Lethal mutagenesis of picornaviruses with N-6-modified purine nucleoside analogues

Abstract

RNA viruses exhibit extraordinarily high mutation rates during genome replication. Nonnatural ribonucleosides that can increase the mutation rate of RNA viruses by acting as ambiguous substrates during replication have been explored as antiviral agents acting through lethal mutagenesis. We have synthesized novel N-6-substituted purine analogues with ambiguous incorporation characteristics due to tautomerization of the nucleobase. The most potent of these analogues reduced the titer of poliovirus (PV) and coxsackievirus (CVB3) over 1,000-fold during a single passage in HeLa cell culture, with an increase in transition mutation frequency up to 65-fold. Kinetic analysis of incorporation by the PV polymerase indicated that these analogues were templated ambiguously with increased efficiency compared to the known mutagenic nucleoside ribavirin. Notably, these nucleosides were not efficient substrates for cellular ribonucleotide reductase in vitro, suggesting that conversion to the deoxyriboucleoside may be hindered, potentially limiting genetic damage to the host cell. Furthermore, a high-fidelity PV variant (G64S) displayed resistance to the antiviral effect and mutagenic potential of these analogues. These purine nucleoside analogues represent promising lead compounds in the development of clinically useful antiviral therapies based on the strategy of lethal mutagenesis.

FIG. 1.

Structures of mutagenic nucleoside analogs. (A) 5-Aza-5,6-dihydro-2′-deoxycytidine (KP-1212), imino tautomer. (B) Ribavirin. (C) P, imino tautomer. R denotes deoxyribose (KP-1212; dP) or ribose (ribavirin; rP).

FIG. 2.

Structures of N-6-modified purine nucleoside analogues.

FIG. 3.

Nucleoside analogues are antiviral against PV and CVB3. HeLa cells were pretreated with nucleoside analogs (at 0.5 or 2 mM) for 1 h prior to infection with PV or CVB3 at an MOI of 5. Infection was allowed to proceed for 6 h, at which time cell-associated virus was collected and the titer was determined. Data were normalized such that the titer of control sample with no nucleoside treatment was set to a value of 100.

FIG. 4.

Cytotoxicity of nucleoside analogs in HeLa cells. Cytotoxicity was measured using an Alamar blue-based assay as described in Materials and Methods. Cells were incubated in the presence of the nucleoside of interest, and Alamar blue reagent was added 6 h prior to measuring absorbance (A) 24 (A) or 48 (B) h postexposure. Data were normalized such that 100% viability was set equal to the percentage of Alamar blue reagent reduced by untreated cells. Data are presented as the means and standard deviations from three independent wells. For each series, the bars represent, from left to right, 0, 0.05, 0.1, 0.5, 1, and 2 mM treatments.

FIG. 5.

Nucleotide analogues are not chain terminators. Complexes of 3D^pol and s/s-U were preassembled and then mixed with either 500 μM nucleotide (− lanes) or with 500 μM nucleotide and 10 μM UTP (+ lanes). The reaction was allowed to proceed at 30°C for 180 s, followed by quenching with EDTA and separation via denaturing PAGE. NTP added is indicated above each lane. The positions of 10-mer substrate and extended products are indicated to the left.

FIG. 6.

Analysis of extracts of HeLa S3 cells by reverse-phase HPLC. (A) Analysis of pure JA28-TP including the full UV spectrum of the JA28-TP peak at 3.6 min (inlay). mAU, milli-absorbance units. (B) Analysis of extracts of untreated HeLa S3 cells including the full UV spectrum of the eluent at 3.6 min. (C) Analysis of extracts of HeLa S3 cells treated with JA28 (0.5 mM) including the full UV spectrum of the eluent at 3.6 min (inlay). The long-wavelength absorption characteristic of JA28-TP is illustrated by the arrows to the right. Peaks were detected by A₃₂₅.

FIG. 7.

Antiviral activity of nucleoside analogues against a high-fidelity PV variant (PV-G64S). Infections were performed at an MOI of 5 and treatment with 2 mM nucleoside analogue. Infection was allowed to proceed for 6 h at 37°C, and cell-associated virus was collected by freeze-thaw. (A) The titer of virus was determined on HeLa S3 cell monolayers. Data were normalized for each virus such that the virus titer resulting from untreated control infection was set equal to 100. Data are plotted as the means and standard deviations from three independent samples. (B) Virus was assayed for the Gua^r phenotype as described in the text. Results are reported as means and standard deviations from three independent samples. Due to the small amount of viable virus recovered from JA30-treated samples, n = 2 for wild-type (WT) PV and n = 1 for PV-G64S for this treatment only.