Figure 4

PACT, ADAR1 and TRBP inhibit PKR activation in HIV-1 producing cells. A) PACT, ADAR1 and TRBP, but not Staufen1, inhibit PKR and eIF2α phosphorylation. HEK 293T cells were mock transfected (lane 1), transfected with 2 μg pNL4-3 (lanes 2–6), and 1.5 μg of pCMV2-Flag-PACT (lane 3), pcDNA3.1-ADARp150-V5 (lane 4), pcDNA3-TRBP2 (lane 5) or pCMV-RSV-Staufen1-HA (lane 6). pcDNA3 was added to reach the same amount of transfected DNA. (Top) RT activity from cell supernatants normalized to 100% in the absence of PKR or dsRBPs. Shown is the average of 6 independent transfections ± SEM. (Bottom) Immunoblot of 150 μg of cell extract of a representative experiment from the same transfected cells using antibodies against P-PKR, PKR, P-eIF2α, eIF2α, PACT, ADAR1, TRBP, Staufen1, HIV-1 p24 and actin as indicated. Probing for P-eIF2α and eIF2α were performed on a separate membrane and the corresponding actin is shown. B) PACT, ADAR1 and TRBP, but not Staufen1, restore PKR inhibition of HIV-1 protein expression and virion production. HEK 293T cells were mock transfected (lane 1), transfected with 2 μg pNL4-3 (lane 2–7), 0.5 μg pcDNA1-PKR (lanes 3–7) and 1.5 μg of pCMV2-Flag-PACT (lane 4), pcDNA3.1-ADARp150-V5 (lane 5), pcDNA3-TRBP2 (lane 6) or pCMV-RSV-Staufen1-HA (lane 7). The empty pCMV2 vector was used to supplement transfections such that the same amount of DNA was transfected into each well. (Top) % RT activity is calculated as in A). Shown is the average of 6 independent transfections ± SEM. (Bottom) 150 μg of each cell extract was analyzed by immunoblot against P-PKR, PKR, P-eIF2α, eIF2α, PACT, ADAR1, TRBP, Staufen1, HIV-1 p24 and actin as indicated. Shown is a representative experiment from the same transfected cells. Probing for P-eIF2α and eIF2α were performed on the same membrane as HIV-1 p24 and have the same actin.