Fig 2.

Phenotypic characterization of H5pg4100 (wt) and H5pm4174 (E1B-P minus) viruses. (A) Viral and cellular protein expression analyses. A549 cells were mock infected or infected with the indicated viruses at an MOI of 20 FFU per cell and harvested at the indicated time points. Total cell extracts were prepared, separated by SDS-PAGE, and immunoblotted for the indicated proteins. (B) Coimmunoprecipitation with Mre11 or p53. A549 cells were mock infected or infected with wt and mutant viruses at an MOI of 20 FFU per cell. Whole-cell lysates were subjected to immunoprecipitation with anti-Mre11 (pNB 100-142) or anti-p53 (DO-1) antibodies. Proteins separated by SDS-PAGE were detected by immunoblotting with anti-E1B antibody 2A6. Steady-state concentrations (inputs) of E1B-55K (2A6), Mre11 (pNB 100-142), β-actin (AC-15), and p53 (DO-1) were determined by immunoblotting of protein extracts with the appropriate antibodies. (C) Virus growth. A549 and H1299 cells were infected with wt and E1B-P minus virus at an MOI of 2 or 20 FFU per cell and harvested at 48 h p.i. The virus yield was determined by quantitative E2A-72K immunofluorescence staining (B6-8) on HEK293 cells. The results represent the average of at least three independent experiments. Bars indicate the standard error of the mean values. Virus yield efficiency is represented as a percentage of H5pg4100 (wt; MOI = 20) yield efficiency. (D) Viral DNA synthesis. A549 cells were infected with H5pg4100 (wt) or H5pm4174 (E1B-P minus) virus at an MOI of 2 or 20 FFU per cell. Total DNA was isolated at the indicated time points and subjected to PCR. PCR products were analyzed by agarose gel electrophoresis. PCR amplification of GAPDH (lanes 21 to 40) served as an internal control.