Fig. 3

PKR-dependent rev/tat mRNA splicing requires eIF2α phosphorylation. Expression of non-phosphorylatable eIF2αS51A, yet not of phosphomimetic eIF2αS51D, inhibits splicing. BHK-21 cells were cotransfected with 1.5 µg of pcDNA-3’HIV DNA together with 1.5 µg DNA of pBS empty vector (EV), eIF2αS51A expression vector (S51A), vector expressing wild type eIF2α (eIF2αwt) or vector expressing eIF2αS51D (S51D). Total RNA was isolated at 20 h post-transfection. Spliced and unspliced rev/tat transcripts were determined by qRT-PCR. Splicing efficiency is expressed as mRNA/pre-mRNA ratio (error bars, SEM; n = 3). A representative experiment is shown