Tradeoffs for a viral mutant with enhanced replication speed

Abstract

RNA viruses exist as genetically heterogeneous populations due to high mutation rates, and many of these mutations reduce fitness and/or replication speed. However, it is unknown whether mutations can increase replication speed of a virus already well adapted to replication in cultured cells. By sequentially passaging coxsackievirus B3 in cultured cells and collecting the very earliest progeny, we selected for increased replication speed. We found that a single mutation in a viral capsid protein, VP1-F106L, was sufficient for the fast-replication phenotype. Characterization of this mutant revealed quicker genome release during entry compared to wild-type virus, highlighting a previously unappreciated infection barrier. However, this mutation also reduced capsid stability in vitro and reduced replication and pathogenesis in mice. These results reveal a tradeoff between overall replication speed and fitness. Importantly, this approach-selecting for the earliest viral progeny-could be applied to a variety of viral systems and has the potential to reveal unanticipated inefficiencies in viral replication cycles.

Keywords: capsid; coxsackievirus; fitness; replication speed.

Fig. 1.

Selection for fast-replicating CVB3. (A) Single-cycle growth curves for the initial stock virus, and viruses passaged 10 times for 4 h or 8 h (4 h P10 and 8 h P10). Viruses were used to infect HeLa cells at MOI = 0.5, and progeny were quantified by plaque assay. Data are mean ± SEM (n ≥ 11, ≥3 independent experiments). *P < 0.05, two-way ANOVA (comparing 4 h P10 and stock). (B) Consensus sequencing revealed that VP1-F106L was the only nonsilent mutation found in the 4 h P10 samples and not the 8 h P10 samples. (C) Sequencing chromatograms showing mixed peaks at nt 2,768 and 3,427. (D) VP1-F106L was cloned into the infectious clone and used to generate VP1-F106L virus. (E) Single-cycle growth curve with WT and VP1-F106L. HeLa cells were infected at MOI = 0.1, and progeny were quantified by plaque assay. Data are mean ± SEM (n ≥ 11, ≥3 independent experiments), *P < 0.05, unpaired t test, ns, not significant.

Fig. 2.

VP1-F106L has WT replication kinetics when entry is bypassed and does not have enhanced binding to cells. (A and B) Entry bypass experiments. Viral RNA from WT and VP1-F106L was in vitro transcribed and transfected into HeLa cells, and progeny was quantified by plaque assay. (A) Time course for entire wells, reflecting both intracellular and extracellular progeny viruses. (B) Time course supernatants reflecting extracellular progeny viruses. Data are mean ± SEM (n ≥ 8, ≥3 independent experiments). Differences were not statistically significant, *P > 0.05, unpaired t test. (C–E) Viral attachment assays. Gradient purified ³⁵S-labeled viruses were incubated with cells followed by washing and quantification by scintillation counting. (C) Binding assay was performed at 4 °C, which facilitates viral binding but not entry. (D) Binding assay was performed at 37 °C, which facilitates both binding and entry. (E) Binding assay was performed at 37 °C after preincubation of virus with 0.1 μg of either BSA, hCAR, or mCAR for 15 min at room temperature. Data are mean ± SEM (n ≥ 8, ≥3 independent experiments), *P < 0.05, two-way ANOVA (multiple comparisons), ns, not significant (P > 0.05).

Fig. 3.

VP1-F106L has early RNA release and less-stable virions. (A) Neutral red virus RNA release assay. Virus stocks were generated with neutral red dye incorporated inside the capsid, which confers light sensitivity unless virion RNA undergoes uncoating prior to light exposure. HeLa cells were infected in the dark and incubated at 37 °C, followed by washing, addition of an agar overlay, and exposure to light versus dark at various time points. Plaques were counted and light titers were divided by dark titers to determine the ratio of virions that had released RNA by that time point. Data are mean ± SEM (n ≥ 12, ≥5 independent experiments), *P < 0.05, unpaired t test. (B) Viral stability assay. Viruses were incubated at 44 °C followed by plaque assay to quantify viable viruses remaining. Titers at 44 °C were compared to control samples incubated at 4 °C. Data are mean ± SEM (n ≥ 8, ≥3 independent experiments), *P < 0.05, unpaired t test. (C) Pleconaril sensitivity assay. Viruses were used to infect HeLa cells treated with or without the capsid stabilizing drug pleconaril, and progeny were collected at 24 h postinfection and quantified by plaque assay. Data are mean ± SEM (n = 9, ≥3 independent experiments), *P < 0.05, unpaired t test, ns, not significant (P > 0.05).

Fig. 4.

VP1-F106L is more fit that WT in HeLa cells. Direct competition experiment between WT and VP1-F106L in HeLa cells. HeLa cells were infected in triplicate with WT and VP1-F106L in a mixed inoculum at an MOI of 0.5. Progeny were harvested at (A) 4 h or (B) 8 h postinfection. After 10 passages, viral RNA was collected from input, P1, and P10, and RT-PCR products were TOPO cloned and sequenced. Data are mean ± SEM (n indicated below each bar).

Fig. 5.

VP1-F106L is attenuated in mice. (A) Titers from orally infected IFNAR^−/− mice. Male mice were orally infected with 1 × 10⁸ PFU of WT or VP1-F106L viruses. At 3 d postinfection, mice were euthanized, tissues were harvested, and titers were determined by plaque assay. Data are mean ± SEM (n ≥ 7 to 8, ≥2 independent experiments), *P < 0.05, unpaired t test. (B) Survival of IFNAR^−/− mice after oral infection. Mice were infected as in A except they were monitored twice daily for survival for 10 d. Data are represented as survival across 10 d (n ≥ 13 to 14, ≥2 independent experiments), *P < 0.05, log-rank test. (C) Survival of IFNAR^−/− mice after IP injection. Male mice were IP injected with 1 × 10³ PFU of WT or VP1-F106L virus. Data are represented as survival across 10 d (n ≥ 11 to 13, ≥3 independent experiments), *P < 0.05, log-rank test. (D) Survival of WT mice after IP injection. Male C57BL/6 mice were IP injected with 1 × 10⁵ PFU of WT or VP1-F106L virus. Data are represented as survival across 10 d (n ≥ 7, ≥2 independent experiments), *P < 0.05, log-rank test.