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. 2018 Sep 20;46(21):11539–11552. doi: 10.1093/nar/gky851

Figure 3.

Figure 3.

DEAD-box RNA helicase eIF4AI interacts with Rev and promotes Gag synthesis from the unspliced mRNA. (A) HeLa cells were transfected with 1 μg pCDNA-Flag-Rev together with 1 μg pCIneo-HA-eIF4E, pCIneo-HA-eIF4G or pCIneo-HA-eIF4AI. At 24 hpt, the interaction between Flag- and HA- tagged eIFs was analyzed by PLA. Red dots indicate interactions between Flag-Rev and the corresponding HA-tagged protein. Scale bar 10 μm. (B) Dots/cell for Rev-4E, Rev-4G and Rev-4A were quantified using ImageJ (****P < 0.0001, Mann–Whitney test). (C) HeLa cells were transfected with 0.3 μg of pNL4.3R-wt or pNL4.3R-ΔRev together with 1 μg of the pCIneo-HA-eIF4AI vector as described in materials and methods (pCIneo-HA-d2EGFP was used as a control). At 24 hpt, cell extracts were prepared for Gag-Renilla activity measurement and for cytoplasmic RNA extraction and RT-qPCR analyzes. Results for Gag synthesis (left panel), cytoplasmic unspliced mRNA (middle panel) and translational efficiency (right panel) were normalized to the wild type provirus (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments (*P < 0.05; **P < 0.01 and NS; non-significant, t-test).