Restriction of HIV-1 infection by Trim5-NUP153_C fusion proteins.

<p>(<b>A</b>) Schematic of Trim-NUP153_C fusion and control constructs. Color code: Trim5 RBCC, brown; rhTrim5α SPRY, auburn; HA-tag, blue; NUP153_C, orange. (<b>B</b>) Western blot of HOS cells stably transduced with HA-tagged Trim-NUP153_C fusion or control constructs, detected with antibody 3F10. (<b>C</b>) Western-blot detection of Trim-NUP153_C fusion proteins with anti-HA monoclonal antibodies 3F10 and 16b12. Antibody 3F10 detects full-length Trim-NUP153_C-HA whereas antibody 16b12 more faithfully detects full-length Trim-HA-NUP153_C. (<b>D</b>) Infectivity of various doses of HIV-1 (left) or MLV (right) GFP reporter viruses on HOS cells stably expressing various Trim-based constructs. The results are an average of two experiments, with error bars denoting standard error. Asterisks in panels B and C mark bands that correspond to the expected mobilities of full length Trim-NUP153_C constructs.</p

The NUP153-CA interaction during HIV-1 infection.

<p>Partially uncoated HIV-1 cores dock at the NPC through engaging NUP358 (light green). Once docked, NUP153 (dark green) FG motifs bind CA through phenylalanine insertion into the hydrophobic pocket of the NTD, forming hydrogen bonds with CA residue Asn57, as well as adjacent polar side-chains (enlarged to the right). CA engagement with NUP153 is required for HIV-1 nuclear import, either directly during PIC translocation, or for completion of a prerequisite uncoating step. Perturbation of NUP153 engagement may affect multiple steps, such as intranuclear trafficking and integration site selection. Cytoplasmic CPSF6 may inhibit HIV-1 nuclear import by antagonizing NUP153 binding to CA.</p

FG motifs determine NUP153_C binding to HIV-1 CA_N.

<p>(<b>A</b>) Pull-down of full-length or quarter deleted HA-NUP153_C by HIV-1, EIAV, or MLV CA_N proteins, detected with antibody 3F10. (<b>B</b>) Pull-down of WT NUP153_C, FG-motif tetra-alanine mutant 1415A, or combinatorial 7×FG/A mutant by beads alone (none, grey), HIV-1 (red), MLV (blue), or EIAV (green) CA_N proteins, as detected by western blot with antibody 3F10. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003693#s2" target="_blank">Results</a> are an average of at least 4 experiments, with error bars denoting standard error. (<b>C</b>) Purified NUP153_C (black circles, solid line), NUP153_CΔ1350–1475 (purple triangles, fine dotted line), and NUP153_C7×FG/A (brown diamonds, coarse dotted line) proteins were incubated with various concentrations of HIV-1 CA_N and a constant amount of Ni-NTA beads. Data points represent the mean and standard error of at least three experiments, fit with non-linear regression curves. The dissociation constant of NUP153_C binding was calculated by averaging concentrations of half-maximal binding for 5 individual experiments, with associated standard error. (<b>D</b>) Sedimentation of WT NUP153_C, FG-motif tetra-alanine mutant 1415A, combinatorial 7×FG/A mutant, or NUP153_CΔ1350–1475 after incubation with buffer alone or assembled CA-NC. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003693#s2" target="_blank">Results</a> are an average of 6 experiments, with error bars denoting 95% confidence intervals. Representative western blotting results are shown.</p

Mode of NUP153_C binding to EIAV CA.

<p>(<b>A</b>) Alignment of residues corresponding to HIV-1 CA Leu56 through Asn74 among various retroviruses. Residues that significantly affected HIV-1 CA_N binding with NUP153_C are highlighted in yellow. (<b>B</b>) Alignment of HIV-1 CA_N (green, pdb: 3mge) and EIAV CA_N (gray, pdb: 1eia), with side-chains surrounding the pocket shown as sticks. (<b>C</b>) Retroviral sensitivities to inhibition by PF74. Color codes: HIV-1, green; SIVmac, black; MLV, red; EIAV, orange; BIV, blue; FIV, purple. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003693#s2" target="_blank">Results</a> are an average of two experiments. (<b>D</b>) Retroviral sensitivities to inhibition by Trim-CPSF6₃₅₈. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003693#s2" target="_blank">Results</a> are an average of 3 experiments. (<b>E</b>) Infectivity of RT-cpm matched EIAV GFP-reporter viruses carrying CA point mutations. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003693#s2" target="_blank">Results</a> are an average of 3 experiments. (<b>F</b>) Pull-down of purified NUP153_C by EIAV point mutant CA_N proteins. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003693#s2" target="_blank">Results</a> are an average of 2 experiments. (<b>G</b>) Sensitivity of EIAV CA point-mutant viruses to Trim-NUP153_C. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003693#s2" target="_blank">Results</a> are an average of 4 experiments. Error bars in each panel denote standard error.</p

The importance of FG motifs for Trim-NUP153_C mediated inhibition of HIV-1 and EIAV infection.

<p>To-scale schematics (<b>A and D</b>), normalized infection data (<b>B and E</b>), and western blotting (<b>C and F</b>) as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003693#ppat-1003693-g004" target="_blank">Figure 4</a>. Infection data are the geometric mean of at least 4 experiments, with error bars denoting 95% confidence intervals. Inverted grey triangle (panels A and D) denotes area of missense mutation.</p

NUP153_C directly binds the HIV-1 CA N-terminal domain.

<p>(<b>A</b>) Schematic of NUP153 protein, with residue numbers of domain boundaries indicated. (<b>B</b>) Full length or truncated fragments of HA-tagged NUP153 extracted from 293T cells were tested for binding to HIV-1 CA-NC. Pelleted proteins were resolved by SDS-PAGE and visualized by western blotting with anti-HA antibody 3F10 (top), or by Coomassie stain (bottom). Input, 20% of binding reaction. CA-NC was included in the binding reactions as indicated. (<b>C</b>) Recombinant, tag-free NUP153_C and GST purified from <i>E. coli</i> were similarly tested for binding to CA-NC; proteins were detected with Coomassie stain. (<b>D</b>) Recombinant NUP153_C pulled down with full length his-tagged wild-type (WT) or W184A/M185A HIV-1 CA, and detected with Coomassie stain. (<b>E</b>) Recombinant NUP153_C pulled down with his-tagged CA_N, and detected with Coomassie stain. Each experiment was repeated at least 3 times, with a single representative result shown.</p

HIV-1 CA mutant-NUP153_C binding and sensitivity to Trim-NUP153_C restriction or NUP153 depletion.

<p>(<b>A</b>) (top) Equal reverse transcriptase (RT) cpm of WT and HIV-1 mutant viruses plated on HOS cells, with resulting infectivities normalized to WT virus. (bottom) Percent infectivity of viruses in Trim-NUP153_C expressing HOS cells, normalized to mock transduced control cells. Graphs show the mean of at least 5 experiments, with error bars denoting 95% confidence intervals. (<b>B</b>) Purified NUP153_C pull-down by WT or the indicated mutant his-tagged HIV-1 CA_N protein, with recovered proteins resolved by SDS-PAGE and detected by SYPRO Ruby stain. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003693#s2" target="_blank">Results</a> are an average of 5 experiments, with error bars denoting 95% confidence intervals. A representative staining result is shown. The dotted line highlights the level of NUP153_C binding to WT CA_N protein. (<b>C</b>) Scatter plot of NUP153_C recovery in pull-down assays (panel B) compared to percent infectivity in Trim-NUP153_C expressing cells (panel A, lower). Points are color-coded based on NUP153_C binding phenotype: grey, not significantly different from WT; white, significantly decreased from WT; black, significantly increased from WT. (<b>D</b>) Scatter plot of normalized infectivity of CA mutant viruses in Trim-NUP153_C expressing cells compared to the average infectivity of three experiments when endogenous NUP153 was knocked down. The comparison exhibited a significant Spearman rank correlation (<i>P</i><0.0001). Points are color-coded as in panel C, except for CA mutants not tested for binding, which are denoted with “x” symbols.</p

PF74 counteracts HIV-1 similarly in the face of Trim-NUP153_C restriction or NUP153 knockdown.

<p>Mock transduced and Trim-NUP153_C expressing (<b>A</b>) or non-targeting control and NUP153 knockdown (<b>B</b>) HOS cells were infected with equal RT-cpm of denoted viruses in the presence of various PF74 concentrations. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003693#s2" target="_blank">Results</a> are shown as infectivity normalized to vehicle only control cells (top), or vehicle only infection for each cell type (bottom) to calculate EC₉₀ values. Dashed lines represent Trim-NUP153_C or NUP153 knockdown results in panels A and B, respectively. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003693#s2" target="_blank">Results</a> are an average of at least 3 experiments, with error bars denoting standard error. Calculated EC₉₀ values are displayed with standard error.</p

Association between NUP153 dependency and cell cycle independence.

<p>(<b>A</b>) Propidium iodide staining of HOS cells untreated (grey) or treated for 24 h with 5 µM Etoposide phosphate (red line). (<b>B</b>) Infectivity of CA mutant viruses in HOS cells arrested with 5 µM Etoposide phosphate, normalized to infectivity in control HOS cells. Error bars denote standard error of 4 experiments. (<b>C</b>) Scatter plot comparison of CA mutant sensitivities in cell cycle arrested HOS cells in the absence or presence of 5 µM cyclosporine (CsA). Mutant viruses most sensitive to cell cycle arrest are indicated. (<b>D to G</b>) Scatter plots comparing sensitivities of mutant viruses to cell cycle arrest versus NUP153 knockdown (D), or restriction by Trim-NUP153_C (E), CPSF6₃₅₈ (F), or Trim-CPSF6₃₅₈ (G). Spearman rank correlation coefficients and measures of significance are indicated. Data points for CA mutant viruses P38A, T54A, A92E, and G94D clustered with the WT virus within these panels, so their labels were omitted to aid legibility.</p

Diverse lentiviruses bind NUP153_C.

<p>(<b>A</b>) Transduction efficiencies of retroviral GFP reporter viruses in Trim-NUP153_C expressing cells normalized to infection in mock transduced cells, which were set to 100%. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003693#s2" target="_blank">Results</a> are the geometric mean of 4 experiments, with error bars denoting 95% confidence intervals. (<b>B</b>) HA-NUP153_C expressed in 293T cells was pulled down by the indicated his-tagged retroviral CA_N proteins. Captured proteins resolved by SDS-PAGE were western blotted with antibody 3F10 alongside a standard curve of input protein. The results are an average of 5 experiments, with error bars denoting 95% confidence intervals. A representative western blot is shown. (<b>C</b>) SYPRO Ruby detection of retroviral CA_N pull-down of purified NUP153_C. The results are an average of two experiments, with error bars denoting standard error; one representative gel is shown. (<b>D</b>) (left) Retroviral infectivities in HOS cells knocked down for NUP153 expression as compared to cells treated with a non-targeting short interfering (si) RNA control <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003693#ppat.1003693-Matreyek1" target="_blank">[19]</a>. The results are the geometric mean of at least 4 experiments, with error bars denoting 95% confidence intervals. (right) Western blot detection of control or NUP153 knockdown HOS cells with antibody mab414, which also detects NUP358. (<b>E</b>) Scatter plot comparing relative retroviral infectivities under each condition.</p