terminal transferase has been reported to function in initiation of poliovirus transcription, no labeling of the WNV RNA by either of these cellular enzymes was detected. Therefore, they appear to play no specific role in flavivirus RNA synthesis.
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PMID:Characterization of West Nile virus RNA-dependent RNA polymerase and cellular terminal adenylyl and uridylyl transferases in cell-free extracts. 302 63

Virus-specific protein and RNA syntheses have been analyzed in chicken embryo fibroblast cells infected with two group IV temperature-sensitive (ts) mutants of influenza A (fowl plague) virus in which the ts lesion maps in RNA segment 8 (J. W. Almond, D. McGeoch, and R. D. Barry, Virology 92:416-427, 1979), known to code to code for two nonstructural proteins, NS1 and NS2. Both mutants induced the synthesis of similar amounts of all the early virus-specific proteins (P1, P2, P3, NP, and NS1) at temperatures that were either permissive (34 degrees C) or nonpermissive (40.5 degrees C) for replication. However, the synthesis of M protein, which normally accumulates late in infection, was greatly reduced in ts mutant-infected cells at 40.5 degrees C compared to 34 degrees C. The NS2 protein was not detected at either temperature in cells infected with one mutant (mN3), and was detected only at the permissive temperature in cells infected with mutant ts47. There was no overall reduction in polyadenylated (A+) complementary RNA, which functions as mRNA, in cells infected with these mutants at 40.5 degrees C compared to 34 degrees C, nor was there any evidence of selective accumulation of this type of RNA within the nucleus at the nonpermissive temperature. No significant differences in ts mutant virion RNA transcriptase activity were detected by assays in vitro at 31 and 40.5 degrees C compared to wild-type virus. Virus-specific non-polyadenylated (A-) complementary RNA, which is believed to act as the template for new virion RNA production, accumulated normally in cells at both 34 and 40.5 degrees C, but at 40.5 degrees C accumulation of new virion RNA was reduced by greater than 90% when compared to accumulation at 34 degrees C.
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PMID:Influenza virus-specific RNA and protein syntheses in cells infected with temperature-sensitive mutants defective in the genome segment encoding nonstructural proteins. 644 1

Hepatitis C virus (HCV) is a positive strand RNA virus with certain similarity to flaviviruses and pestiviruses. To examine the processing and possible assembly of HCV proteins, we constructed a recombinant vaccinia virus that expresses a full-length genomic RNA, infected chimp liver cells with the virus, and analyzed HCV-related protein products by immunofluorescent antibody staining and Western blot detection with mouse monoclonal antibodies. The putative core, envelope, and NS1 and NS3 proteins that yielded from this recombinant were 22, 32, 53 to 58, and 65 kDa in size, respectively. The NS4 protein was unexpectedly small, with an estimated molecular weight of 7 kDa, and the NS5 protein was found to be further cleaved into 52-kDa NS5a and 58-kDa NS5b proteins, the latter of which contains a hallmark of RNA replicase. A point mutation in the putative protease domain of NS3 resulted in a failure in the production of NS3, NS4, NS5a, and NS5b, but coexpression of NS3 restored the proper processing of these proteins, demonstrating that NS3, the putative viral protease, is essential for the production of these nonstructural proteins. Thus, HCV strikingly resembles pestiviruses in the size and the processing mode of the nonstructural proteins, particularly NS4 and NS5.
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PMID:Production of nonstructural proteins of hepatitis C virus requires a putative viral protease encoded by NS3. 829 Dec 45

We recently developed a model for flavivirus infection in mice and hamsters using the Modoc virus (MODV), a flavivirus with no known vector (P. Leyssen, A. Van Lommel, C. Drosten, H. Schmitz, E. De Clercq, and J. Neyts, 2001, Virology 279, 27-37). We now present the coding and noncoding sequence of MODV. The Modoc virus genome was determined to be 10,600 nucleotides in length with a single open reading frame extending from nucleotides 110 to 10,234, encoding 3374 amino acids. The deduced gene order of the single open reading frame is C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5, which is exactly the same as that of the mosquito- and tick-borne flaviviruses. It is flanked by a 5'- and 3'-untranslated region (UTR) of 109 and 366 nucleotides, respectively. Alignment of the MODV amino acid sequence with that of 20 other flaviviruses revealed several regions with high sequence similarity corresponding to functionally important domains (e.g., the serine protease/helicase/NTPase of NS3 and the methyltransferase/RNA-dependent RNA polymerase of NS5) and conserved sites for proteolytic cleavage by viral and cellular proteases. Phylogenetic analysis of the entire coding region confirmed the classification of MODV within the flaviviruses with no known vector, which is in agreement with previous findings based on partial NS5 sequences. A detailed comparative analysis of the putative folding patterns of the 5'- and 3'-UTR of MODV and of the tick- and mosquito-borne viruses was carried out. Structural elements in the 5'- and 3' UTR of MODV that are preserved among vector-borne flaviviruses were noted and so were structural elements distinguishing the MODV UTRs from mosquito-borne and tick-borne flaviviruses. Also the putative secondary structure of circularized MODV RNA is presented.
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PMID:Complete genome sequence, taxonomic assignment, and comparative analysis of the untranslated regions of the Modoc virus, a flavivirus with no known vector. 1185 6

The Kunjin virus (KUNV) has provided a useful laboratory model for Flavivirus RNA replication. The synthesis of progeny RNA(+) strands occurs via asymmetric and semiconservative replication on a template of recycling double-stranded RNA (dsRna) or replicative form (RF). Kinetics of viral RNA synthesis indicated a cycle period of about 15 min during which, on average, a single nascent RNA (+) strand displaces the pre-existing RNA(+) strand in the replicative intermediate. Data on the composition of the replication complex (RC) in KUNV-infected cells were obtained from several sources, including analyses of the partially-purified still active RC, immunogold labeling of cryosections using monospecific antibodies to the nonstructural proteins and to the dsRNA, radioimmunoprecipitations of cell lysates using antibodies to dsRNA and to an RC-associated cell marker, and pull-down assays of cell lysates using fusion proteins GST-NS2A and GST-NS4A. These results yeilded a consensus composition of NS1, NS2A, NS3, NS4A, and NS5 strongly associated with the dsRNA template. The RC was located in induced membranes described as vesicle packets. The RNA-dependent RNA polymerase activity late in infection did not require continuing protein synthesis. Replication of genomic RNA was completely dependent on the presence of conserved complementary or cyclization sequences near the 5' and 3' ends. Assembly of the RC during translation in cis and the relationships, particularly those of NS1 and NS5 among the components, were deduced from an extensive set of complementation experiments in trans involving mutations/deletions in all the nonstructural proteins and use of KUN or alphahavirus replicons as helpers. The KUN replicon has found useful applications also as a noncytopathic vector for the continuing expression of foreign genes, delivered either as packaged RNA or as plasmid DNA.
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PMID:Kunjin RNA replication and applications of Kunjin replicons. 1469 28

The review gives recent data on the structure of hepatitis C virus genome. Linear RNA comprises one translated and two nontranslated regions: 5'UTR and 3'UTR. Translation of viral RNA gives rise to a single polyprotein precursor that contains structural and nonstructural regions. The structural region comprises one nucleocapsid core-protein and two envelope proteins known as E1 and E2. E2 protein includes two hypervariable regions: HVR1 and HVR2. The nonstructural region consists of 7 proteins: p7 (NS1), NS (metalloproteinase), NS (serine protease/ helicase), NS4A (co-factor protease, co-factor RNA-dependent RNA polymerase), and NS4B, NS5A, NS5B (RNA-dependent RNA polymerase). NS5A includes ISDR (interferon-sensitivity-determining region).
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PMID:[Structural and functional organization of hepatitis C virus genome]. 1675 71

Influenza viruses replicate and transcribe their segmented negative-sense single-stranded RNA genome in the nucleus of the infected host cell. All RNA synthesising activities associated with influenza virus are performed by the virally encoded RNA-dependent RNA polymerase (RdRp) that consists of three subunits, PA, PB1 and PB2. However, viral transcription is critically dependent on on-going cellular transcription, in particular, on activities associated with the cellular DNA-dependent RNA polymerase II (Pol II). Thus, the viral RdRp uses short 5' capped RNA fragments, derived from cellular Pol II transcripts, as primers for viral mRNA synthesis. These capped RNA primers are generated by cleavage of host Pol II transcripts by an endonuclease activity associated with the viral RdRp. Moreover, some viral transcripts require splicing and since influenza virus does not encode splicing machinery, it is dependent on host splicing, an activity also related to Pol II transcription. Despite these functional links between viral and host Pol II transcription, there has been no evidence that a physical association existed between the two transcriptional machineries. However, recently it was reported that there is a physical interaction between the trimeric viral RdRp and cellular Pol II. The viral RdRp was found to interact with the C-terminal domain (CTD) of initiating Pol II, at a stage in the transcription cycle when capping takes place. It was therefore proposed that this interaction may be required for the viral RNA (vRNA) polymerase to gain access to capped RNA substrates for endonucleolytic cleavage. The virus not only relies on cellular factors to support its own RNA synthesis, but also subverts cellular pathways in order to generate an environment optimised for viral multiplication. In this respect, the interaction of the viral NS1 protein with factors involved in cellular pre-mRNA processing is of particular relevance. The virus also alters the distribution of Pol II on cellular genes, leading to a reduction in elongating Pol II thereby contributing to the phenomenon known as host shut-off.
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PMID:Functional association between viral and cellular transcription during influenza virus infection. 1693 65

Influenza A virus transcribes its segmented negative sense RNA genome in the nuclei of infected cells in a process long known to require host RNA polymerase II (RNAP-II). RNA polymerase II synthesizes pre-mRNAs whose 5'-cap structures are scavenged by the viral RNA-dependent RNA polymerase during synthesis of viral mRNAs. Drugs that inhibit RNAP-II therefore block viral replication, but not necessarily solely by denying the viral polymerase a source of cap-donor molecules. We show here that 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), a compound that prevents processive transcription by RNAP-II, inhibits expression of the viral HA, M1 and NS1 genes at the post-transcriptional level. Abundant quantities of functionally and structurally intact viral mRNAs are made in the presence of DRB but with the exception of NP and NS2 mRNAs, are not efficiently translated. Taking M1 and NP mRNAs as representatives of DRB-sensitive and insensitive mRNAs, respectively, we found that the block to translation operates at the level of nuclear export. Furthermore, removal of DRB reversed this block unless a variety of chemically and mechanistically distinct RNAP-II inhibitors were added instead. We conclude that influenza A virus replication requires RNAP-II activity not just to provide capped mRNA substrates but also to facilitate nuclear export of selected viral mRNAs.
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PMID:Nuclear export of influenza A virus mRNAs requires ongoing RNA polymerase II activity. 1713 45

The organization of flaviviral replicase proteins within the membrane-bound replication complexes of West Nile (WNV), dengue (DENV) and Japanese encephalitis viruses (JEV) was probed by investigating the combined effect of detergents and trypsin on both viral replicase activity and profile of metabolically labelled viral proteins. While trypsin treatment of virus-induced membrane fractions degraded the vast majority of replicase proteins, viral RNA-dependent RNA polymerase (RdRp) activity remained completely unaffected. Solubilization of the membranes with deoxycholate (DOC) however rendered the replicase accessible to trypsin. Triton X-100 (TX100) treatment reduced RdRp activity by half in WNV but totally destroyed RdRp activity in JEV. TX100 also dissociated NS1' in addition to NS1 from NS5 and NS3 inJEV. Antibodies to NS3 coprecipitated NS1' along with NS5 only from DOC-solubilized but not from TX100-treated extracts, the former of which alone retained RdRp activity. Exogenous addition of recombinant NS1' to TX100 treated JEV-induced membranes restored the defect in the release step of RNA synthesis. Our results suggest for the first time a direct role for JEV NS1' in viral RNA synthesis in vitro.
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PMID:Organization of flaviviral replicase proteins in virus-induced membranes: a role for NS1' in Japanese encephalitis virus RNA synthesis. 1731 59


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