The rate-limiting step for nucleotide incorporation in the presteady state for most nucleic acid polymerases is thought to be a conformational change. As a result, very little information is available on the role of active-site residues in the chemistry of nucleotidyl transfer. For the poliovirus RNA-dependent RNA polymerase (3D pol ), chemistry is partially (Mg 2؉ ) or completely (Mn 2؉ ) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pK a values of 7.0 or 8.2 and 10.5, depending upon the divalent cation used in the reaction. A solvent deuterium isotope effect of three to seven was observed on the rate constant for nucleotide incorporation in the pre-steady state; none was observed in the steady state. Proton-inventory experiments were consistent with two protons being transferred during the rate-limiting transition state of the reaction, suggesting that both deprotonation of the 3 -hydroxyl nucleophile and protonation of the pyrophosphate leaving group occur in the transition state for phosphodiester bond formation. Importantly, two proton transfers occur in the transition state for nucleotidyl-transfer reactions catalyzed by RB69 DNA-dependent DNA polymerase, T7 DNA-dependent RNA polymerase and HIV reverse transcriptase. Interpretation of these data in the context of known polymerase structures suggests the existence of a general base for deprotonation of the 3 -OH nucleophile, although use of a water molecule cannot be ruled out conclusively, and a general acid for protonation of the pyrophosphate leaving group in all nucleic acid polymerases. These data imply an associative-like transition-state structure.general-acid-base catalysis ͉ phosphoryl transfer ͉ two-metal-ion mechanism N ucleic acid polymerases are essential for the maintenance and expression of the genomes of all organisms. All classes of polymerases use the same five-step kinetic scheme for nucleotide incorporation (1-6). The kinetic mechanism for the RNAdependent RNA polymerase (RdRp 1 ) from poliovirus (3D pol ) is shown in Scheme 1. One of the advantages of this system is that once 3D pol assembles onto the primer-template substrate, this complex has a half-life of Ͼ2 h (7), greatly simplifying kinetic analysis. In step one, the enzyme-nucleic acid complex (ER n ) binds the nucleoside triphosphate forming a ternary complex (ER n NTP).Step two involves a conformational change (*ER n NTP) that orients the triphosphate for catalysis. In step three, nucleotidyl transfer occurs (*ER nϩ1 PP i ), followed by a second conformational-change step (ER nϩ1 PP i ) and pyrophosphate release (ER nϩ1 ).Although the sequence of events occurring during the nucleotide-addition cycle is identical for all polymerases, the ratelimiting step appears to be different. In most cases, the first conformational-change step (step two) is rate-limiting (2, 8, 9). In one, chemistry (step three) is rate-limiting (10), and in some (e.g., T4 and RB69 DNA polymerases), the rate-limiting step has not been established. For 3D pol , bot...

Synthetic small molecules that promote viral mutagenesis represent a promising new class of antiviral therapeutics. Ribavirin is a broad-spectrum antiviral nucleoside whose antiviral mechanism against RNA viruses likely reflects the ability of this compound to introduce mutations into the viral genome. The mutagenicity of ribavirin results from the incorporation of ribavirin triphosphate opposite both cytidine and uridine in viral RNA. In an effort to identify compounds with mutagenicity greater than that of ribavirin, we synthesized 1-beta-D-ribofuranosyl-3-nitropyrrole (3-NPN) and the corresponding triphosphate (3-NPNTP). These compounds constitute RNA analogues of the known DNA nucleoside 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole. The 3-nitropyrrole pseudobase has been shown to maintain the integrity of DNA duplexes when placed opposite any of the four nucleobases without requiring hydrogen bonding. X-ray crystallography revealed that 3-NPN is structurally similar to ribavirin, and both compounds are substrates for adenosine kinase, an enzyme critical for conversion to the corresponding triphosphate in cells. Whereas ribavirin exhibits antiviral activity against poliovirus in cell culture, 3-NPN lacks this activity. Evaluation of 3-NPNTP utilization by poliovirus RNA-dependent RNA polymerase (RdRP) revealed that 3-NPNTP was not accepted universally. Rather, incorporation was only observed opposite A and U in the template and at a rate 100-fold slower than the rate of incorporation of ribavirin triphosphate. This diminished rate of incorporation into viral RNA likely precludes 3-NPN from functioning as an antiviral agent. These results indicate that hydrogen bonding substituents are critical for efficient incorporation of ribonucleotides into RNA by viral RdRPs, thus providing important considerations for the design of improved mutagenic antiviral nucleosides.

Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses.

Studies of the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV), 3Dpol, have shown that Asn-297 permits this enzyme to distinguish ribose from 2 -deoxyribose. All animal RNA viruses have Asn at the structurally homologous position of their polymerases, suggesting a conserved function for this residue. However, all prokaryotic RNA viruses have Glu at this position. In the presence of Mg 2؉ , the apparent affinity of Glu-297 3Dpol for 2 -deoxyribonucleotides was decreased by 6-fold relative to wild type without a substantial difference in the fidelity of 2 -dNMP incorporation. The fidelity of ribonucleotide misincorporation for Glu-297 3Dpol was reduced by 14-fold relative to wild type. A 4-to 11-fold reduction in the rate of ribonucleotide incorporation was observed. Glu-297 PV was unable to grow in HeLa cells due to a replication defect equivalent to that observed for a mutant PV encoding an inactive polymerase. Evaluation of the protein-(VPg)-primed initiation reaction showed that only half of the Glu-297 3Dpol initiation complexes were capable of producing VPg-pUpU product and that the overall yield of uridylylated VPg products was reduced by 20-fold relative to wild-type enzyme, a circumstance attributable to a reduced affinity for UTP. These studies identify the first RdRp derivative with a mutator phenotype and provide a mechanistic basis for the elevated mutation frequency of RNA phage relative to animal RNA viruses observed in culture. Although protein-primed initiation and RNA-primed elongation complexes employ the same polymerase active site, the functional differences reported here imply significant structural differences between these complexes.RNA viruses cause a variety of acute and chronic diseases in humans: common cold, summer flu, hepatitis, severe acute respiratory syndrome, and liver cancer, to name a few (1-5). The genomes of these viruses are transcribed and replicated by a virus-encoded RNA-dependent RNA polymerase (RdRp) 2 (4, 6, 7). Like other viral polymerases, the RdRp represents an important target for antiviral drug development (8 -11). The RdRp from poliovirus (PV), 3Dpol, has emerged as an important model for understanding the structure, function, and mechanism of this class of nucleic acid polymerases (12-15).PV 3Dpol will incorporate a ribonucleotide (rNMP) with an incorrect base at a frequency of ϳ10 Ϫ7 to 10 Ϫ4 (16, 17). However, this enzyme is much more tolerant of nucleotides with an incorrect sugar configuration. Both 2Ј-and 3Ј-deoxynucleotides (dNMPs) are incorporated at a frequency of ϳ10 Ϫ2 (15). It is known that incorporation of more than one incorrect ribonucleotide per PV genome decreases the specific infectivity of the RNA (16, 18). Whether or not 2Ј-dNMP incorporation has an effect on viral RNA infectivity is not known.Several factors likely limit 2Ј-dNMP incorporation into the genomes of RNA viruses of eukaryotes. First, dNTP pools in cells are thought to be low, in the 5-30 M range (19). Low dNTP levels are maintained by regulating the activity and localizat...

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