Fig. 5

The 2B coding region of the poliovirus genome is a hotspot for the accumulation of mutations following cell culture adaptation. a High-density genetic screen procedure to identify adaptive mutations on cell culture conditions. WT, GD, or WT were passaged in HeLa cells or HeLa cells pre-treated with interferon β (IFN β). Using circular resequencing (CirSeq), the mutation composition, allele frequency distribution, and fitness of specific alleles were determined. b Scatter plot showing the position and fitness of identified adaptive mutations. Adaptive alleles with fitness values >2 standard deviations (s.d.s) above neutrality are shown. Alleles with fitness > 25 s.d.s above neutrality are labeled. The details of the specific mutations labeled in the plot are shown in Supplementary Table 1. The 2B coding region of the genome is shaded grey. c Allele frequency spectrums for 4 independently passaged of poliovirus (PV) populations. Alleles are ranked by frequency and plotted to show the relative diversity of passaged populations. All populations shown are from passage 6 of parallel passage experiments. d Interferon (IFN) sensitivity assay for specific 2B mutations identified in the passage screen. One-tailed Student’s t-test. *P ≤ 0.05, **P ≤ 0.01, n = 3. Data were presented as logarithm(mean ± s.d.), PFU per ml. n = 3, three replicates for each time, for each viral strain. e,f Comparison of immune signaling in MG63 cells infected by WT or 2B mutants at m.o.i=20. e Supernatant was collected at the indicated times to measure IFN β induction by ELISA. IFN β level < 2.3 pg /ml is undetectable. f mRNA expression level of IL6, IL8, RIG-I, TNFα measured by qRT-PCR at postinfected 5 h by 2B mutants and WT. Data shows mean ± s.d., * P < 0.05, ** P < 0.01, n = 3, Student’s t-test. g Growth kinetics of poliovirus carrying 2B mutations identified in HeLa cells. Viruses replication was examined for 2B mutants and WT by one-step growth curves in HeLa cells (m.o.i=10). (Data are shown as mean ± s.d., ***P < 0.0001, n = 3, three replicates for each time, for each viral strain, Student’s t-test). h. SEAP reporter assay to assess the ability of 2B mutants to inhibit protein secretion activity. One-tailed Student’s t-test. *P ≤ 0.05, n = 3, three replicates for each time, for each viral strain