Figure 3

PACT increases HIV-1 protein expression and virion production in HEK 293T cells by PKR and eIF2α inhibition. A) PACT inhibits endogenous PKR and eIF2α phosphorylation and increases pNL4-3 expression. HEK 293 T cells were mock transfected (lane 1), transfected with 2 μg pNL4-3 (lanes 2–5) and 0.5 μg (lane 3), 1.0 μg (lane 4) or 1.5 μg (lane 5) of pCMV2-Flag-PACT. pCMV2 was added to reach the same amount of transfected DNA. (Top) % RT activity is the ratio between the RT level in the presence of PKR and PACT versus pNL4-3 alone normalized to 100%. Shown is the average of 6 independent transfections ± SEM. (Middle) Immunoblot of cell extracts of a representative experiment from the same transfected cells using antibodies against P-PKR, PKR, Flag, HIV-1 p24, P-eIF2α, eIF2α and actin. (Bottom) Ratio of phosphorylated PKR (P-PKR) versus PKR and P-eIF2α versus eIF2α. The band intensity was digitalized using Adobe Photoshop software from the bands shown above. P-PKR/PKR and P-eIF2α/eIF2α ratio was calculated by dividing the P-PKR or P-eIF2α intensity by the total PKR or eIF2α intensity of each band. B) PACT reverses PKR inhibition of pNL4-3. HEK 293 T cells were mock transfected (lane 1), transfected with 2 μg pNL4-3 (lanes 2–6), 0.5 μg pcDNA1-PKR (lanes 3–6), 0.5 μg (lane 4), 1.0 μg (lane 5) or 1.5 μg (lane 6) of pCMV2-Flag-PACT. Empty corresponding plasmids were added to reach the same amount of transfected DNA. (Top) % RT activity is calculated as in A). Shown is the average of 6 independent transfections ± SEM. (Middle) Immunoblot of cell extracts of a representative experiment from the same transfected cells using the same antibodies as in A). (Bottom) Ratio of phosphorylated PKR versus PKR and P-eIF2α versus eIF2α. The band intensities were calculated as in A).