AUC-SV data for NPM1^FL, NPM1^OD, and US11^FL, respectively.

<p>MW, molecular weight; S20,w (S), sedimentation rate at 20°C; f/f0, frictional coefficient. In all three cases the values refer to a single, dominant species, which represented more than 90% of the sample.</p><p>AUC-SV data for NPM1^FL, NPM1^OD, and US11^FL, respectively.</p

Direct NPM1 interaction with HIV-1 Rev.

<p>(A) Qualitative interaction analysis by GST pull-down assay and subsequent CBB staining. NPM1 FL, OD and HRBD, but not RBD, displayed a selective interaction with HIV-1 Rev (upper panel), which was also observed after an RNase A treatment (lower panel). (B) Quantitative interaction analysis by ITC. The binding parameters for the interaction between NPM1^FL and Rev were obtained using ITC. Titration of NPM1^FL (750 μM) to Rev^FL (35 μM) showed an exothermic response (negative peaks) indicating that Rev selectively interacts with NPM1^FL. The upper graph shows calorimetric changes plotted versus the time and the lower graph represents the changes in temperature according to the molar ratio of the interacting proteins. (C) No interaction was observed in a control experiments by titrating NPM1^RBD (300 μM) to Rev^FL (30 μM).</p

NPM1 association with HSV-1 US11 in the cell.

<p>(A) Nucleolar colocalization of endogenous NPM1 with myc-US11. Confocal images of HeLa cells transfected with myc-US11 were obtained by staining endogenous NPM1 (Mouse anti-NPM1 (ab10530)), myc-US11 (anti-myc antibody), and filamentous actin (rhodamine-phalloidin). For clarity, a boxed area in the merged panel shows colocalization of NPM1 and US11 in the nucleolus as pointed by arrows. Scale bar: 20 μm. (B) Myc-US11 associates with endogenous NPM1 in COS-7 cells. NPM1 was co-immunoprecipitated with myc-US11 overexpressed in COS-7 cells using anti-myc antibody. A normal Rabbit IgG and sample without antibody were used as IP controls. Input, 5% of total cell lysate; IP, immunoprecipitation; IB, immunoblotting. (C) Myc-US11^FL displayed an interaction with NPM1^FL. Myc-US11^FL was pulled down with the GST-fusion NPM1^FL, but not with GST, which was used as a negative control. Samples prior pull-down (PD) analysis were used as input control.</p

CIGB300 treatment interferes with HIV-1 production.

<p>CIGB-300 treated or untreated HOS.CD4.CXCR4 cells were infected with NL4.3 virus at an MOI of 1. Culture supernatant was collected 48 and 72 h post infection and virus titer was determined. The figure shows one representative experiment out of four, in which virus quantification was performed by TZM-bl cell titration. Values are the means ± S.D. of three measurements. Statistical significance (P) was calculated by the Student`s t-test: ***P<0.002; **P<0.02.</p

The synthetic peptide CIGB-300 competes with Rev and US11 by binding NPM1^OD with high-affinity.

<p>(A) CIGB-300 consists of the cyclic P15 (blue) and the Tat (purple) peptides, and labeled with fluorescein (green; FITC). (B) Fluorescence polarization experiments conducted by titrating increasing amounts of NMP1 variants, Rev, US11, and GST to 0.1 μM FITC-labelled CIGB-300 (f CIGB-300). A high affinity interaction with the peptide was only observed for NPM1^FL and NPM1^OD, resulting from an increase of polarization, but not for Rev, US11, GST, and the other NPM1 variants. (C-D) Contrary to US11, Rev only displaced NPM1^OD from its fCIGB-300 complex. Displacement experiments were performed by adding increasing amounts of Rev or US11 to the NPM1^FL-fCIGB-300 complex (C) or to the NPM1^OD-fCIGB-300 complex (D). (E) A proposed NPM1^OD-CIGB-300 docking model of pentameric NPM1^OD structure in the complex with CIGB-300. Cyclic part (blue) and basic part (purple) of the peptide shown as sticks and ribbons wraps around several monomeric units of NPM1 represented by surfaces in different colors shown in top view (left), rotated orientation (middle), and the bottom view (right).</p

Physical interaction of HSV-1 US11 with NPM1.

<p>(A) C-terminal region of US11 largely contributes to NPM1 interaction. Pull-down experiments were conducted with purified proteins in the presence of RNase A by using GST-fused US11^FL, US11^Nterm, US11^Cterm, and GST as a negative control. For the detection of NPM1 variants two different antibodies were used, ab52644 recognized an N-terminal epitope containing in NPM1^FL and NPM1^OD, and ab10530 recognized a C-terminal epitope containing in NPM1^HRBD and NPM1^RBD. The same pattern of interaction was obtained for the N-terminal and the C-terminal parts of US11, although the interaction between NPM1^FL and NPM1^OD with US11^Nterm was much weaker than with US11^Cterm. The exposure time was 1 min for all the blots. (B-C) US11 binds NPM1 with a binding constant in the low micromolar range. To measure the binding parameter for the NPM1-US11 interaction, 1.2 mM NPM1^FL (B) and buffer (C) were titrated to 60 μM US11^FL. Both NPM1 and US11 were treated with RNase A. Conditions were the same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143634#pone.0143634.g002" target="_blank">Fig 2</a>. US11 binding to NPM1 is an endothermic reaction. (D) US11 binds to a pentameric NPM1. aSEC-MALS/RI analysis of NPM1^OD, US11^FL, and a mixture of both proteins revealed an oligomeric nature of NPM1^OD with a molecular weight (MW) of 66.1 kDa corresponding to the pentameric form. Obtained MW for US11 was 16.6 kDa, which matches the theoretical MW of 16.7 kDa for a monomeric US11 (upper panel). SDS-PAGE and CBB staining of the aSEC (Superdex 200, 10/300) elution fractions of NPM1^OD, US11^Fl, and a mixture of both clearly revealed a NPM1-US11 complex formation (lower panel). Both NPM1 and US11 were treated with RNase A. The MW of this complex corresponds to 76.6 kDa for a pentameric NPM1^OD, and a monomeric US11^FL. A MW of 21.8 kDa was measured that is estimated to an unbound US11^FL.</p

Schematic representation of domain organization, various constructs and proteins of NPM1, HSV-1 US11, and HIV-1 Rev.

<p>(A) Domains and various constructs of NPM1, US11 and Rev. The numbers indicate the N- and C-terminal amino acids of the respective constructs used in this study. A1-A3, acidic regions 1–3; Cterm, C-terminal; ED, effector domain; FL, full-length; HRBD, histone and RNA-binding domains; HBD, histone binding domain; NES, nuclear export signal; NLS, nuclear localization signal; NoLS, nucleolar localization signal; Nterm, N-terminal; OD, oligomerization domain; RBD, RNA-binding domain. (B) Coomassie brilliant blue (CBB) stained SDS-PAGE of purified proteins used in this study.</p

ITC data for HIV-1 Rev^FL interaction with NPM1 variants.

<p>K_a^, association constant; K_d^, dissociation constant; ΔH, enthalpy; n, binding stoichiometry (number of binding sites). HIV-1 Rev^FL did not show any binding to the RNA-binding domain (RBD) of NPM1. All measurements were performed at 25°C.</p><p>^a K_d values were calculated from K_d = 1/K_a.</p><p>ITC data for HIV-1 Rev^FL interaction with NPM1 variants.</p