Ribosome Profiling Reveals Translational Upregulation of Cellular Oxidative Phosphorylation mRNAs During Vaccinia Virus-induced Host Shutoff

The experiment procedures were described previously (13). Briefly, ribosome profiling was carried out as described elsewhere with minor modifications (10). VACV (at an multiplicity of infection [MOI] of 10)- and mock-infected HeLa S3 cells pretreated with translational inhibitor cycloheximide (CHX) were lysed and treated with DNase (Thermo Fisher Scientific, MA), and the lysate was clarified. Messenger RNA was isolated from a portion of the lysate using oligo(dT) and fragmented using RNaseIII (New England Biolab, MA). The mRNA fragments between 50 and 80 nucleotides (nt) were extracted. The ribosome-protected RNA fragments (RPFs) were separated by electrophoresis after the lysate was digested with RNase I (Thermo Fisher Scientific) and the RPFs between 28 and 34 nts were isolated. The purified mRNA and RPFs were used to generate libraries for deep sequencing as described previously (10). The purified libraries were sequenced using a HiSeq 2000 system.