FIG. 8.

Effects of dnMEK and caMEK overexpression on R3616 viral recovery and PKR function during R3616 infection. (A) Replicate cultures of HT-dnMEK, HT1080, and HT-caMEK cells were exposed to 1 PFU of R3616 virus per cell in serum-free medium for 2 h, after which medium containing virus was removed and fresh medium containing 1% calf serum was added. At 36 h after infection, R3616 viral recovery was determined by standard plaque assay. (C) R3616 viral recovery from replicate cultures of Mia-dnMEK, MiaPaCa2, and Mia-caMEK at 36 h after infection. (B) To determine the influence of mutant MEK expression on PKR activation, replicate cultures of HT-dnMEK, HT1080, and HT-caMEK cells were exposed to 10 PFU of R3616 virus per cell. Cells were harvested at 12 h after infection and processed as described in Materials and Methods. Electrophoretically separated proteins were immunoblotted with antibodies for total and the phosphorylated forms of ERK1 and ERK2 on threonine 202 and tyrosine204, PKR on threonine 446, and eIF-2α on serine 51. The same lysates were immunoblotted with antibodies for immediate-early [α(ICP27)] and late [γ(gC)] viral proteins. (D) Immunoblotting was performed on replicate lysates of Mia-dnMEK, MiaPaCa2, and Mia-caMEK cells.