Coxsackievirus B3 mutator strains are attenuated in vivo

Abstract

Based on structural data of the RNA-dependent RNA polymerase, rational targeting of key residues, and screens for Coxsackievirus B3 fidelity variants, we isolated nine polymerase variants with mutator phenotypes, which allowed us to probe the effects of lowering fidelity on virus replication, mutability, and in vivo fitness. These mutator strains generate higher mutation frequencies than WT virus and are more sensitive to mutagenic treatments, and their purified polymerases present lower-fidelity profiles in an in vitro incorporation assay. Whereas these strains replicate with WT-like kinetics in tissue culture, in vivo infections reveal a strong correlation between mutation frequency and fitness. Variants with the highest mutation frequencies are less fit in vivo and fail to productively infect important target organs, such as the heart or pancreas. Furthermore, whereas WT virus is readily detectable in target organs 30 d after infection, some variants fail to successfully establish persistent infections. Our results show that, although mutator strains are sufficiently fit when grown in large population size, their fitness is greatly impacted when subjected to severe bottlenecking, which would occur during in vivo infection. The data indicate that, although RNA viruses have extreme mutation frequencies to maximize adaptability, nature has fine-tuned replication fidelity. Our work forges ground in showing that the mutability of RNA viruses does have an upper limit, where larger than natural genetic diversity is deleterious to virus survival.

Fig. 1.

Targeted mutations sites on CV3B polymerase (26) superimposed on the RNA from the homologous poliovirus polymerase elongation complex (22). (A) Details of the buried N terminus (blue sphere) that is locked in place by hydrogen bonds with the backbone carbonyls of residues 64, 239, and 241, positioning Asp238 in the active site for interactions with NTPs. The terminal cytosine of the RNA, which sits in the NTP binding pocket of the postcatalysis poliovirus elongation complex (Protein Data Bank ID code 3OL7), is shown in orange. (B) The three groups of residues comprising 50 separate mutations engineered to alter fidelity by affecting active site closure for catalysis (green; I230, F232, Y234, D238, I306, and L344), the linkage between the palm and fingers domains (orange; S240, F246, Y268, L269, and M287), and the positioning of the template RNA and bound NTP (red; R174, I176, and S289) are shown. (C) Location of the seven stable and viable mutants (blue) from the set of 50 mutants tested in infectious virus studies. The locations of other compensatory mutations commonly observed in the viruses emerging from the mutation studies are shown in dark red.

Fig. 2.

Viral production (infectious virus and total genomic RNA) of CVB3 polymerase variants in cell culture. (A–D) Production of infectious virus measured by one-step growth kinetics of (A and B) WT, P48K, S164P, L241I, A239G, and A239S viruses and (C and D) WT, I176V, I230F, F232V, F232Y, Y268H, and Y268W viruses. HeLa cells were infected at MOI of 10, and progeny virus was quantified at different hours postinfection by TCID₅₀ assay. Mean titers (TCID₅₀/mL) ± SEM are shown (n = 3 independent experiments); no significant difference was found comparing WT and each of the CVB3 variants, except A239S (*P < 0.05 and ***P < 0.0001 by two-way ANOVA with Bonferroni posttest). (E–H) Total genomic RNA content of the same virus populations examined above (A–D). The number of RNA genomes was determined by real-time RT-PCR at different times postinfection. Mean values (genome copies per milliliter) ± SEM are shown (n = 3 independent experiments); significant differences with respect to WT are indicated by *P < 0.05 or ***P < 0.0001 by two-way ANOVA with Bonferroni posttest.

Fig. 3.

CVB3 polymerase variants are mutator strains. (A) Average mutation frequencies of WT and other polymerase variants are shown as the mean number of mutations per 10⁴ nt sequenced by TopoTA cloning of total RNA from passage 3 virus stock populations. Between 75 and 150 clones (78,750–157,500 nt total) were sequenced per population (ns, not significant; *P < 0.05, **P < 0.01, and ***P < 0.001 compared with WT by Mann–Whitney u test). (B) The CVB3 fidelity variants are more sensitive to treatment with the RNA mutagen ribavirin. HeLa cells, treated with either 200 or 400 μM ribavirin or mock-treated, were infected at MOI of 0.01; 48 h postinfection, the loss of infectivity in the progeny virus was titered by TCID₅₀ assay. The percentage of each CVB3 variant surviving the treatment with either 200 or 400 μM ribavirin relative to the untreated control is shown. The mean values ± SEM are shown (n = 5; P < 0.001 for all samples compared with WT by Student t test).

Fig. 4.

In vitro fidelity assay used to determine how efficiently the Coxsackievirus polymerases use CTP and 2′-deoxy-CTP as substrates. (A) The 5′ fluorescein-labeled primer template RNA was designed with a terminal sequence such that ∼40% of the maximal change in the fluorescein signal occurs before the CTP addition, whereas the remaining signal increase to 100% is rate-limited by the CTP or dCTP addition itself. (B) Data from the I176V mutant polymerase showing the similar kinetic behavior on the addition of nanomolar CTP (Left) or micromolar dCTP (Right). Kinetic parameters (lag phase time, K_m, and k_cat) obtained by fitting the data are listed in Table S1. Insets show gels with an analogous elongation of a 10 + 1 − 12 RNA (42), where a locked +1 complex yields a +7 product in the absence of CTP and a full-length +13 product in the presence of nanomolar CTP or micromolar dCTP. (C) Plot showing the correlation between the observed infectious virus mutation rates (Fig. 3) and the CTP vs. dCTP discrimination factors based on NTP use efficiencies (Table S1). F232L and F232V mutants were not stable, and the plotted mutation rates represent a lower limit based on preliminary sequencing from a mixed population of virus in the process of reverting. (D) Plot showing the correlation between polymerase elongation rates and RNA genome production during virus infection (fold change between 3 and 5 h in Fig. 2 E–H). Faster polymerases use less time to replicate through the lag phase sequence indicated in A.

Fig. 5.

Low-fidelity CVB3 variants are attenuated in vivo. (A–D) Virus titers (genome copies per milliliter) measured by qRT-PCR of mice infected with 10⁵ TCID₅₀ and killed at 3 d after infection are shown. Each panel presents a different organ from the same groups of mice. Box plots show median values ± SEM (n = 8–12; *P < 0.05, **P < 0.01, and ***P < 0.001 by Kruskal–Wallis test). (E–I) In vivo replication kinetics of selected CVB3 variants. Mice were inoculated with WT, I230F, Y268W, and A239G and killed on days 3, 5, and 7 postinoculation (n = 4). Virus was quantified as above. (J) Mice were inoculated with each variant or WT virus, and 35 d after infection, virus was quantified in the spleen (n = 8; *P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 6.

Mutator strains are prone to extinction during extreme bottlenecking in tissue culture. (A–G) WT virus (solid lines) and each polymerase variant (dashed lines) were subjected to extreme bottlenecking by plaque-to-plaque passage on HeLa cells. Triplicate passage of each virus was performed through nine passages (x axis). Some samples extinguished at the first passage (dashed lines along x axis). The mean surfaces are square millimeters of plaques, and SEM values are shown for each triplicate passage series (n = 20–52; ***P < 0.001 by Mann–Whitney u test). (H) Larger population size passage of polymerase variants; 1 × 10⁶ HeLa cells were infected with 1,000 virus particles of each variant, and 48 h after infection, virus was quantified by qRT-PCR to determine the genome copies per milliliter (y axis) at each passage number (x axis).

Fig. P1.

Coxsackie virus polymerase mutations targeting four different RNA polymerase domains that were expected to alter replication fidelity: a domain involved in RNA template and NTP binding, an H-bond network found to alter fidelity in the closely related poliovirus polymerase and accompanying compensatory mutations affecting NTP positioning, an interaction domain between the palm and finger regions of the polymerase, and the domain involved in active site closure. The figure shows the residue positions in reference to the thumb (purple), plam (gray), and finger (red) domain structures; also, the RNA template (cyan) and incoming base (green) are shown. All nine variants present replication kinetics comparable with WT virus. The mutation frequencies (mutations per 10,000 nt sequenced) confirm that these variants possess mutator phenotypes. Variants with the highest mutation frequencies are the most attenuated in vivo, which correlates with a compromised ability to survive severe population bottlenecks, likely because of more rapid accumulation of detrimental mutations.