High-Order Epistasis and Functional Coupling of Infection Steps Drive Virus Evolution toward Independence from a Host Pathway

FIG 1. Analysis of high-order epistasis of the 2B mutations in viral spread from the infected cells in PI4KB/OSBP-independent infection. (A, Top) Schematic view of PV1(ΔPI4KB resistant) genome. (Bottom) Plaque phenotypes of PV mutants in RD(WT) cells or in RD(ΔPI4KB) cells. RD(WT) cells or RD(ΔPI4KB) cells were inoculated with diluted PV mutants and then stained at 2 days p.i. or at 3 days p.i., respectively. Dilution of WT and mutant 3 were 1/105, 1/106, and 1/107. Dilution of mutants 1 and 2 were 1/104, 1/105, and 1/106. Filled diamonds represent nucleotide mutations derived from PV(ΔPI4KB resistant), and open diamonds represent nucleotides of the WT. The data are representative of three independent experiments with two biological replicates. (B) Virus titer determined in RD(WT) cells or in RD(ΔPI4KB) cells. Ratio of virus titer represents log10[CCID50 in RD(WT) cells/CCID50 in RD(ΔPI4KB) cells]. Standard deviation is shown in parentheses. The data represent mean and standard deviation of three independent experiments with two biological replicates.

FIG 2. Analysis of high-order epistasis of the 2B mutations in infectivity and viral growth in PI4KB/OSBP-independent infection. RD(WT) cells or RD(ΔPI4KB) cells were infected with PV mutants at a multiplicity of infection (MOI) of 10 and then harvested at 24 h p.i. Virus titer was measured in RD(WT) cells or in RD(ΔPI4KB) cells. Virus titer and the ratio of virus titer measured in RD(WT) cells to that in RD(ΔPI4KB) cells are shown. Standard deviation is shown in parentheses. Filled diamonds represent nucleotide mutation derived from PV(ΔPI4KB resistant), and open diamonds represent nucleotides of the WT. The data represent mean and standard deviation of three independent experiments with two biological replicates. n.s., not significant.

FIG 3. Replication kinetics of PV(EGFP)pv mutants in PI4KB/OSBP-independent infection. The cells were infected with PV(EGFP)pv at an MOI of 10 and then observed for 24 h by a time-lapse fluorescence microscopy (LCV110; Olympus). Percentages of infected cells are shown. Maximum number of the infected cells observed in each infection was taken as 100%. Kinetics of the infection was analyzed by a paired t test. n.d., not detectable. The data represent mean and standard deviation of two independent experiments with two biological replicates.

FIG 4. Flow cytometry analysis of viral replication level, cell death, and infectivity in PI4KB/OSBP-independent infection. Flow cytometry analysis of RD(WT) cells or RD(ΔPI4KB) cells infected with PV(EGFP)pv mutants at an MOI of 0.5 at 8, 16, or 24 h p.i. is shown. Infected cells were identified by EGFP produced by the infection. Membrane-compromised cells (dead cells) were identified by a membrane-impermeable dye for nuclei staining (SYTOX red dye). The data are representative of two independent experiments with three biological replicates.

FIG 5. Quantification of effects of the mutations on viral replication level, cell death, and infectivity in PI4KB/OSBP-independent infection. Quantification of the data shown in Fig. 3 is shown. Normalized EGFP signal per cell (EGFP signal in noninfected cells is taken as 1), population of infected cells with compromised membrane (total number of infected cells is taken as 100%), and relative infectivity in RD(ΔPI4KB) cells (total number of infected RD(WT) cells is taken as 100%) are shown. For the measurement of the relative infectivity in RD(ΔPI4KB) cells, infected RD(WT) cells and infected RD(ΔPI4KB) cells were counted at 16 h p.i. and 20 h p.i., respectively. The data represent mean and standard deviation of two independent experiments with three biological replicates. Statistical significance to PV(EGFP)pv(mutant 1) infection was analyzed. n.d., not detectable.

FIG 6. Ratio of viral plus-strand RNA to the minus-strand RNA in PI4KB/OSBP-independent infection. (Top) Number of copies of viral RNA in the cells infected with PV(Fluc)pv mutants determined by strand-specific RT-PCR is shown. (Bottom) Ratio of the plus-strand RNA and minus-strand RNA at indicated time after the infection is shown. The data represent mean and standard deviation of two independent experiments with three biological replicates.

FIG 7. Model of the PV evolutionary pathway toward independence from the host PI4KB/OSBP pathway.