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)

In order to investigate the possible function of the potexviral conserved hexanucleotide sequence (ACc/uUAA) found in the 3' untranslated region of bamboo mosaic potexvirus (BaMV) genomic RNA, each nucleotide in the hexamer motif was substituted. Transcripts derived from wild-type and mutants with a loop deletion or a single-nucleotide substitution were inoculated into protoplasts. The accumulation levels of viral coat protein and RNAs detected from Western and Northern blots of each inoculation were examined after a 48-h incubation. Our data revealed that the nucleotides at positions 4-6 of the hexamer motif cannot be replaced by other nucleotides; the first position of this hexamer is purine specific, and the second position is restricted to pyrimidine. Substitution at the third position has less effect on viral accumulation in protoplasts. In addition to the results reported previously that the E. coli over-expressed BaMV RNA-dependent RNA polymerase could specifically interact with the hexamer motif, the hexanucleotides were thought to serve as a recognition site of viral replicase and the specificity may be derived from the functional groups of each nucleotide of this hexamer motif.
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PMID:Specificity analysis of the conserved hexanucleotides for the replication of bamboo mosaic potexvirus RNA. 1186 48

The hepatitis C virus (HCV)-encoded NS5B protein is an RNA-dependent RNA polymerase which plays a substantial role in viral replication. We expressed and purified the recombinant NS5B of an HCV genotype 3a from Esherichia coli, and we investigated its ability to bind to the viral RNA and its enzymatic activity. The results presented here demonstrate that NS5B interacts strongly with the coding region of positive-strand RNA, although not in a sequence-specific manner. It was also determined that more than two molecules of polymerase bound sequentially to this region with the direction 3' to 5'. Also, we attempted to determine the initiation site(s) of de novo synthesis by NS5B on X RNA, which contains the last 98 nucleotides of HCV positive-strand RNA. The initiation site(s) on X RNA was localized in the pyrimidine-rich region of stem I. However, when more than five of the nucleotides of stem I in X RNA were deleted from the 3' end, RNA synthesis initiated at another site of the specific ribonucleotide. Our study also showed that the efficiency of RNA synthesis, which was directed by X RNA, was maximized by the GC base pair at the penultimate position from the 3' end of the stem. These results will provide some clues to understanding the mechanism of HCV genomic RNA replication in terms of viral RNA-NS5B interaction and the initiation of de novo RNA synthesis.
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PMID:Template requirements for de novo RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase on the viral X RNA. 1207 95

De novo RNA synthesis by hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase has been investigated using short RNA templates. Various templates including those derived from the HCV genome were evaluated by examining the early steps of de novo RNA synthesis. NS5B was shown to be able to produce an initiation dinucleotide product from templates as short as 4-mer and from the 3'-terminal sequences of both plus and minus strands of the HCV RNA genome. GMP, GDP, and guanosine were able to act as an initiating nucleotide in de novo RNA synthesis, indicating that the triphosphate moiety is not absolutely required by an initiating nucleotide. Significant amounts of the initiation product accumulated in de novo synthesis, and elongation from the dinucleotide was observed when large amounts of dinucleotide were available. This result suggests that NS5B, a template, and incoming nucleotides are able to form an initiation complex that aborts frequently by releasing the dinucleotide product before transition to an elongation complex. The transition is rate limiting. Furthermore, we discovered that the secondary structure of a template was not essential for de novo initiation and that 3'-terminal bases of a template conferred specificity in selection of an initiation site. Initiation can occur at the +1, +2, or +3 position numbered from the 3' end of a template depending on base composition. Pyrimidine bases at any of the three positions are able to serve as an initiation site, while purine bases at the +2 and +3 positions do not support initiation. This result implies that HCV possesses an intrinsic ability to ensure that de novo synthesis is initiated from the +1 position and to maintain the integrity of the 3' end of its genome. This assay system should be an important tool for investigating the detailed mechanism of de novo initiation by HCV NS5B as well as other viral RNA polymerases.
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PMID:Selection of 3'-template bases and initiating nucleotides by hepatitis C virus NS5B RNA-dependent RNA polymerase. 1207 3

Nucleoside chain terminators represent one of the most promising classes of antiviral drug for DNA viruses and retroviral infection; however, they have not been fully explored against RNA viral polymerases. In this report, we investigate the notion of employing canonical 3'-deoxyribonucleoside triphosphates (3'-dNTPs) as a chain terminator for hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp). Using a HCV RNA transcript-dependent RNA elongating assay, we found that they inhibit NS5B RdRp with K(i) ranged from 0.7 to 23 microM. Additional structure-activity relationship studies showed that removal of 2'-hydroxyl group, elimination of ribose's 2',3'-carbon-carbon bond, or addition of 5-methyl group to a pyrimidine base is detrimental to 3'-dNTP's potency. Direct evidence was obtained that all four canonical 3'-dNTP are incorporated into elongating RNA chains and the incorporation terminates NS5B RdRp-catalyzed RNA synthesis. The K(i) values for each of 3'-dNTPs were determined in the single nucleotide incorporation experiments. The nucleoside form of 3'-dNTPs was further evaluated in a cell culture-based HCV subgenomic replicon assay. The discrepancy between the potent in vitro activity and the weak cellular activity of these chain terminators was discussed in the context of nucleoside metabolism. This proof of concept study demonstrates that canonical 3'-dNTPs can function as an effective chain terminator for HCV NS5B RdRp with cytidine as the preferred nucleoside scaffold. Our results further sheds light on the potential hurdles that need to be overcome for successful development of active nucleoside chain terminators in vivo for a viral RNA polymerase, especially the HCV NS5B RdRp.
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PMID:Canonical 3'-deoxyribonucleotides as a chain terminator for HCV NS5B RNA-dependent RNA polymerase. 1276 72

Point mutations that resulted in a substitution of the conserved 3'-penultimate cytidine in genomic RNA or the RNA negative strand of the self-amplifying replicon of the Flavivirus Kunjin virus completely blocked in vivo replication. Similarly, substitutions of the conserved 3'-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively. The same preference for cytidine in the 3'-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis. The Kunjin virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of phi6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3'-penultimate cytidines and a 3'-terminal pyrimidine. A previously untested substitution of the conserved pentanucleotide at the top of the 3'-terminal stem-loop of all Flavivirus species also blocked detectable in vivo replication of the Kunjin virus replicon RNA.
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PMID:Significance in replication of the terminal nucleotides of the flavivirus genome. 1297 Apr 46

Replication of nearly all RNA viruses depends on a virus-encoded RNA-dependent RNA polymerase (RdRp). Our earlier work found that purified recombinant hepatitis C virus (HCV) RdRp (NS5B) was able to initiate RNA synthesis de novo by using purine (A and G) but not pyrimidine (C and U) nucleotides (G. Luo et al., J. Virol. 74:851-863, 2000). For most human RNA viruses, the initiation nucleotides of both positive- and negative-strand RNAs were found to be either an adenylate (A) or guanylate (G). To determine the nucleotide used for initiation and control of HCV RNA replication, a genetic mutagenesis analysis of the nucleotides at the very 5' and 3' ends of HCV RNAs was performed by using a cell-based HCV replicon replication system. Either a G or an A at the 5' end of HCV genomic RNA was able to efficiently induce cell colony formation, whereas a nucleotide C at the 5' end dramatically reduced the efficiency of cell colony formation. Likewise, the 3'-end nucleotide U-to-C mutation did not significantly affect the efficiency of cell colony formation. In contrast, a U-to-G mutation at the 3' end caused a remarkable decrease in cell colony formation, and a U-to-A mutation resulted in a complete abolition of cell colony formation. Sequence analysis of the HCV replicon RNAs recovered from G418-resistant Huh7 cells revealed several interesting findings. First, the 5'-end nucleotide G of the replicon RNA was changed to an A upon multiple rounds of replication. Second, the nucleotide A at the 5' end was stably maintained among all replicon RNAs isolated from Huh7 cells transfected with an RNA with a 5'-end A. Third, initiation of HCV RNA replication with a CTP resulted in a >10-fold reduction in the levels of HCV RNAs, suggesting that initiation of RNA replication with CTP was very inefficient. Fourth, the 3'-end nucleotide U-to-C and -G mutations were all reverted back to a wild-type nucleotide U. In addition, extra U and UU residues were identified at the 3' ends of revertants recovered from Huh7 cells transfected with an RNA with a nucleotide G at the 3' end. We also determined the 5'-end nucleotide of positive-strand RNA of some clinical HCV isolates. Either G or A was identified at the 5' end of HCV RNA genome depending on the specific HCV isolate. Collectively, these findings demonstrate that replication of positive-strand HCV RNA was preferentially initiated with purine nucleotides (ATP and GTP), whereas the negative-strand HCV RNA replication is invariably initiated with an ATP.
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PMID:Effects of mutations of the initiation nucleotides on hepatitis C virus RNA replication in the cell. 1501 84

We reported earlier the isolation and characterization of a Sindbis virus mutant, SV(PZF), that can grow in mosquito cells treated with pyrazofurin (PZF), a compound that interferes with pyrimidine biosynthesis (Y. H. Lin, P. Yadav, R. Ravatn, and V. Stollar, Virology 272:61-71, 2000; Y. H. Lin, H. A. Simmonds, and V. Stollar, Virology 292:78-86, 2002). Three amino acid changes in nsP4, the viral RNA polymerase, were required to produce this phenotype. We now describe a mutant of Sindbis virus, SVCPC, that is resistant to cyclopentenylcytosine (CPC), a compound that interferes only with the synthesis of CTP. Thus, in contrast to SVPZF, which was selected for its ability to grow in mosquito cells with low levels of UTP and CTP, SVCPC was selected for its ability to grow in cells in which only the level of CTP was reduced. Although SV(PZF) was cross-resistant to CPC, SVCPC was not resistant to PZF. Only one amino acid change in nsP4, Leu 585 to Phe, was required for the CPC resistance phenotype. The viral replicase/transcriptase generated in SVCPC-infected mosquito cells had a lower Km for CTP (but not for UTP) than did the enzyme made in SVSTD-infected mosquito cells. SV(PZF) and SVCPC represent the first examples of viral mutants selected for the ability to grow in cells with low levels of ribonucleoside triphosphates (rNTPs). Further study of these mutants and determination of the structure of nsP4 should demonstrate how alterations in an RNA-dependent RNA polymerase permit it to function in cells with abnormally low levels of rNTPs.
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PMID:A mutant of Sindbis virus which is able to replicate in cells with reduced CTP makes a replicase/transcriptase with a decreased Km for CTP. 1533 97

The aim of the present study was to estimate the expression of mRNA, specific for thymidine kinase 1 (TK1), deoxycytidine kinase (dCK), and thymidine phosphorylase (dThdPase), i.e. enzymes involved in pyrimidine and purine metabolism in human papillary thyroid carcinoma (PTC) tissue. Additionally, the expression of dCK was estimated, in medullary thyroid carcinoma (MTC). For control, the RNA expression levels for all the enzymes were measured in macroscopically unchanged thyroid tissue. Reverse transcriptase-polymerase chain reaction (RT-PCR) and densitometry were employed for mRNA expression measurements, with the beta-actin gene as a control housekeeping gene. The levels of mRNA expression for TK1, dCK and dThdPase in human PTC, as well as mRNA expression for dCK in MTC, were significantly higher than mRNA expressions for those enzymes found in macroscopically unchanged thyroid tissue. It is concluded that an increased expression of mRNA, specific for TK1, dCK and dThdPase, may be involved in carcinogenic processes in the human thyroid.
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PMID:Increased expression of mRNA specific for thymidine kinase, deoxycytidine kinase or thymidine phosphorylase in human papillary thyroid carcinoma. 1597 30

The pyrimidine nucleoside beta-d-2'-deoxy-2'-fluoro-2'-C-methylcytidine (1) was designed as a hepatitis C virus RNA-dependent RNA polymerase (HCV RdRp) inhibitor. The title compound was obtained by a DAST fluorination of N(4)-benzoyl-1-(2-methyl-3,5-di-O-benzoyl-beta-d-arabinofuranosyl]cytosine to provide N(4)-benzoyl-1-[2-fluoro-2-methyl-3,5-di-O-benzoyl-beta-d-ribofuranosyl]cytosine. The protected 2'-C-methylcytidine was obtained as a byproduct from the DAST fluorination and allowed for the preparation of two biologically active compounds from a common precursor. Compound 1 and 2'-C-methylcytidine were assayed in a subgenomic HCV replicon assay system and found to be potent and selective inhibitors of HCV replication. Compound 1 shows increased inhibitory activity in the HCV replicon assay compared to 2'-C-methylcytidine and low cellular toxicity.
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PMID:Design, synthesis, and antiviral activity of 2'-deoxy-2'-fluoro-2'-C-methylcytidine, a potent inhibitor of hepatitis C virus replication. 1610 49

The 5'-cloverleaf of the picornavirus RNA genome is essential for the assembly of a ribonucleoprotein replication complex. Stem-loop D (SLD) of the cloverleaf is the recognition site for the multifunctional viral protein 3Cpro. This protein is the principal viral protease, and its interaction with SLD also helps to position the viral RNA-dependent RNA polymerase (3Dpol) for replication. Human rhinovirus-14 (HRV-14) is distinct from the majority of picornaviruses in that its SLD forms a cUAUg triloop instead of the more common uYACGg tetraloop. This difference appears to be functionally significant, as 3Cpro from tetraloop-containing viruses cannot bind the HRV-14 SLD. We have determined the solution structure of the HRV-14 SLD using NMR spectroscopy. The structure is predominantly an A-form helix, but with a central pyrimidine-pyrimidine base-paired region and a significantly widened major groove. The stabilizing hydrogen bonding present in the uYACGg tetraloop was not found in the cUAUg triloop. However, the triloop uses different structural elements to present a largely similar surface: sequence and underlying architecture are not conserved, but key aspects of the surface structure are. Important structural differences do exist, though, and may account for the observed cross-isotype binding specificities between 3Cpro and SLD.
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PMID:NMR structure of stem-loop D from human rhinovirus-14. 1719 19


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