Combinatorial Approaches to Viral Attenuation

Differential gene expression for promoter knockout strains against wild type. Shown is RNA abundance (measured as transcripts per million [tpm], rescaled to a range of 0 to 1) for promoter knockout strains (y axis) versus wild type (x axis). Each point represents the RNA abundance for a single gene (genes 9 and 10A are labeled). Each panel shows a different comparison of mutant versus wild type, where columns indicate promoter knockout and rows represent and are colored by genetic background (orange, wt; blue, 10_deop; green, 8_Δstop). Panels marked “NA” represent knockout-background combinations for which no data were collected. Samples were taken at 9 min postinfection.

RNA abundances for genes 8 to 12. Each bar represents the mean mRNA expression level from promoter knockout strains for genes immediately surrounding the ϕ9 and ϕ10 locations. Each point represents a single measurement. Lines connecting points indicate single batches (samples collected and sequenced together). Promoter knockouts are indicated along the x axis with columns organized by genetic background (column names) distinguished by color (orange, wt; blue, 10_deop; green, 8_Δstop). Samples were taken at 9 min postinfection.

Relative RNA abundance for differentially expressed genes. Each panel represents a different strain, where columns indicate promoter knockout and rows represent genetic background. Each bar indicates the relative RNA abundance for a given gene. Genes for which a significant difference was found (FDR of <0.05) are shown as solid bars, and all other genes are shown as partially transparent bars. Panels marked as “NA” represent knockout-background combinations for which no data were collected. Colors indicate the genetic background for each strain (orange, wt; blue, 10_deop; green, 8_Δstop). The horizontal lines provide a reference to the ancestor strain (orange, wt; blue, 10_deop). Bar heights below these lines indicate reduced expression, and bar heights above indicate increased expression. (A) RNA abundance relative to wild type. Adjusted P values for each comparison are provided in Table S2. (B) RNA abundance for 10_deop strains relative to wt_10deop. Adjusted P values for each comparison are provided in Table S3 in the supplemental material. Samples were taken 9 min postinfection.

Fitness recovery for promoter knockout strains. Initial and final fitness for evolved lines after ~160 to 180 generations. Promoter knockout lines are indicated along the x axis, colored by genetic background (wt, orange; 10_deop, blue; 8_Δstop, green). Desaturated bars (on the right of each pair) indicate evolved fitness. The orange horizontal line indicates the mean fitness for the wt ancestor strain—the standard against which the magnitude of fitness recovery of attenuation is ultimately measured. Fitness increases are seen in all four lines, but all remain below wt (P < 0.05, two-sample t test). All P values are provided in Table S4 in the supplemental material.

RNA abundance increases in evolved population. Shown are initial and evolved transcript abundance (measured as transcripts per million [tpm], rescaled to a range of 0 to 1) for genes 8 to 12 (rows) in 2 evolved lines (evo-Δϕ9/ϕ10_wt, orange; evo-Δϕ9/ϕ10_8Δstop, green). Each point represents a single measurement, with the bars indicating the mean RNA abundances. We include transcript abundances for the wt strain (orange) as a reference. Significant increases in abundance are indicated with a star, and nonsignificant ones are labeled “NS.” Expression increases in genes 9 and 10A in evo-Δϕ9/ϕ10_wt and in genes 9 to 12 in evo-Δϕ9/ϕ10_8Δstop (FDR < 0.05, two-sample t test), but each of these genes remains well below wt levels (FDR < 0.001). Samples were taken at 9 min postinfection.