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)

The active RNA-dependent RNA polymerase of poliovirus, 3Dpol, is generated by cleavage of the 3CDpro precursor protein, a protease that has no polymerase activity despite containing the entire polymerase domain. By intentionally disrupting a known and persistent crystal packing interaction, we have crystallized the poliovirus polymerase in a new space group and solved the complete structure of the protein at 2.0 A resolution. It shows that the N-terminus of fully processed 3Dpol is buried in a surface pocket where it makes hydrogen bonds that act to position Asp238 in the active site. Asp238 is an essential residue that selects for the 2' OH group of substrate rNTPs, as shown by a 2.35 A structure of a 3Dpol-GTP complex. Mutational, biochemical, and structural data further demonstrate that 3Dpol activity is exquisitely sensitive to mutations at the N-terminus. This sensitivity is the result of allosteric effects where the structure around the buried N-terminus directly affects the positioning of Asp238 in the active site.
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PMID:Structural basis for proteolysis-dependent activation of the poliovirus RNA-dependent RNA polymerase. 1530 52

Improved treatments for chronic hepatitis C virus (HCV) infection are needed due to the suboptimal response rates and deleterious side effects associated with current treatment options. The triphosphates of 2'-C-methyl-adenosine and 2'-C-methyl-guanosine were previously shown to be potent inhibitors of the HCV RNA-dependent RNA polymerase (RdRp) that is responsible for the replication of viral RNA in cells. Here we demonstrate that the inclusion of a 7-deaza modification in a series of purine nucleoside triphosphates results in an increase in inhibitory potency against the HCV RdRp and improved pharmacokinetic properties. Notably, incorporation of the 7-deaza modification into 2'-C-methyl-adenosine results in an inhibitor with a 20-fold-increased potency as the 5'-triphosphate in HCV RdRp assays while maintaining the inhibitory potency of the nucleoside in the bicistronic HCV replicon and with reduced cellular toxicity. In contrast, while 7-deaza-2'-C-methyl-GTP also displays enhanced inhibitory potency in enzyme assays, due to poor cellular penetration and/or metabolism, the nucleoside does not inhibit replication of a bicistronic HCV replicon in cell culture. 7-Deaza-2'-C-methyl-adenosine displays promising in vivo pharmacokinetics in three animal species, as well as an acute oral lethal dose in excess of 2,000 mg/kg of body weight in mice. Taken together, these data demonstrate that 7-deaza-2'-C-methyl-adenosine is an attractive candidate for further investigation as a potential treatment for HCV infection.
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PMID:A 7-deaza-adenosine analog is a potent and selective inhibitor of hepatitis C virus replication with excellent pharmacokinetic properties. 1538 57

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has several distinct biochemical activities, including initiation of RNA synthesis by a de novo mechanism, extension from a primed template, nontemplated nucleotide addition, and synthesis of a recombinant RNA product from two or more noncovalently linked templates (template switch). All of these activities require specific interaction with nucleoside triphosphates (NTPs). Based on the structure of the HCV RdRp bound to NTP (S. Bressanelli, L. Tomei, F. A. Rey, and R. DeFrancesco, J. Virol. 76:3482-3492, 2002), we mutated the amino acid residues that contact the putative initiation GTP and examined the effects on the various activities. Although all mutations retained the ability for primer extension, alanine substitution at R48, R158, R386, R394, or D225 decreased de novo initiation, and two or more mutations abolished de novo initiation. While the prototype enzyme had a K(m) for GTP of 3.5 microM, all of the mutations except one had K(m)s that were three- to sevenfold higher. These results demonstrate that the affected residues are functionally required to interact with the initiation nucleotide. Unexpectedly, many of the mutations also affected the addition of nontemplated nucleotide, indicating that residues in the initiating NTP (NTPi)-binding pocket are required for nontemplated nucleotide additions. Interestingly, mutations in D225 are dramatically affected in template switch, indicating that this residue of the NTPi pocket also interacts with components in the elongation complex. We also examined the interaction of ribavirin triphosphate with the NTPi-binding site.
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PMID:De novo initiation pocket mutations have multiple effects on hepatitis C virus RNA-dependent RNA polymerase activities. 1550 7

Several nucleotide analogues have been described as inhibitors of NS5B, the essential viral RNA-dependent RNA polymerase of hepatitis C virus. However, their precise mode of action remains poorly defined at the molecular level, much like the different steps of de novo initiation of viral RNA synthesis. Here, we show that before elongation, de novo RNA synthesis is made of at least two distinct kinetic phases, the creation of the first phosphodiester bond being the most efficient nucleotide incorporation event. We have studied 2'-O-methyl-GTP as an inhibitor of NS5B-directed RNA synthesis. As a nucleotide competitor of GTP in RNA synthesis, 2'-O-methyl-GTP is able to act as a chain terminator and inhibit RNA synthesis. Relative to GTP, we find that this analogue is strongly discriminated against at the initiation step ( approximately 150-fold) compared with approximately 2-fold at the elongation step. Interestingly, discrimination of the 2'-O-methyl-GTP at initiation is suppressed in a variant NS5B deleted in a subdomain critical for initiation (the "flap," encompassing amino acids 443-454), but not in P495L NS5B, which shows a selective alteration of transition from initiation to elongation. Our results demonstrate that the conformational change occurring between initiation and elongation is dependent on the allosteric GTP-binding site and relaxes nucleotide selectivity. RNA elongation may represent the most probable target of 2'-modified nucleotide analogues, because it is more permissive to inhibition than initiation.
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PMID:A relaxed discrimination of 2'-O-methyl-GTP relative to GTP between de novo and Elongative RNA synthesis by the hepatitis C RNA-dependent RNA polymerase NS5B. 1556 9

Infectious pancreatic necrosis virus (IPNV) is a bi-segmented, dsRNA virus of the Birnaviridae family. The structural protein VP1 has been postulated as the RNA-dependent RNA polymerase (RdRp), but its transcriptional activity has not been unequivocally identified from viral particles. Here, we assayed partially purified IPNV in an in vitro RNA synthesis system. To test the RdRp, dialdehyde-nucleotide analogs were used to covalently inhibit the polymerase-associated activity. Our results showed that dialdehyde-nucleotide analogs completely abrogated IPNV in vitro RNA synthesis. The protein involved in this process was identified as viral VP1, since: (a) after incubation of IPNV with [alpha-(32)P]2',3'-dialdehyde-UTP, labeled VP1 protein was identified and (b) VP1 was unable to bind [alpha-(32)P]GTP when particles were preincubated with 2',3'-dialdehyde-ATP. Thus, within viral particles, inhibition of the transcriptional activity is a result of the binding of 2',3'-dialdehyde-nucleotide analogs to the RdRp, VP1.
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PMID:Inhibition of virion-associated IPNV RNA polymerase, VP1, by radiolabeled nucleotide analogs. 1602 7

The kinetic properties of Qbeta replicase, an RNA-dependent RNA polymerase, were investigated experimentally. The reaction at the A-incorporation site was inhibited by UTP and CTP with inhibition constants of 3.2 and 2.7 mM, respectively, while the reactions at the U-, G-, and C-incorporation sites were inhibited by ATP with inhibition constants of 1.09, 1.25, and 1.48 mM, respectively. When nucleotide concentrations were low, C was incorporated at the fastest rate and G at the slowest. Accordingly, the G-incorporation step largely limits the overall reaction rate. From the obtained kinetic parameters, calculations showed that the optimum ratio of the concentrations of the four nucleotides could be achieved by increasing the ratio of GTP concentration with a concomitant decrease in the ratios of CTP and ATP concentrations. Consequently, a 60 to 140% increase in the reaction rate is expected as compared to the rate with equimolar ratio of the four nucleotides.
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PMID:Kinetic properties of Qbeta replicase, an RNA dependent RNA polymerase. 1623 8

The crystal structure of the recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) revealed extensive interactions between the fingers and the thumb subdomains, resulting in a closed conformation with an established template channel that should specifically accept single-stranded templates. We made circularized RNA templates and found that they were efficiently used by the HCV RdRp to synthesize product RNAs that are significantly longer than the template, suggesting that RdRp could exist in an open conformation prior to template binding. RNA synthesis using circular RNA templates had properties similar to those previously documented for linear RNA, including a need for higher GTP concentration for initiation, usage of GTP analogs, sensitivity to salt, and involvement of active-site residues for product formation. Some products were resistant to challenge with the template competitor heparin, indicating that the elongation complexes remain bound to template and are competent for RNA synthesis. Other products were not elongated in the presence of heparin, indicating that the elongation complex was terminated. Lastly, recombinant RdRps from two other flaviviruses and from the Pseudomonas phage phi6 also could use circular RNA templates for RNA-dependent RNA synthesis, although the phi6 RdRp could only use circular RNAs made from the 3'-terminal sequence of the phi6 genome.
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PMID:Recombinant viral RdRps can initiate RNA synthesis from circular templates. 1637 81

Here we report a detailed characterization of the biochemical and kinetic properties of the hepatitis C virus (HCV, genotype-1b, J4 consensus) RNA-dependent RNA polymerase NS5B, by performing comprehensive RNA binding, nucleotide incorporation, and protein/protein oligomerization studies. By applying equilibrium fluorescence titrations, we determined a surprisingly high dissociation constant (K(d)) of approximately 250 nM for single-stranded as well as for partially double-stranded RNA. A detailed analysis of the nucleic acid binding mechanism using pre-steady-state techniques revealed the association reaction to be nearly diffusion controlled. It occurs in a single step with a second-order rate constant (k(on)) of 0.273 nM(-)(1) s(-)(1). The dissociation of the nucleic acid-polymerase complex is fast with a dissociation rate constant (k(off)) of 59.3 s(-)(1). With short, partially double-stranded RNAs, no nucleotide incorporation could be observed, while de novo RNA synthesis with short RNA templates showed nucleotide incorporation and end-to-end template switching events. Single-turnover, single-nucleotide incorporation studies (representing here the initiation and not processive polymerization) using dinucleotide primers revealed a very slow incorporation rate (k(pol)) of 0.0007 s(-)(1) and a K(d) of the binary enzyme-nucleic acid complex for the incoming ATP of 27.7 microM. Using dynamic laser light scattering, it could be shown for the first time that oligomerization of HCV NS5B is a dynamic and monovalent salt concentration dependent process. While NS5B is highly oligomeric at low salt concentrations, monomers were only observed at NaCl concentrations above 300 mM. Binding of short RNA substrates led to a further increase in oligomerization, whereas GTP did not show any effect on protein/protein interactions. Furthermore, nucleotide incorporation studies indicate the oligomerization state does not correlate with enzymatic activities as previously proposed.
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PMID:Biochemical and pre-steady-state kinetic characterization of the hepatitis C virus RNA polymerase (NS5BDelta21, HC-J4). 1653 43

Flavivirus protein NS5 harbors the RNA-dependent RNA polymerase (RdRp) activity. In contrast to the RdRps of hepaci- and pestiviruses, which belong to the same family of Flaviviridae, NS5 carries two activities, a methyltransferase (MTase) and a RdRp. RdRp domains of Dengue virus (DV) and West Nile virus (WNV) NS5 were purified in high yield relative to full-length NS5 and showed full RdRp activity. Steady-state enzymatic parameters were determined on homopolymeric template poly(rC). The presence of the MTase domain does not affect the RdRp activity. Flavivirus RdRp domains might bear more than one GTP binding site displaying positive cooperativity. The kinetics of RNA synthesis by four Flaviviridae RdRps were compared. In comparison to Hepatitis C RdRp, DV and WNV as well as Bovine Viral Diarrhea virus RdRps show less rate limitation by early steps of short-product formation. This suggests that they display a higher conformational flexibility upon the transition from initiation to elongation.
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PMID:Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases. 1663 Dec 21

A membrane-bound RNA-dependent RNA polymerase (RdRp) complex was isolated by differential sedimentation from oat plants infected with cereal yellow dwarf virus (CYDV). When incubated with 32P-labelled UTP, unlabelled ATP, CTP and GTP, and Mg2+ ions, the RdRp preparation catalysed the synthesis of double-stranded (ds) RNAs corresponding in size to the virus genomic RNA (5.7 kbp) and two putative subgenomic RNAs (2.8 and 0.7 kbp). Hybridisation using strand-specific hybridization targets showed that the 5.7-kbp dsRNA was labelled mainly in the plus strand, whereas the 2.8- and 0.7-kbp dsRNAs were labelled only in the minus strand. Genomic-length single-stranded, plus-strand RNA of 5.7 kb and single-stranded, plus-strand subgenomic RNAs of 2.8 and 0.7 kbp were detected in RNA isolated from oat plants infected with CYDV. Mapping experiments were consistent with the genomic and subgenomic RNAs having common 3' ends, but different 5' ends, whether produced in vitro or in vivo. The RdRp-encoding region of the CYDV genome was cloned and expressed in Escherichia coli, and the purified protein was used to raise antibodies in a rabbit. In immunoblots, the antibodies detected a protein of about 68 kDa in RdRp preparations from CYDV-infected oat plants, but not from equivalent preparations from healthy oats. As far as we are aware, this is the first report of an in vitro RNA synthesis system for a phloem-limited virus.
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PMID:Synthesis of genomic and subgenomic RNAs by a membrane-bound RNA-dependent RNA polymerase isolated from oat plants infected with cereal yellow dwarf virus. 1675 73


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