PACT overexpression reverses PKR-mediated inhibition of HIV-1 LTR expression. (A) PACT reverses PKR activity on HIV-1 LTR expression in HeLa cells. HeLa cells were transfected with 0.05 μg of pGL2-LTR-Luc (lanes 1 to 7) and 1 μg of pcDNA1-PKRwt (PKR) as indicated. Cotransfection was performed with 0.05 (lane 3), 0.1 (lane 4), 0.25 (lane 5), 0.5 (lane 6), or 0.95 (lane 7) μg of pCMV-Flag-PACT. In each experiment the empty vector (pCMV-Flag) was added to reach the same amount of transfected DNA. The activation is the ratio between the Luc level in the presence of PACT and/or PKR versus LTR-Luc alone. Whole-cell extracts (150 μg) (lanes 1 to 7) were subjected to SDS-PAGE and blotted with anti-PACTeg2 and antiactin antibodies as indicated. The lower band of PACT is the endogenous form, whereas the upper band is the transfected Flag-PACT. The graph represents the average of three independent transfections ± standard error of the means. (B) PACT does not increase the RNA level of an LTR-Luc reporter gene inhibited by PKR. Cells were transfected as described in panel A. RT-PCR was performed on RNA extracted from the transfected cells as indicated in Materials and Methods. Lane M, molecular weight markers; lane C, control with no cDNA. The Luc- and the GAPDH-amplified DNAs are indicated on the right.

PACT activation of PKR in HeLa cells requires deletion of 13 amino acids (Δ13) and IFN induction. (A) Schematic representation of PACT and PACTΔ13 and their domains. Domain C is also called the Medipal domain of PACT by homology with TRBP (46). (B) Translation inhibition assay with PACT and PACTΔ13. (Top) HeLa cells were transfected with 800 ng of pGL2C (Promega) encoding Luc (SV40-Luc) and without plasmid (lane 1) or with 0.15 μg (lane 2), 0.3 μg (lane 3), 0.45 μg (lane 4), or 0.6 μg (lane 5) of Flag-PACT-pCB6+ (light bars) or Flag-PACTΔ13-pCB6+ (dark bars). At 24 h posttransfection, cells were treated with 100 U/ml of IFN-β for 24 h and then harvested for Luc activity measurement. Luc activity was normalized for the total protein present in the extract, and the error bars represent the standard error calculated from six independent values. (Bottom) Extracts from lane 1 (no PACT) above or lane 5 (PACT or PACTΔ13) were separated by SDS-PAGE and blotted with an anti-Flag or antiactin antibody. (C) PACT or PACTΔ13 does not activate PKR in the absence of IFN. HeLa cells were transfected alone (lanes 1, 8, and 9) or with 0.1 (lanes 2 and 5), 0.5 (lanes 3 and 6), and 1 (lanes 4 and 7) μg of pCMV2-Flag-PACT or pCMV2-Flag-PACTΔ13, as indicated, or with 2 μg of pcDNA1-PKR and 0.02 μg of poly(I)·poly(C) (lane 9). Whole-cell extracts (200 μg) were subjected to SDS-PAGE and blotted with anti-P-PKR, anti-PKR, anti-Flag, and antiactin antibodies as indicated.

PACTΔ13 weakly interacts with either TRBP or PKR. (A) PACTΔ13 interacts weakly with TRBP and PKR. HeLa cells, not treated (lanes 1 to 3 and 7 to 9) or treated by IFN-α (lanes 4 to 6 and 10 to 12), were transfected alone (lanes 1 and 7) or with 10 μg of pCMV2-Flag-PACT (lanes 2, 5, 8, and 11) or 10 μg of pCMV2-Flag-PACTΔ13 (lanes 3, 6, 9, and 12). IP was performed with 1.5 mg of protein and anti-Flag antibody. A total of 200 μg of proteins from each lysate (input; lanes 1 to 6) and the immunoprecipitated complexes (lanes 7 to 12) was run on a 12% SDS-PAGE gel and blotted using anti-P-PKR, anti-PKR, anti-TRBP672, anti-Flag, and antiactin antibodies. (B) The TRBP Medipal domain does not bind PACT-CΔ13. Shown is the two-hybrid assay for the TRBP-C and PACT-C fragments with the PACT-C and PACT-CΔ13 fragments in the indicated vectors. Tat-CycT interaction is a positive control. CycT is a negative control for PACT-C and PACT-CΔ13.

PACT activity on PKR activation is restored in cells when TRBP expression is decreased by RNAi. (A) RNAi against TRBP. HeLa cells were transfected with 2 μg of pcDNA3-TRBP1 (lane 1) or pcDNA3-TRBP2 (lane 2) without siRNAs (lane 3) or with 80 nM nonspecific siRNA (lane 4) or TRBP siRNA571 (lane 5). Whole-cell extracts in the amount of 25 (lanes 1 and 2) or 200 μg (lanes 3 to 5) were subjected to SDS-PAGE and blotted with anti-TRBP672 or antiactin antibodies as indicated. (B and C) siRNAs against TRBP restore PACT activation of PKR. HeLa (B) or HEK293T (C) cells were transfected without siRNAs (lanes 1 and 2) or with 80 nM nonspecific siRNA (lanes 3 and 4) or 80 nM siRNA571 (lanes 5 and 6) 24 h prior to transfection with 2 μg of pCMV2-Flag (lanes 1, 3, and 5) or pCMV2-Flag-PACT (lanes 2, 4, and 6). Whole-cell extracts (200 μg) were subjected to SDS-PAGE and blotted with anti-P-PKR, anti-PKR, anti-Flag, and antiactin antibodies as indicated. ns, nonspecific.

Stresses dissociate TRBP-PACT interaction and decrease PACT-induced LTR-Luc expression. (A) Stresses dissociate the TRBP-PACT interaction in HEK293T cells. HEK293T cells were transfected alone (lanes 1, 4, 7, and 11) or with 2 μg of pCMV2-Flag-PACT (lanes 2, 3, 5, 6, 8 to 10, and 12 to 14) and not treated (lanes 1, 2, 4, 5, 7, 8, 11, and 12) or treated with 0.5 mM sodium arsenite (As) for 1 h (lanes 3 and 6) or 10 mM hydrogen peroxide (H₂O₂) for 1 h (lanes 9 and 13) or subjected to serum starvation (SS) for 24 h (lanes 10 and 14). IP was performed with 1.5 mg of protein and anti-Flag antibody. Proteins (150 μg) from each lysate (input; lanes 1 to 3 and 7 to 10) and the immunoprecipitated complexes (lanes 4 to 6 and 11 to 14) were run on a 10% SDS-PAGE gel and blotted using anti-P-PKR, anti-PKR, anti-TRBPjbx, anti-Flag, and antiactin antibodies. Anti-TRBP672 and anti-TRBPjbx were both tested and gave the same results. (B) Stresses decrease PACT-induced LTR-Luc expression. HEK293T cells were transfected alone (lane 1) or with 2 μg of pCMV2-Flag-PACT (lanes 2 to 5) and not treated (lanes 1 and 2) or treated with 0.5 mM sodium arsenite (lane 3) or 10 mM H₂O₂ (lane 4) or subjected to SS (lane 5) as described in panel A. Bars represent the amount of firefly Luc activity per μg of total protein and are the average of three independent experiments (± standard error of the mean). (C) Stresses dissociate the TRBP-PACT interaction in HeLa cells. HeLa cells were transfected alone (lanes 1 and 6) or with 2 μg of pCMV2-Flag-PACT (lanes 2 to 5 and 7 to 10) and not treated (lanes 1, 2, 6, and 7) or treated with 0.5 mM sodium arsenite (lanes 3 and 8) or 10 mM H₂O₂ (lanes 4 and 9) or subjected to SS (lanes 5 and 10) as described in panel A. IP was performed with 1.5 mg of protein and anti-Flag antibody. Proteins (150 μg) from each lysate (input; lanes 1 to 5) and the immunoprecipitated complexes (lanes 6 to 10) were run and blotted as described in panel A.

PACT overexpression reverses IFN-induced inhibition of SV40 promoter expression in HeLa, COS-7, NIH 3T3, and MEF cells. (A) Jurkat and HeLa cells are activated by IFN treatment. Jurkat or HeLa cells were incubated without IFN (0) or with 1,000 U/ml of IFN-α2b or IFN-β as indicated. Fifty micrograms of total protein was separated by SDS-PAGE and blotted with anti-P-Stat1 antibodies. (B) PACT reverses IFN-mediated SV40-Luc repression in HeLa cells. HeLa cells were transfected with 0.8 μg of SV40-Luc and 0.1 (lane 3), 0.2 (lane 4), 0.4 (lane 5), of 0.8 (lane 6) μg of pCMV-Flag-PACT. pCMV-Flag was added to reach the same amount of transfected DNA. Cells (lanes 2 to 6) were stimulated with 100 U/ml IFN-β as indicated. The Luc level was normalized to 1 for cells treated with IFN. Fifty micrograms of whole-cell extracts (lanes 1 to 6) was subjected to SDS-PAGE and blotted with anti-Flag and antiactin antibodies as indicated. (C) PACT mildly increases the RNA level of an SV40-Luc reporter gene inhibited by IFN. Cells were transfected as described in panel B (lanes 1 to 6). RT-PCR (top) was performed on RNA extracted from the transfected cells as indicated in Materials and Methods. Lane M, molecular weight markers; lane C, control with no cDNA. The Luc- and the GAPDH-amplified DNAs are indicated on top. Whole-cell extracts (200 μg) were subjected to SDS-PAGE and blotted (bottom) with anti-Luc and anti-actin antibodies as indicated. (D) PACT reverses IFN-mediated SV40-Luc repression in COS-7, NIH 3T3, and MEF cells. COS-7, NIH 3T3, MEF, and PKR^−/− MEF cells were transfected as described in panel B with 0.8 μg of SV40-Luc plasmid (all) and 0.2 μg of pCMV-Flag-PACT (Flag-PACT) as indicated. Cells were treated with 100 U/ml IFN-β as indicated. The Luc level was normalized to 1 for cells treated with IFN (black bars) compared to untreated cells (open bars). The activation is calculated as the ratio between Luc expression in the presence of PACT versus in the absence of PACT in the IFN-treated cells (gray bars). Graphs represent the averages of three to five independent transfections ± standard error of the mean.

PACT and PACTΔ13 induce phosphorylation of PKR in astrocytes. (A) Translation inhibition assay with PACT and PACTΔ13. U251MG cells were transfected with 800 ng of pGL2C (SV40-Luc) without plasmid (lane 1) or with 0.15 μg (lane 2), 0.3 μg (lane 3), 0.45 μg (lane 4), or 0.6 μg (lane 5) of Flag-PACT-pCB6+ (light bars) or Flag-PACTΔ13-pCB6+ (dark bars). Cells were treated with IFN-β and harvested as described in the legend of Fig. 3B. Luc activity was normalized for the total protein present in the extract, and the error bars represent the standard error calculated from two independent experiments performed in triplicate. (B) PACT and PACTΔ13 activate PKR in astrocytes. U251MG cells were transfected alone (lane 1) or with 0.1 (lanes 2 and 5), 0.5 (lanes 3 and 6), and 1 (lanes 4 and 7) μg of pCMV2-Flag-PACT or pCMV2-Flag-PACTΔ13 as indicated. Whole-cell extracts (200 μg) were subjected to SDS-PAGE and blotted with anti-P-PKR, anti-PKR, anti-Flag, and antiactin antibodies as indicated. (C) PACT-induced phosphorylation of PKR in astrocytes is suppressed by TRBP2 overexpression. U251MG cells that overexpress pcDNA3-TRBP2 were transfected alone (lane 1) or with 0.1 (lane 2), 0.5 (lane 3), and 1 (lane 4) μg of pCMV2-Flag-PACT. Whole-cell extracts (150 μg) were subjected to SDS-PAGE and blotted with anti-P-PKR, anti-PKR, anti-Flag, anti-TRBP672, and antiactin antibodies as indicated.

PACT activates PKR in Tarbp2^−/− cells. (A) Northern blot for Tarbp2 mRNA. A blot with equal amounts of RNA from Tarbp2^+/− (+/−) and Tarbp2^−/− (−/−) fibroblasts (TEFs) and testis was probed with a 1.5-kb Tarbp2 cDNA fragment as indicated. (B) PACT-induced PKR activation in Tarbp2^−/− cells. Tarbp2^+/− and Tarbp2^−/− TEFs were transfected with 2 μg of pCMV2-Flag (lanes 1 and 3) or pCMV2-Flag-PACT (lanes 2 and 4) and 2 μg of pEGFP-N3 (lanes 1 to 4). Whole-cell extracts (200 μg) were subjected to SDS-PAGE and blotted with anti-P-PKR, anti-PKR, anti-PRBP, anti-Flag, and antiactin antibodies as indicated. Transfection efficiency is the average of the percentage of EGFP-expressing cells versus total number of cells in three representative experiments.

Stresses activate PKR but do not modify TRBP levels significantly. (A) Stress mediated by arsenite treatment induces higher PKR phosphorylation in Tarbp2^−/− cells. Tarbp2^+/− and Tarbp2^−/− TEFs were either untreated (lanes 1 and 3) or treated with 0.5 mM sodium arsenite (As) for 1 h (lanes 2 and 4). Whole-cell extracts (200 μg) were subjected to SDS-PAGE and blotted with anti-P-PKR, anti-PKR, anti-PACTeg2, and antiactin antibodies as indicated. RAX, the murine homolog of PACT, was detected with the anti-PACT. The lower-migrating band revealed with anti-P-PKR in the absence of sodium arsenite treatment (lanes 1 and 3) corresponds to the size of PKR shown just below and might represent a different phosphorylated form in murine cells. (B) Stresses do not modify TRBP levels significantly. HEK293T cells were transfected without plasmid (lanes 1 to 4) or with 2 μg of pCMV2-Flag-PACT (lanes 5 to 8) and not treated (lanes 1 and 5) or treated with 0.5 mM sodium arsenite (As) for 1 h (lanes 2 and 6) or 10 mM hydrogen peroxide (H₂O₂) for 1 h or subjected to serum starvation (SS) for 24 h. A total of 150 μg of proteins from each lysate was run on a 10% SDS-PAGE gel and blotted using anti-TRBPjbx and antiactin antibodies.

Model for the controlled activation of PKR by TRBP, PACT, and stress. (A) PKR activation in cells with low levels of TRBP. In cells where TRBP concentration is low, PACT can activate PKR, which in turn phosphorylates eIF2α (eIF2α-P). eIF2α-P blocks viral and cellular translation initiation. (B) Absence of PKR activation in cells with high levels of TRBP. When TRBP concentration is high, PACT forms heterodimers with TRBP that inhibit PKR phosphorylation (P) and overcome its translation block. PACT is unable to activate PKR and, therefore, mRNA translation remains active. (C) Overexpression of PACTΔ13 strongly activates PKR. When PACTΔ13 is overexpressed in cells, the heterodimer with TRBP is unstable. As a consequence, PACTΔ13 strongly activates PKR. P-PKR has a decreased affinity for PACTΔ13 and becomes available to activate eIF2α, which blocks translation initiation. (D) Stress treatment dissociates TRBP-PACT interaction and activates PKR. A stress treatment induces a separation between TRBP and PACT. Therefore, PACT becomes available for PKR activation either as a monomer or as a dimer. In all panels, A, B, and C represent regions that encompass dsRBD1, dsRBD2, and the Medipal domains, respectively.