hHR23A influences cleavage of polyUb chains by the Josephin domain.

<p><b>A)</b> Full length ataxin-3 (ataxin-3(Q22); 100 nM) was incubated with K63-Ub5 (250 nM) chains and either full length GST-tagged hHR23A (100–250 nM) or its GST-tagged UBL domain (100–250 nM). Fractions were collected at the indicated times. <b>B</b>) Isolated Josephin domain (ataxin-3(1-182); 100 nM) was incubated with K48-Ub5 chains (250 nM) and either GST-tagged hHR23A (100–250 nM) or its GST-tagged UBL domain (100–250 nM). Samples were collected at the indicated times. Block arrows highlight differences in cleavage activity among samples. <b>C</b>) Full length ataxin-3 (ataxin-3Q22(WT); 100 nM) or the isolated Josephin domain (ataxin-3(1-182); 100 nM) were incubated with mixed-linkage polyUb chains (K48-K63-K18-Ub4; 250 nM) and either full-length GST-hHR23A (100–250 nM) or its GST-tagged UBL domain (100–250 nM). Block arrows highlight differences in cleavage among samples.</p

Relevance of Ub-binding sites 1 and 2 to polyUb chain cleavage by the Josephin domain.

<p><b>A</b>) Josephin structure (1yzb). The side chains of the mutated residues and of the catalytic triad are indicated in yellow and cyan, respectively. Arrows denote the positions of the two Ub binding sites. <b>B–D</b>) GST-tagged, isolated Josephin domain samples of wild-type ataxin-3 (ataxin-3(1-182)-WT), mutated in Ub-binding site 1 (ataxin-3(1-182)-I77K-Q78K), or mutated in Ub-binding site 2 (ataxin-3(1-182)-W87K) were incubated with the following Ub chains: <b>B</b>) K63-linked hexa-Ub (K63-Ub6), <b>C</b>) K48-linked penta-Ub (K48-Ub5), or <b>D</b>) mixed-linkage tetra-Ub (K48-K63-K48-Ub4). Fractions were collected at the indicated times, electrophoresed on SDS-PAGE gels, and probed as indicated. HMW: high molecular weight Ub chain species that are thought to be dimers and trimers of the respective chains (14). Graphs below each blot represent semi-quantification of data from three independently run experiments. Shown are means +/− standard deviation. Asterisks: P<0.05 (*) or P<0.01 (**) when comparing reaction products from WT ataxin-3 to site-1 or site-2 mutated ataxin-3.</p

Relevance of Ub-binding sites 1 and 2 to polyUb chain cleavage by full-length ataxin-3.

<p>Full-length, untagged ataxin-3 protein samples that were wild type (ataxin-3(Q22)-WT), mutated in Ub-binding site 1 (ataxin-3(Q22)-I77A-Q78A), mutated in Ub-binding site 2 (ataxin-3(Q22)-W87A), or mutated in both sites (ataxin-3(Q22)-I77A-Q78A-W87A), were incubated with the following polyUb chains: <b>A</b>) K63-linked hexa-Ub (K63-Ub6), or <b>B</b>) K48-linked penta-Ub (K48-Ub5). Fractions were collected at the indicated times, electrophoresed on SDS-PAGE gels, and probed as indicated. Q22 denotes 22 glutamine residues in the polyQ region of ataxin-3. Lower panel for each blot represents semi-quantification of data from at least three independent experiments. Means +/− SD. Asterisks: P<0.05 (*) or P<0.01 (**) when comparing reaction products from WT ataxin-3 to ataxin-3 mutated at site 1, site 2 or both sites 1 and 2.</p

Modelling of diUb complexes with Josephin.

<p><b>A</b>) Structural superposition of representative models of the Josephin complexes with K48- (left) and K63-linked (right) diUb. The structures are superposed on Josephin backbone atoms to enhance the similarities/differences of the Ub relative positions. The C-terminus (residue G76) of the Ub in site 1 is indicated by spheres. The side chains of the catalytic triad are shown in cyan. The side chains of the cross-linking lysines are also shown explicitly. <b>B</b>) Comparison of the Josephin/K48-linked diUb model and the known structures of polyUb chains in isolation and in a complex. Top line: the structures of diUb from Ub₂ (1aar in cyan, 2bgf in red) and from Ub₄ chains (2o6v in green, 1tbe in magenta). Bottom line: the structure diUb from a different crystal form (1f9j in purple); the structure of the UBA domain in complex with Ub₂ (1zo6 in gold) and the structure of the Josephin complex (2jri, in blue). Residues L8, I44 and V71, which are often involved in Ub interfaces, are indicated by green spheres to provide a direct comparison of the relative orientations of the two subunits. With the sole exception of 1f9j, which was suggested to provide evidence of an open-to-closed equilibrium, the Ub subunits are in a closed conformation when not in a complex. In the two complexes, the conformation is open with the linkers differently stretched to adapt to the binding sites.</p

NMR titrations and models of Josephin/Ub complexes.

<p><b>A</b>) and <b>B</b>) CSPs (Δδ) as a function of the amino acid sequences for the titration of the ¹⁵N labelled Josephin with unlabelled K48 (upper panel) and K63 (lower panel) diUb chains ([Josephin]/[diUb] 1/3). Asterisks denote residues whose resonances are attenuated upon binding. <b>C</b>) Comparison of the correlation times measured during titration of ¹⁵N labeled Josephin with K48- and K63-linked diUb chains.</p

Cleavage of diUb chains by ataxin-3.

<p><b>A</b>) Isolated Josephin domain species were incubated with K48-linked diUb chains (K48-Ub2) for the indicated times. Left and right panels are representative of independent trials with little or no detectable DUB activity, respectively. <b>B</b>) Isolated Josephin domain species were incubated with K63-linked diUb chains (K63-Ub2) for the indicated periods of time. <b>C</b>) GST-tagged USP28 was incubated with K48-linked or K63-linked diUb chains for the indicated times. <b>D</b>) Equal amounts of penta-Ub K48 or K63-linked chains (K48-Ub5; K63-Ub5) were incubated with the isolated Josephin domain. Fractions were collected at the indicated times. Lower panel: Quantification of data from the left panel and other similar experiments (N = 3). Shown are means +/− SD. <b>E</b>) Equal amounts of K48-linked di-, tri-, tetra-, or pentaUb chains were incubated with the isolated Josephin domain, and fractions were collected at the indicated time points.</p

MOESM4 of HIV-1 capsid is involved in post-nuclear entry steps

Additional file 4: Fig. S4. Conditions for the CsA washout assay. (A) H126Q cells were infected with WT HIV-1GFP vector by spinoculation in the presence of CsA (1 μM) or C-A1 (3 μM). The drug was washed out at the indicated time points (time of washout) and cells were analysed by FACS 48 h later to determine the percentage of infected (GFP+) cells. (B) Same as (A) but T5Cyp cells expressing functional human TRIMCyp were used. (C) A prolonged time of uncoating assay, in which CsA was washed out at the indicated time points. Rescue of infection reaches a plateau after 10 h. Average ± SD of three independent experiments are shown in (A-C)

MOESM4 of HIV-1 capsid is involved in post-nuclear entry steps

Additional file 4: Fig. S4. Conditions for the CsA washout assay. (A) H126Q cells were infected with WT HIV-1GFP vector by spinoculation in the presence of CsA (1 μM) or C-A1 (3 μM). The drug was washed out at the indicated time points (time of washout) and cells were analysed by FACS 48 h later to determine the percentage of infected (GFP+) cells. (B) Same as (A) but T5Cyp cells expressing functional human TRIMCyp were used. (C) A prolonged time of uncoating assay, in which CsA was washed out at the indicated time points. Rescue of infection reaches a plateau after 10 h. Average ± SD of three independent experiments are shown in (A-C)

MOESM5 of HIV-1 capsid is involved in post-nuclear entry steps

Additional file 5: Fig. S5. CsA washout assays in cells depleted of Nup153 or control DsRed cells. T5Cyp cells expressing an shRNA against Nup153 or DsRed (control) were infected at an MOI of 0.01–0.05 with an HIV-1GFP vector (WT) in the presence of CsA (1 μM). The drug was washed out at the indicated time points (time of washout) and cells were analysed by FACS 48 h later to determine the percentage of infected (GFP+) cells. To control for specificity, cells were infected in the same way using the N74D mutant virus. Raw data of three independent experiments are shown. The data have been used to compile fold rescue levels shown in Figure 6

Analysis of genome content of PFV-Gag central domain mutants.

<p>Quantification of particle-associated PFV genomic RNA (vgRNA) and DNA (vgDNA) by qPCR. Mean values and standard deviation (n = 2) normalized for Gag content are shown as relative values compared to the <i>wt</i> control.</p