1. Volume 25, Issue 3, Pages 369-383 (February 2007)
The Proteasome Regulates HIV-1 Transcription by Both Proteolytic and Nonproteolytic Mechanisms 
Irina Lassot, Daniel Latreille, Emilie Rousset, Marion Sourisseau, Laetitia K. Linares, Christine Chable-Bessia, Olivier Coux, Monsef Benkirane, Rosemary E. Kiernan 
Molecular Cell 
Volume 25, Issue 3, Pages (February 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
The 19S and 20S Subunits of the Proteasome Have Distinct Effects on Transcription from the HIV-1 Promoter
(A) Ablation of proteasome components by RNA interference. HeLa-LTR-luc cells were subjected to three consecutive rounds of transfection with siRNAs directed against proteasome components or a control siRNA (Sc) as indicated on the figure. Following the final round of transfection, cells were transduced by overnight treatment with GST-Tat. Cell extracts were harvested 24 hr later and analyzed by direct western blotting using the antibodies indicated.
(B) Subunits of the 19S particle are required for Tat-mediated transactivation. Cells transfected with siRNAs as in (A) were assayed for luciferase activity. Fold Tat transactivation was calculated relative to transfection in the absence of Tat.
(C) 20S α4 regulates basal transcription from the HIV-1 promoter. HeLa-LTR-luc cells were transfected as described in (A) and assayed for luciferase activity. Shown are luciferase values normalized to the value obtained in control cells, which was attributed a value of 1.
(D) Inhibition of proteasome activity increases HIV-1 transcription. HeLa-LTR-luc cells were treated with GST or GST-Tat as indicated followed by treatment with MG132 or carrier as indicated below the figure. Values represent the amount of luciferase reporter gene mRNA normalized to the amount of GAPDH mRNA in each sample. Values shown above the columns represent fold transactivation by GST-Tat relative to GST alone.
(E) Overexpression of 19S subunits enhances Tat-mediated transcription. HeLa-LTR-luc cells were transfected with 500 ng of plasmid expressing HA-Rpt1-6, HA-Rpn9, or Flag-20S β4, as indicated, in the presence or absence of 100 ng of pTat101Flag. Shown is coactivation of Tat-dependent transcription where transactivation by Tat in control cells was attributed a value of 1. Western blot of cell extracts using anti-HA, anti-Flag, and anti-tubulin antibodies is shown below the graph. All graphs represent mean and standard error obtained from at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
Proteasomal ATPases Are Recruited to the HIV-1 Promoter in Response to Tat
(A) Schematic diagram showing the locations of primers used to amplify sequences present in chromatin immunoprecipitates by Q-PCR. HIV-1 DNA sequences are depicted as a black line; NF-κB and Sp1 sites within the HIV-1 promoter are indicated by hatched and striped boxes, respectively; transcription start site is shown by the arrowhead; and luciferase reporter gene is shown as a white box. TAR RNA stem loop is indicated at the 5′ end of the transcript in gray. Primers used to amplify promoter-proximal and coding region sequences are indicated on the figure.
(B) HeLa-LTR-luc cells were treated with GST (gray bars) or GST-Tat101 (black bars) and then analyzed by ChIP by using the antibodies indicated on the figure. Regions proximal to the promoter (top panel) and within the coding sequence (bottom panel) were amplified by Q-PCR.
(C) Tat induces a redistribution of cellular proteasome components. Nuclear extracts (NE) of S3 and S101 cells, depicted as − and +Tat, respectively, were analyzed by gel filtration chromatography followed by SDS/PAGE and western blot using the antibodies indicated. An aliquot of each fraction was analyzed for peptidase activity, as indicated below the figure. Elution positions of molecular weight markers are shown above the figure.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3
A Proteasome-Associated Protein, PAAF1, Is a Coactivator of Tat
(A) PAAF1 induces proteasome dissociation in vitro. Purified 26S proteasome (denoted as RP2CP on the figure) was incubated with GST or GST-PAAF1 for 1 hr at 37°C. Products were analyzed by nondenaturing PAGE followed by in-gel peptidase activity assay (left panel). Products were transferred to PVDF and analyzed by western blot using anti-Rpt4 antibody. A faster-migrating complex that represents 19S RP is denoted by an asterisk on the figure (right panel).
(B) Tat interacts with PAAF1. Tat-HA was immunoprecipitated with anti-HA antibody, and immunoprecipitates were analyzed by western blot using anti-Flag (top panel). An aliquot of cell extract was analyzed by direct western blot using anti-Flag and anti-HA antibodies (middle and bottom panels).
(C) PAAF1 is a coactivator of Tat-mediated transcription. HeLa-LTR-luc cells were transfected with or without plasmid expressing Tat101Flag (100 ng) in the presence of plasmid expressing HA-PAAF1 (500 ng) or empty vector. Extracts were assayed for luciferase activity. Fold activation refers to the increase in Tat transactivation over that seen with Tat101Flag alone, which was normalized to 1. Expression of HA-PAAF1 in cell extracts is shown below.
(D) Endogenous PAAF1 is required for optimal Tat-mediated transactivation. HeLa-LTR-luc cells were transfected with control (Sc) or PAAF1-specific (PAAF1) siRNA. Following treatment with GST or GST-Tat, cell extracts were analyzed for luciferase activity, which was normalized to protein content (left panel). An aliquot of cells was analyzed by Q-RT-PCR for the amount of PAAF1 mRNA, which was normalized to GAPDH mRNA in the same samples (right panel).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4 Formation of a 19S-like Complex Is Dependent on PAAF1
(A) Nuclear extracts from control (−) or Flag-PAAF1-expressing (+) S3 cells were analyzed by gel filtration chromatography followed by SDS/PAGE and western blot using the indicated antibodies. Fractions containing peak peptidase activity are indicated below.
(B) Formation of a 19S-like complex is dependent on PAAF1. S101 cells were transfected with control or PAAF1-specific siRNA as indicated and analyzed by gel filtration chromatography followed by SDS/PAGE and western blot using anti-Rpt4 and anti-20S α4 antibodies. Fractions containing peak peptidase activity are indicated below.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5
Tat-Mediated Recruitment of a 19S Subcomplex to the HIV-1 Promoter Is Dependent on PAAF1
(A) Recruitment of Tat to the LTR is unaffected by PAAF1. HeLa-LTR-luc cells, transfected with pFlag-PAAF1 or empty vector, then treated with GST or GST-Tat101 as indicated, were analyzed by ChIP by using anti-GST antibodies. Sequences within the promoter-proximal and coding regions were amplified by Q-PCR. Shown is fold enrichment relative to immunoprecipitations performed in the absence of Tat.
(B) Tat recruits PAAF1 to HIV-1 DNA sequences. The same samples analyzed in (A) were immunoprecipitated by using anti-Flag antibodies and amplified by Q-PCR. Shown is fold enrichment relative to immunoprecipitations performed in the absence of Flag-PAAF1.
(C) PAAF1 is required for the specific recruitment of a 19S subcomplex to the HIV-1 promoter in the presence of Tat. HeLa-LTR-luc cells were transfected with control (Sc) or PAAF1-specific (PAAF1) siRNA and then treated with GST or GST-Tat as indicated. Samples were analyzed by ChIP using the indicated antibodies followed by Q-PCR of the promoter-proximal region (left panel). The amount of PAAF1 mRNA in control and PAAF1 knockdown cells was quantitated by Q-RT-PCR and normalized to the amount of GAPDH mRNA in the same samples (right panel).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6
Knockdown of Proteasome Subunits and Inhibition of Proteasome Activity Affect Different Stages of HIV-1 Transcription
(A) Schematic diagram showing the locations of the different primers used in Q-RT-PCR analysis. HIV-1 promoter is shown as a gray line, and luciferase reporter gene is shown as a white box. The RNA transcript is shown as a black line. TAR RNA hairpin is indicated at the 5′ end of the transcript. The location of the RNAPII pause site is indicated. Forward and reverse primers used to amplify TAR sequences are located within TAR; to amplify early elongated transcripts, a forward primer corresponding to the stem sequence of TAR was used together with a reverse primer corresponding to the 5′ end of luciferase; midelongated transcripts were amplified by using forward and reverse primers within luciferase.
(B) Knockdown of a 19S or 20S subunit affects transcriptional elongation. HeLa-LTR-luc cells were transfected with control siRNA or siRNAs specific for a 19S subunit (Rpt1) or a 20S subunit (20S α4), then treated with GST or GST-Tat as indicated. Total RNA was isolated, and reverse transcripts were analyzed by Q-PCR using the primer pairs indicated on the figure. Values were normalized to the quantity of GAPDH in each sample. Knockdown of Rpt1 and 20S α4 was verified by western blot of cell extracts using the antibodies indicated as shown in the top panel.
(C) Inhibition of proteasome activity increases initiation of transcription. HeLa-LTR-luc cells were treated with MG132 or carrier in the presence or absence of GST or GST-Tat, as indicated on the figure. Total RNA was purified, reverse transcribed, and analyzed by Q-PCR using the primer pairs indicated. Values were normalized to the quantity of GAPDH in each sample.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7
19S Subunits Are Required for Clearance of RNAPII and Cdk9 from the HIV-1 Promoter
(A) HeLa-LTR-luc cells were transfected with control or Rpt1-specific siRNA and treated with GST or GST-Tat as indicated on the figure. Samples were analyzed by ChIP using anti-GST, anti-RNAP II, or anti-Rpt1 antibodies as indicated. Promoter-proximal and coding region sequences were amplified by Q-PCR from immunoprecipitates. An aliquot of cell extract was analyzed by western blot using anti-Rpt1 and anti-tubulin antibodies (bottom right panel).
(B) Experiment was performed as described in (A) except that cells were transfected with pHA-CDK9 and pCyclinT1 and samples were immunoprecipitated by using anti-HA antibody.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions