FIG. 5.

Analysis of initiation translation complexes formed during rotavirus infection. MA104 cells were infected with rotavirus RRV at an MOI of 3, treated with thapsigargin (400 nM), or transfected for 1 h with 5 μg/ml poly(I:C) or purified rotavirus dsRNA. Six hours after the indicated treatment, cells were lysed by homogenization in the presence of 100 μg/ml cycloheximide and were loaded into discontinuous sucrose gradients (10 to 50%). (A) The sucrose gradients were fractionated using an absorbance monitor, and the OD₂₆₀ profiles were obtained. (B) The presence of the indicated ribosomal complexes was confirmed by visualizing the 28S and 18S ribosomal RNAs by the ethidium bromide staining of agarose gels. (C and E) The indicated fractions or the total, nonfractionated lysates (D) were tested for the presence of several translation initiation factors by immunoblotting using antibodies against phospho-eIF2α (eIF2α-P), total eIF2α, PKR, eIF2Bɛ, eIF2A, S6, or phospho-S6 (S6-P) as indicated.