Figure 5. Effect of Bacterial Strains on Poliovirus Recombination Frequency.

(A) Diagram of recombination between Drug^R/Temp^S and Drug^S/Temp^R parental polioviruses. Triangle denotes mutation conferring guanidine (Drug) resistance and x denotes site of temperature sensitive mutations inhibiting replication at 39.5°C (Kirkegaard and Baltimore, 1986). The dotted line indicates the location of recombination events that create progeny that are able to grow at 39.5°C in the presence of 1 mM guanidine (Drug^R/Temp^R). (B) Schematic of recombination assay. 1×10⁵ PFU of each parental virus was mixed and incubated with 1×10⁸ CFU bacteria prior to infection of 1×10⁷ HeLa cells (MOI of 0.01). Infection proceeded for 8 h under permissive conditions for both viruses (33°C without drug) and progeny viruses were quantified by plaque assay at both permissive and restrictive conditions to determine yields of individual parental viruses and recombinants. (C) Recombination frequencies after exposure to bacterial strains. Recombination frequencies are shown as the viral titers (PFU/mL) at 39.5°C+Drug divided by viral titers at 33°C−drug. HeLa cells were also infected in parallel with each parental virus alone as controls to calculate the frequency of reversion and de novo mutation acquisition. Data are represented as the mean ± SEM (n=4–12), from at least 2 independent experiments. *p<0.01 versus the mix of parental viruses in PBS (Student’s unpaired t test). (D) Scatter plot to test for correlation between the percentage of dual infected cells and the recombination frequency. Data points are the mean values presented in Figures 5C, 3A and 3C. p<0.0001, R²=0.93, r=0.97 (Pearson’s correlation coefficient calculation). See also Figure S5.