Experimental and mathematical insights on the interactions between poliovirus and a defective interfering genome

Figures

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  Fig 1. DI genomes inhibit WT virus production via genomic replication and encapsidation. (A) Structure of the WT poliovirus, DI(Δ1175–2956), and DI(Δ1175–2956)(Rep-). The DI genome has an in-frame deletion in its P1-encoding region. The PvuII-cut DI genome is used for a non-replicating RNA control (Rep-). (B) Growth curves of WT poliovirus after transfection of WT genomes and/or DI genomes (sampling time-points: 0, 3, 6 and 9 hours after transfection). (C, D) Copy numbers of total (C) or encapsidated (D) genomes over time after transfection (sampling time-points: 0, 2, 3.5, 5, 7 and 9 hours post transfection). Blue and red squares indicate copy numbers of WT and DI genomes, respectively. Solid lines indicate single-transfected (WT or DI genome-transfected) samples, while dotted lines indicate double-transfected (both WT and DI genomes-transfected) samples. n = 3, mean ± standard deviations (SD) (B-D).

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  Fig 2. Flow diagram of the model (Eqs (1)–(6)). State variables: resource units number R; wild-type (WT) poliovirus naked genome number GWT; defective interfering (DI) naked genome number GDI; WT-produced capsid proteins number C; encapsidated WT genome number CWT; encapsidated DI genome number CWT. Model parameters are defined in Table 1. Color code is blue for WT, red for DI and brown for resources. Segments represent genomes and diamonds represent capids.

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  Fig 3. Experimental data and model fit for the naked and encapsidated wild-type (WT) and defective interfering (DI) genomes. Evolution of the number of WT and DI (A-B) naked genome copies and (C-D) encapsidated genome copies with time, from 2 to 9 hours post transfection (hpt). (A & C) show data in dually infected cells whereas (B & D) show data in singly infected cells. WT and DI results are shown in blue and red color, respectively. Crosses indicate experimental data for 3 replicates per sampling time at 2, 3.5, 5, 7 and 9 hpt (data drawn from Fig 1C and 1D, see Material and methods for details). Dashed curves represent the fit of the reduced model with logistic equation and solid curves show the fit of the full model (Eqs (1)–(6)).

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  Fig 4. Sensitivity indices for the proportion of WT virions at cell lysis (ΦWT) and the impact of the most important parameters. A: Main, total and most important interaction indices for ΦWT. Main indices correspond to single parameter effect (black parts), and total indices add the effect of the factor in interaction with all other factors (second-order interactions, full bars). Hatched areas represent the importance of the strongest pairwise interaction between defective interfering (DI) to WT encapsidation ratio ω and DI to WT replication ratio P. B: Heat map representing the impact of P and ω on ΦWT, all other parameters being fixed to their best estimated value. The cross and lines in (B) show estimated parameter values from fitting the model to the experimental data.

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  Fig 5. Investigating DI mutant genomes. A: PV3 and PV1-DI particles were serially passaged 8 times. B: Titers of PV3 (PFU/mL) and PV1-DI particles (IU/mL) included in the samples were determined. C: Mutations in the DI genomes positively selected over passages. D: One-step replication cycle. Dynamics of the ratio of PV1-DI to (wild-type) WT PV1 naked genome copies (total RNA) over time (3, 4.33, 6.33 and 8.17 hours post transfection), for distinct DI mutants (Ori: original non-mutant DI genome). E: Estimation of the ratio of PV1-DI to WT PV1 replication rate P from a least square regression between experimental and model predicted PV1-DI to WT PV1 naked genome ratios. Confidence intervals represent values of P at 10% of the minimum least square value. Model predictions of P against experimental DI to WT naked genome ratios at the last sampling time-point.

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  Fig 6. Model-predicted impact of infection delay and initial multiplicity of infection (MOI) on WT and DI burst sizes. A: WT (blue) and DI (red) predicted burst sizes (encapsidated genomes at 9 hours post infection) for various delays in DI infection of a cell. Negative delays correspond to cases where DI infects first and positive delays to cases where WT infects first. The dashed gray line marks the case of simultaneous infection by WT and DI. B-C: Heat maps representing the predicted relative burst sizes of (B) WT, BWT, and (C) DI, BDI, as a function of initial WT (x-axis) and DI (y-axis) MOIs (input). WT burst sizes were normalized by WT burst size resulting from WT:DI = 1:0 MOIs and DI burst sizes by DI burst size resulting from WT:DI = 1:1 MOIs (black squares).