Jamie J. Arnold

Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population

An RNA virus population does not consist of a single genotype; rather, it is an ensemble of related sequences, termed quasispecies. Quasispecies arise from rapid genomic evolution powered by the high mutation rate of RNA viral replication. Although a high mutation rate is dangerous for a virus because it results in nonviable individuals, it has been hypothesized that high mutation rates create a ‘cloud’ of potentially beneficial mutations at the population level, which afford the viral quasispecies a greater probability to evolve and adapt to new environments and challenges during infection. Mathematical models predict that viral quasispecies are not simply a collection of diverse mutants but a group of interactive variants, which together contribute to the characteristics of the population. According to this view, viral populations, rather than individual variants, are the target of evolutionary selection. Here we test this hypothesis by examining the consequences of limiting genomic diversity on viral populations. We find that poliovirus carrying a high-fidelity polymerase replicates at wild-type levels but generates less genomic diversity and is unable to adapt to adverse growth conditions. In infected animals, the reduced viral diversity leads to loss of neurotropism and an attenuated pathogenic phenotype. Notably, using chemical mutagenesis to expand quasispecies diversity of the high-fidelity virus before infection restores neurotropism and pathogenesis. Analysis of viruses isolated from brain provides direct evidence for complementation between members in the quasispecies, indicating that selection indeed occurs at the population level rather than on individual variants. Our study provides direct evidence for a fundamental prediction of the quasispecies theory and establishes a link between mutation rate, population dynamics and pathogenesis.

Nucleic acid polymerases use a general acid for nucleotidyl transfer.

Nucleic acid polymerases catalyze the formation of DNA or RNA from nucleoside-triphosphate precursors. Amino acid residues in the active site of polymerases are thought to contribute only indirectly to catalysis by serving as ligands for the two divalent cations that are required for activity or substrate binding. Two proton-transfer reactions are necessary for polymerase-catalyzed nucleotidyl transfer: deprotonation of the 3′-hydroxyl nucleophile and protonation of the pyrophosphate leaving group. Using model enzymes representing all four classes of nucleic acid polymerases, we show that the proton donor to pyrophosphate is an active-site amino acid residue. The use of general acid catalysis by polymerases extends the mechanism of nucleotidyl transfer beyond that of the well-established two-metal-ion mechanism. The existence of an active-site residue that regulates polymerase catalysis may permit manipulation of viral polymerase replication speed and/or fidelity for virus attenuation and vaccine development.

Structure-Function Relationships Among RNA-Dependent RNA Polymerases

RNA-dependent RNA polymerases (RdRPs) play key roles in viral transcription and genome replication, as well as epigenetic and post-transcriptional control of cellular gene expression. In this article, we review the crystallographic, biochemical, and molecular genetic data available for viral RdRPs that have led to a detailed description of substrate and cofactor binding, fidelity of nucleotide selection and incorporation, and catalysis. It is likely that the cellular RdRPs will share some of the basic structural and mechanistic principles gleaned from studies of viral RdRPs. Therefore, studies of the viral RdRP establish a framework for the study of cellular RdRPs, an important yet understudied class of nucleic acid polymerases.

Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases

The rate-limiting step for nucleotide incorporation in the pre-steady 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 (3Dpol), chemistry is partially (Mg2+) or completely (Mn2+) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pKa 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.

Poliovirus RNA-dependent RNA Polymerase (3Dpol) ASSEMBLY OF STABLE, ELONGATION-COMPETENT COMPLEXES BY USING A SYMMETRICAL PRIMER-TEMPLATE SUBSTRATE (sym/sub)

Detailed studies of the kinetics and mechanism of nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus, 3Dpol, have been limited by the inability to assemble elongation complexes that permit activity to be monitored by extension of end-labeled primers. We have solved this problem by employing a short, symmetrical, heteropolymeric RNA primer-template that we refer to as “sym/sub.” Formation of 3Dpol-sym/sub complexes is slow owing to a slow rate of association (0.1 μm −1 s−1) of 3Dpol and sym/sub and a slow isomerization (0.076 s−1) of the 3Dpol-sym/sub complex that is a prerequisite for catalytic competence of this complex. Complex assembly is stoichiometric under conditions in which competing reactions, such as enzyme inactivation, are eliminated. Inactivation of 3Dpol occurs at a maximal rate of 0.051 s−1 at 22 °C in reaction buffer lacking nucleotide. At this temperature, ATP protects 3Dpol against inactivation with aK 0.5 of 37 μm. Once formed, 3Dpol-sym/sub elongation complexes are stable (t 1 2 = 2 h at 22 °C) and appear to contain only a single polymerase monomer. In the presence of Mg2+, AMP, 2′-dAMP, and 3′-dAMP are incorporated into sym/sub by 3Dpol at rates of 72, 0.6, and 1 s−1, respectively. After incorporation of AMP, 3Dpol-sym/sub product complexes have a half-life of 8 h at 22 °C. The stability of 3Dpol-sym/sub complexes is temperature-dependent. At 30 °C, there is a 2–8-fold decrease in complex stability. Complex dissociation is the rate-limiting step for primer utilization. 3Dpoldissociates from the end of template at a rate 10-fold faster than from internal positions. The sym/sub system will facilitate mechanistic analysis of 3Dpol and permit a direct kinetic and thermodynamic comparison of the RNA-dependent RNA polymerase to the other classes of nucleic acid polymerases.

Incorporation fidelity of the viral RNA-dependent RNA polymerase: a kinetic, thermodynamic and structural perspective.

Abstract Positive-strand RNA viruses exist as a quasi-species due to the incorporation of mutations into the viral genome during replication by the virus-encoded RNA-dependent RNA polymerase (RdRP). Therefore, the RdRP is often described as a low-fidelity enzyme. However, until recently, a complete description of the kinetic, thermodynamic and structural basis for the nucleotide incorporation fidelity of the RdRP has not been available. In this article, we review the following: (i) the steps employed by the RdRP to incorporate a correct nucleotide; (ii) the steps that are employed by the RdRP for nucleotide selection; (iii) the structure-based hypothesis for nucleotide selection; (iv) the impact of sites remote from the active site on polymerase fidelity. Given the recent observation that RNA viruses exist on the threshold of error catastrophe, the studies reviewed herein suggest novel strategies to perturb RdRP fidelity that may lead ultimately to the development of antiviral agents to treat RNA virus infection.

Determinants of RNA-Dependent RNA Polymerase (In)fidelity Revealed by Kinetic Analysis of the Polymerase Encoded by a Foot-and-Mouth Disease Virus Mutant with Reduced Sensitivity to Ribavirin

ABSTRACT A mutant poliovirus (PV) encoding a change in its polymerase (3Dpol) at a site remote from the catalytic center (G64S) confers reduced sensitivity to ribavirin and forms a restricted quasispecies, because G64S 3Dpol is a high-fidelity enzyme. A foot-and-mouth disease virus (FMDV) mutant that encodes a change in the polymerase catalytic site (M296I) exhibits reduced sensitivity to ribavirin without restricting the viral quasispecies. In order to resolve this apparent paradox, we have established a minimal kinetic mechanism for nucleotide addition by wild-type (WT) FMDV 3Dpol that permits a direct comparison to PV 3Dpol as well as to FMDV 3Dpol derivatives. Rate constants for correct nucleotide addition were on par with those of PV 3Dpol, but apparent binding constants for correct nucleotides were higher than those observed for PV 3Dpol. The A-to-G transition frequency was calculated to be 1/20,000, which is quite similar to that calculated for PV 3Dpol. The analysis of FMDV M296I 3Dpol revealed a decrease in the calculated ribavirin incorporation frequency (1/8,000) relative to that (1/4,000) observed for the WT enzyme. Unexpectedly, the A-to-G transition frequency was higher (1/8,000) than that observed for the WT enzyme. Therefore, FMDV selected a polymerase that increases the frequency of the misincorporation of natural nucleotides while specifically decreasing the frequency of the incorporation of ribavirin nucleotide. These studies provide a mechanistic framework for understanding FMDV 3Dpol structure-function relationships, provide the first direct analysis of the fidelity of FMDV 3Dpol in vitro, identify the β9-α11 loop as a (in)fidelity determinant, and demonstrate that not all ribavirin-resistant mutants will encode high-fidelity polymerases.

Poliovirus RNA-dependent RNA Polymerase (3Dpol) STRUCTURAL, BIOCHEMICAL, AND BIOLOGICAL ANALYSIS OF CONSERVED STRUCTURAL MOTIFS A AND B

We have constructed a structural model for poliovirus RNA-dependent RNA polymerase (3Dpol) in complex with a primer-template (sym/sub) and ATP. Residues found in conserved structural motifs A (Asp-238) and B (Asn-297) are involved in nucleotide selection. Asp-238 appears to couple binding of nucleotides with the correct sugar configuration to catalytic efficiency at the active site of the enzyme. Asn-297 is involved in selection of ribonucleoside triphosphates over 2′-dNTPs, a role mediated most likely via a hydrogen bond between the side chain of this residue and the 2′-OH of the ribonucleoside triphosphate. Substitutions at position 238 or 297 of 3Dpolproduced derivatives exhibiting a range of catalytic efficiencies when assayed in vitro for poly(rU) polymerase activity or sym/sub elongation activity. A direct correlation existed between activity on sym/sub and biological phenotypes; a 2.5-fold reduction in polymerase elongation rate produced virus with a temperature-sensitive growth phenotype. These data permit us to propose a detailed, structural model for nucleotide selection by 3Dpol, confirm the biological relevance of the sym/sub system, and provide additional evidence for kinetic coupling between RNA synthesis and subsequent steps in the virus life cycle.

Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-Dependent RNA Polymerase

ABSTRACT Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3Cpro and the viral polymerase 3Dpol and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4-Å resolution and the G64S fidelity mutant of 3Dpol at a 3.0-Å resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3Dpol G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domains N terminus to the VPg binding site suggest how 3Dpol makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.

Poliovirus RNA-dependent RNA polymerase (3D(pol)). Divalent cation modulation of primer, template, and nucleotide selection.

We have analyzed the divalent cation specificity of poliovirus RNA-dependent RNA polymerase, 3Dpol. The following preference was observed: Mn2+ > Co2+ > Ni2+ > Fe2+ > Mg2+ > Ca2+ > Cu2+, and Zn2+ was incapable of supporting 3Dpol-catalyzed nucleotide incorporation. In the presence of Mn2+, 3Dpol activity was increased by greater than 10-fold relative to that in the presence of Mg2+. Steady-state kinetic analysis revealed that the increased activity observed in the presence of Mn2+ was due, primarily, to a reduction in the K M value for 3Dpol binding to primer/template, without any significant effect on the K M value for nucleotide. The ability of 3Dpol to catalyze RNA synthesis de novo was also stimulated approximately 10-fold by using Mn2+, and the enzyme was now capable of also utilizing a DNA template for primer-independent RNA synthesis. Interestingly, the use of Mn2+ as divalent cation permitted 3Dpolactivity to be monitored by following extension of 5′-32P-end-labeled, heteropolymeric RNA primer/templates. The kinetics of primer extension were biphasic because of the enzyme binding to primer/template in both possible orientations. When bound in the incorrect orientation, 3Dpol was capable of efficient addition of nucleotides to the blunt-ended duplex; this activity was also apparent in the presence of Mg2+. In the presence of Mn2+, 3Dpol efficiently utilized dNTPs, ddNTPs, and incorrect NTPs. On average, three incorrect nucleotides could be incorporated by 3Dpol. The ability of 3Dpol to incorporate the correct dNTP, but not the correct ddNTP, was also observed in the presence of Mg2+. Taken together, these results provide the first glimpse into the nucleotide specificity and fidelity of the poliovirus polymerase and suggest novel alternatives for the design of primer/templates to study the mechanism of 3Dpol-catalyzed nucleotide incorporation.