Identification of Tat-interacting proteins by mass spectrometry. A. Flag-immunoprecipitated material from Tat-Flag- and mock-transfected HEK 293T cells was resolved by 6–10% gradient SDS-PAGE gel, followed by silver staining. Protein bands present exclusively in the sample transfected with Tat-Flag were excised from the gel and their identification attempted by ESI-MS/MS. The identified proteins, in addition to hNAP-1 and Tat-Flag, are indicated (1: B23/nucleophosmin; 2: pre-mRNA splicing factor SF2p32 – Tat-associated protein TAP; 3: ribosomal protein S4). B. Amino acid sequence of the human NAP-1 protein (locus NP_631946) – 391 aa. The underlined amino acid sequences correspond to peptides obtained from MS/MS analysis of three independent preparations (P = 7.8 × 10^-19).

Mapping of hNAP-1 and Tat interacting domains. A. Schematic representation of hNAP-1 protein and of its deletion mutants obtained as GST fusion proteins. The capacity of binding to Tat – see experiment in panel B – is indicated on the right side of each mutant. The two dotted boxes indicate the hNAP-1 domains interacting with Tat. B. Representative GST pulldown experiment using the indicated hNAP-1 mutants and radiolabelled Tat101 protein. The autoradiography shows the amount of Tat binding to each mutant; the histogram on top shows densitometric quantification of data, expressed as fold binding with respect to background binding to GST alone (set as 1). The lower panel shows the Coomassie stained gel at the end of the binding experiment. The experiment was repeated at least three times with similar results. C. Schematic representation of HIV-1 Tat protein and of its mutants obtained as GST fusion proteins. The capacity of binding to hNAP-1 – see experiment in panel D – is indicated on the right side of each mutant. The dotted box corresponds to the basic domain of Tat, which binds hNAP-1. D. Representative GST pulldown experiment using the indicated Tat mutants (obtained as GST fusion proteins) and in vitro transcribed and translated hNAP-1 protein. The autoradiography shows the amount of hNAP-1 binding to each mutant; the histogram on top shows densitometric quantification of data, expressed as fold binding with respect to background binding to GST alone (set as 1). The lower panel shows the Coomassie stained gel at the end of the binding experiment. The experiment was repeated at least three times with similar results.

Effect of hNAP-1 on HIV-1 infection. A. Overexpression of hNAP-1 enhances LTR transcription upon HIV-1 infection. HeLa cells were transfected with an expression vector for HA-hNAP-1 or with a control vector, and then infected with VSG-luciferase HIV-1 vector. Luciferase activity was measured after 24 h post-infection. The mean ± s.d. of at least three different experiments is shown. The panel on the right side shows anti-HA western blottings to assess HA-hNAP-1 expression in a representative experiment. B. Silencing of hNAP-1 impairs LTR transcription upon HIV-1 infection. HeLa cells were treated with an siRNA directed against hNAP-1 or a control siRNA. Forty-eight hours after the beginning of siRNA treatment, cells were infected with the luciferase reported virus, and luciferase assays were performed on cell lysates 24 hours later. The mean ± s.d. of at least three different experiments is shown. The panel on the right side shows anti-hNAP-1 western blottings to assess the levels of endogenous hNAP-1 and tubulin expression in a representative experiment.

Co-immunoprecipitation of Tat with transfected and endogenous hNAP-1. A. Schematic representation of hNAP-1 structure. The acidic domains of the protein are shown by black boxes, with the indication of their boundary amino acids. The localization of nuclear export and nuclear localization signals (NES and NLS respectively) are indicated. B. Schematic representation of the regions of amino acid homology between hNAP-1 and hSET/TAF-I. C. Co-immunoprecipitation of transfected hNAP-1 with Tat. The plasmids indicated on top of the figure were transfected into HEK 293T cells. The upper two panels show western blots with the indicated antibodies after immunoprecipitation using an anti-Flag antibody; the lower two panels show western blotting controls from whole cell lysates (WCL) from transfected cells to show the levels of expression of the transfected proteins. D. Co-immunoprecipitation of endogenous hNAP-1 with Tat. The experiment was performed by transfecting HEK 293T cells with plasmids encoding GFP-Tat or GFP alone, followed by co-immunoprecipitation with anti-GFP antibody. GFP-Tat retains full transcriptional and trafficking capacities as wt Tat [69, 74, 75]. E. GST-pulldown experiment using GST-Tat and HEK 293T whole cell lysates. GST-Tat, but not control GST protein, pulled down endogenous hNAP-1.

hNAP-1 cooperates with Tat in LTR transactivation. A. hNAP-1 synergizes with Tat in transcriptional activation. HeLa cells were cotransfected with a reporter construct containing the HIV-1 LTR upstream of the luciferase gene, and with vectors for HA-tagged hNAP-1 (100 ng) and HIV-1 Tat (5 and 25 ng), as indicated. The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. The co-expression of hNAP-1 significantly increased Tat transactivation of the LTR promoter. The western blot at the bottom shows the levels of transfected hNAP-1 protein in a representative experiment. B. hNAP-1 knock down decreases Tat transactivation. HeLa cells were transfected with a specific siRNA against hNAP-1 or a control siRNA, and then transfected with the LTR-luciferase reporter together with Tat (5 and 25 ng). The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. The western blot at the bottom shows the levels of endogenous hNAP-1 protein and of tubulin as a control in a representative experiment. C. hNAP-1, Tat and the acetyltransferase p300 synergistically activate viral transcription. HeLa cells were transfected with LTR-luciferase reporter plasmid and with vectors for HIV-1 Tat (5 ng), HA-hNAP-1 (100 ng) and p300 (100 ng), as indicated. After 24 h from transfection, luciferase assays were performed. The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. D. p300 enhances Tat-hNAP-1 interaction in vivo. The plasmids indicated on top of the figure were transfected into HEK 293T cells. The upper panel shows western blots with the indicated antibodies after immunoprecipitation using an anti-Flag antibody; the lower three panels show western blotting controls from whole cell lysates (WCL) from transfected cells to show the levels of expression of the transfected proteins.