Lethal mutagenesis of poliovirus mediated by a mutagenic pyrimidine analogue

Abstract

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.

FIG. 1.

P base pairs with adenine and guanine. P exists as two tautomers: imino-P and amino-P. The former hydrogen bonds with adenine; the latter hydrogen bonds with guanine. The imino-P/amino-P ratio is approximately 11:1 (20).

FIG. 2.

RTP is not an efficient substrate for T7 RNAP. (A) Substrates used for the T7 RNAP extension assay are illustrated with DNA template (top strand) and RNA primer (bottom strand). The templating nucleotide is indicated in bold. (B) Primer extension by T7 RNAP is shown after 30 and 180 seconds when either RTP or the correct nucleotide was provided as substrate. RMP was incorporated inefficiently compared to the correct (natural) substrate. The asterisk indicates an 11-mer extended RNA substrate.

FIG. 3.

rPMP is incorporated into genomic RNA during transcription with T7 RNAP. (A) HPLC separation of RNA transcribed in the presence of rPTP. RNA was digested to component nucleosides as described in the text. rP was observed along with the four natural nucleosides. (B) rPMP composition of RNA (as determined by HPLC of digested RNA) is plotted against the concentration of rPTP present in the in vitro transcription reaction mixture. The PV genome is 7.5 kb in length. (C) RNA transcribed in the presence of increasing concentrations of rPTP was run on a 1% agarose gel and stained with ethidium bromide. Differences in RNA quality or length were not observed when rPTP was added to the transcription reaction mixture at up to 20% of the total nucleotide pool. L, DNA ladder.

FIG. 4.

rPMP incorporation into PV genomic RNA results in a dose-dependent decrease in specific infectivity. (A) HeLa S3 cells were transfected with various concentrations of in vitro-transcribed RNA and serially diluted on HeLa S3 monolayers. Resulting plaques increased linearly up to ∼5 μg. (B) PV genomic RNA was transcribed in vitro in the presence of various concentrations of rPTP, and infectivity was determined in an infectious center assay. The number of rPMP incorporations per genome is plotted on the x axis, as determined by extrapolating the data in Fig. 3B for transcription in the presence of various amounts of rPTP. Specific infectivity was normalized such that for each experiment the number of plaques resulting from RNA transcribed in the absence of rPTP was set to 100. The mean and standard deviation of at least three independent samples are shown for each data point.

FIG. 5.

PV polymerase incorporates rPMP opposite both adenine and guanine in RNA. Either s/s-A (A) or s/s-G (B) was employed. Utilization of rPTP was compared directly to utilization of the correct nucleotide: UTP (A) or CTP (B). Polymerase-s/s complexes were assembled and nucleotide added to a final concentration of 500 μM. Product formed after 15, 30, 45, 60, 120, 300, and 600 s was separated from substrate by denaturing PAGE and visualized by phosphorimaging. Extended RNA product is indicated by the asterisks.

FIG. 6.

PV 3D^pol utilizes rPTP more efficiently than ribavirin triphosphate. (A) Poliovirus polymerase 3D^pol was incubated with s/s for 120 s prior to initiating the reaction by addition of the appropriate nucleotide to a final concentration of 500 μM. The solid and dashed lines represent fits of the data to a single exponential with k_obs of 0.30 ± 0.02 s⁻¹ and 0.44 ± 0.05 s⁻¹ for rPMP incorporation opposite A (•) and G (▪) and k_obs of 0.008 ± 0.001 s⁻¹ and 0.009 ± 0.004 s⁻¹ for rRMP incorporation across U (○) and C (□), respectively. (B) A chain termination experiment was performed with s/s-A as described in the legend for Fig. 6A. Incorporation was monitored in the presence of rPTP alone (left panel) or in the presence of rPTP and ATP, the next correct nucleotide (right panel). Quantitative incorporation of AMP was observed (the n + 2 product is indicated by the asterisk).

FIG. 7.

rPTP is not detected in HeLa-TK cells treated with rP. (A) Separation of extracts from untreated HeLa-TK cells (100-μl injection). (B) Separation of extracts from HeLa-TK cells that were treated with 2 mM rP for 3 h (100-μl injection). rP (retention time [t_R], 11.8 min) was clearly observed in treated extracts. No new peaks with rPTP retention (t_R, 2.2 min) or characteristic UV trace were observed. The absorbance wavelength for all traces was 295 nm.

FIG. 8.

P exhibits mild antiviral and mutagenic properties. (A) HeLa cells were pretreated for 1 hour with P and then infected with PV (MOI, 5), followed by a 6-h incubation at 37°C. Cell-associated virus was recovered by freeze-thaw, and the titer was determined. Experiments were performed in triplicate. (B) A guanidine resistance assay was performed in triplicate as described in the text.

FIG. 9.

Cell-free translation of PV genomic RNA is unaffected by the presence of rPTP. (A) Cell-free reactions were programmed with PV replicon RNA containing luciferase in place of the capsid-coding sequence. Translation was measured after 12 h via a luciferase activity assay. (B) Cell-free reactions were programmed with PV vRNA. After 12 h of translation, products were separated via SDS-PAGE. (C) Extracts were programmed with PV vRNA, and viable virus produced was quantitated by plaque assay.

FIG. 10.

The PV replicon containing rPMP shows a reduction in active luciferase reporter activity. The PV replicon contains the luciferase reporter in place of the capsid-coding sequence. Replicons were transcribed under conditions previously shown to result in 0, 16.5, 33, or 82.5 rPMP substitutions per RNA molecule. (A) Replicon-transfected cells were incubated in the presence (open symbols) or absence (closed symbols) of 3 mM guanidine hydrochloride for 8 h. (B) Replicon-transfected cells were incubated for up to 3 h in the presence of 3 mM guanidine hydrochloride. Activity of the luciferase reporter was reduced by a maximum of 10-fold.

FIG. 11.

rPMP substitution in genomic RNA is more deleterious to a high-fidelity PV variant (G64S). Cells were transfected by T7 RNAP-transcribed RNA containing a known number of rPMP substitutions, followed by incubation at 37°C for 6 h. Cell-associated virus was collected by freeze-thaw, and the titer was determined. Titers were normalized such that the titer of virus produced by RNA with no rPMP substitutions was set to 100. The titer resulting from transfection of wild-type (WT) genomes containing no PTP was 2.3 ×10⁶ PFU/ml; for G64S, the titer was 6.6 ×10⁶ PFU/ml.