Dependence of various measures for protein stability over the simulation time after removal of the center-of-mass (c.o.m.) constraint (set to 0 ns).

<p>From top to bottom: Root mean square deviation (RMSD) of the protein backbone, <i>z</i> coordinate (membrane normal) of the c.o.m. of the protein (corrected by removing the total membrane drift), the protein's radius of gyration (<i>R_g</i>), and the helical fraction recognized for the fold.</p

Alignment of predicted amino acid sequences of PB1-F2 proteins.

<p>The proteins from A/Puerto Rico/8/34 (H1N1) strain (PB1-F2_pr8), the Spanish flu isolate (PB1-F2_sf) and the bird flu virus (H5N1) (PB1-F2_bf) have an overall identity (*) of ca 60%. The domains, which are predicted by structural prediction algorithms to have a high propensity for α-helixes are marked in gray. The truncated peptide sPB1-F2_pr8^50–87 is underlined.</p

<i>s</i>PB1-F2 generates Ca²⁺ and anion fluxes into liposomes.

<p>(A) Fluorescence of liposomes with Ca²⁺ sensitive dye Fluo3 was recorded before and after adding (at arrow) ionophore Valinomycin (triangle), sPB1-F2_pr8 alone (filled squares) or together with Valinomycin (open squares). Peptide and ionophore were added during the time gap of ca. 1 min indicated in the graph. The presence of the peptide results in an increase in fluorescence indicating an influx of Ca²⁺ into the liposomes. The ionophore enhances Ca²⁺ influx because it prevents building up of a charge, which hinders net Ca²⁺ influx. (B) Fluorescence of liposomes filled with Ca²⁺ sensitive dye Fluo-3 before and after addition (at arrow) of 1 µM peptide to incubation medium. The truncated peptide sPB1-F2_pr8^50–87 results in a fast rise in Fluo3 fluorescence. (C) Fluorescence of liposomes filled with anion sensitive dye lucigenin was measured before and after adding of anion specific ionophore TBT (filled squares, added at arrow 1), sPB1-F2_pr8 (open triangle, arrow 2). The control was left untreated (filled circles); the stepwise drop of the control signal is due to an unspecific drift of the signal. Both ionophore and sPB1-F2_pr8 generate a strong quenching of the lucigenin fluorescence well beyond the control indicating an influx of anions. Peptide and ionophore were added during the time gap of ca. 1 min indicated in the graph.</p

Snapshots of the simulation system after removal of the center-of-mass constraint (set to 0 ns).

<p>The protein is shown in cartoon representation with explicit depiction of positively charged residues (arginine: blue, lysine: red). Lipid molecules have been removed except for the head groups that are depicted as grey spheres. Potassium ions are shown in green, chloride ions in blue. The c-terminus is located on the bottom side.</p

<i>s</i>PB1-F2 evoked membrane conductance.

<p>(A) Addition of <i>s</i>PB1-F2_PR8 protein at 1 µM to the trans side of a planar lipid bilayer results in current fluctuations. In the present case only occasionally clear channel like fluctuations (see expanded trace) are resolvable on the background of many unresolved fluctuations. (B) In a majority of experiments the conductance fluctuates in a channel like manner between a closed (c) and two defined conductance levels o₁ and o₂. Transitions between the two conductance levels (*) are expanded in the left inset. The channel like fluctuations are occasionally interrupted by burst like events (**), which reveal also at higher magnification (inset on the right) no resolvable conductance levels.</p

I/V relation of the small (o₁) and large (o₂) <i>s</i>PB1-F2 generated current fluctuation.

<p>(A) Unitary currents were recorded in bilayer with 500 mM KCl on <i>trans</i> side and 500 mM NaCl on trans (open circles) or with 500 mM KCl on cis and 500 mM K-gluconate on trans (filled squares). (B) I/V relation obtained with 500 mM KCl on trans side and 50 mM KCl on cis side. (C) I/V relation obtained with 500 mM KCl on cis and 500 mM CaCl₂ on trans side. Currents were elicited upon adding <i>s</i>PB1-F2_pr8 (in A-C) and sPB1-F2_sf (in C) to trans side.</p

In Vitro Tat Deacetylation by Human SIRT Proteins

<div><p>(A) Scheme of Tat deacetylation assay with immunoprecipitated SIRT1–7 proteins. Expression vectors for FLAG-tagged SIRT proteins were transfected into HEK 293 cells, immunoprecipitated, and incubated with synthetic Tat (72 amino acids) carrying an N-terminal biotin label and an acetyl group at position 50 (AcTat) in the presence of NAD⁺. Immunoprecipitated material was also analyzed in a radioactive (³H) histone deacetylase assay using an H3 peptide as a substrate.</p> <p>(B) WB analysis of deacetylation reactions with antibodies specific for acetylated lysine 50 in Tat (α-AcTat), with SA-HRP, or with α-FLAG antibodies.</p> <p>(C) WB of Tat deacetylation by immunoprecipitated SIRT1 in the presence or absence of NAD⁺, TSA, or nicotinamide (Nic).</p></div

Inhibition of HIV Gene Expression by a Small Molecule Inhibitor of SIRT1

<div><p>(A) In vitro histone deacetylation assays including recombinant SIRT1, radioactively labeled histone H3 peptide, and indicated concentrations of splitomicin or HR73. The average (± SEM) of two experiments performed in duplicate is shown for each point.</p> <p>(B) Chemical structures of splitomicin and its derivative HR73.</p> <p>(C) Inhibition of Tat transactivation by HR73. RSV-Tat (0, 20, and 200 ng) and HIV LTR luciferase (200 ng) or RSV-luciferase (200 ng) vectors were transfected into HeLa cells. Transfected cells were treated with indicated concentrations of HR73 or DMSO for 8 h.</p> <p>(D) Inhibition of HIV gene expression by HR73. GFP expression in Jurkat T cells infected with HIV_NL4–3 containing the GFP open reading frame in place of the viral <i>nef</i> gene or with an HIV-based lentiviral vector expressing GFP from the heterologous EF-1α promoter. Treatment with HR73 (1 μM in DMSO) or DMSO was performed for 36 h after overnight infection. The average (± SEM) of four experiments is shown.</p></div

SIRT1 Is a Positive Cofactor for Tat Transactivation

<div><p>(A) Cotransfection of SIRT1 or the catalytically inactive SIRT1 mutant SIRT1H363Y with the HIV LTR luciferase construct and increasing amounts of a Tat expression vector (RSV-Tat: 0, 2, 20, and 200 ng), an HIV LTR luciferase construct containing mutated binding sites for the transcription factor NF-κB and RSV-Tat (20 ng), or with an RSV-luciferase construct (200 ng) in HeLa cells. The average of three experiments is shown (± standard error of the mean [SEM]).</p> <p>(B) WB analysis of HeLa cells 72 h after transfection of siRNAs directed against SIRT1 or GL3 control siRNAs.</p> <p>(C) Cotransfection of the HIV LTR luciferase construct with increasing amounts of CMV-Tat or CMV-TatK50R (0, 50, 100, 200, 400, and 800 ng) 48 h after transfection of double-stranded siRNAs directed against SIRT1 or GL3 control siRNAs in HeLa cells. Luciferase activity was measured 24 h after plasmid transfection and 72 h after siRNA transfection. Note that all luciferase reporter vectors used in this study are based on the pGL2 luciferase vector, which is not affected by GL3-specific siRNAs [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030041#pbio-0030041-b36" target="_blank">36</a>]. The average of three experiments is shown (± SEM).</p> <p>(D) The transcriptional activity of increasing amounts of the CMV-luciferase reporter (0, 50, 100, 200, 400, and 800 ng) was similar in SIRT1 knockdown or GL3-treated control cells. The average of two experiments performed in duplicate is shown (± SEM).</p> <p>(E) WB of endogenous SIRT1 or actin 72 h after transfection of siRNA directed against SIRT1 or mutated SIRT1 siRNA.</p> <p>(F) Cotransfection of the HIV LTR luciferase with increasing amounts of CMV-Tat (0, 2, 20, and 200 ng) in HeLa cells pretransfected with wild-type or mutant SIRT1 siRNA oligonucleotides as described in (C). WT, wild-type.</p></div

Transcriptional Activity of AcTat Depends on Deacetylation by SIRT1

<div><p>(A) AcTat functions through TAR and cyclinT1 binding. Nuclear microinjection of increasing amounts of synthetic Tat or AcTat together with wild-type (wt TAR), TAR Δbulge, or TAR Δloop mutant HIV LTR luciferase constructs into HeLa cells. Cells were coinjected with CMV-GFP, and luciferase activity was calculated per GFP-positive cell. An average of three experiments is shown (± SEM).</p> <p>(B) AcTat transactivation requires CDK9. HeLa cells microinjected with Tat or AcTat (each 30 ng/μl) and the HIV LTR luciferase reporter were treated with increasing amounts of DRB, a known CDK9 inhibitor, for 4 h.</p> <p>(C) AcTat transcriptional activity is inhibited by nicotinamide, but not TSA. HeLa cells injected with HIV LTR luciferase and increasing amounts of AcTat were treated with TSA (400 nM) or nicotinamide (5 mM) for 4 h. The average of two experiments is shown.</p> <p>(D) SIRT1 is necessary for AcTat, but not Tat function. HeLa cells were transfected with siRNAs specific for SIRT1 or GL3 control siRNAs 48 h before microinjection of HIV LTR luciferase and Tat or AcTat (each 30 ng/μl). The average of three experiments is shown (± SEM).</p></div