Figure 9. Stable knock down of PKR can rescue the replication of M029-defective MYXV in human cells.

HeLa cell lines with constitutive knockdown of PKR (shPKR) were constructed by infecting parental HeLa with a lentivirus packaged with shRNAs targeting PKR mRNA. A control HeLa cell line (shControl) was also constructed by infecting HeLa cells with a lentivirus packaged with control shRNAs. Single step growth curves of MYXV infection in A) shControl B) shPKR cells. The indicated cells were infected with vMyx-GFP, vMyx-M029KO or vMyx-M029ID at an MOI of 5, and then cells were collected at 0, 6, 24, and 48 h p.i. The virus titers were determined in triplicate following serial dilution onto RK13-E3 cells. Data are representative of three independent experiments. C) Fluorescence microscope images of infected shControl (columns 1 and 3), and shPKR (columns 2 and 4) cells. The images were taken using an inverted fluorescence microscope using a lens with ×10 magnification at 48 h p.i. The cells were infected with a low MOI of 0.5 (columns 1 and 2) and high MOI of 3.0 (columns 3 and 4) with vMyx-GFP (top panels), vMyx-M029ID (middle panels) and vMyx-M029KO (bottom panels). D) Expression of viral early (M-T7) and late (Serp-1) proteins in the infected shControl and shPKR cell lines. Cells were left uninfected (M) (lanes 1 and 8) or were infected with vMyx-GFP (top panels) or vMyx-M029ID (middle panels) at an MOI of 3. Cells were collected at 2, 4, 8, 12, 24 and 24a in the presence of AraC (lanes 7 and 14) h p.i. The membranes were first probed with anti-Serp-1 antibody, stripped and probed for actin (loading control).