Nucleoporin NUP153 Phenylalanine-Glycine Motifs Engage a Common Binding Pocket within the HIV-1 Capsid Protein to Mediate Lentiviral Infectivity

Lentiviruses can infect non-dividing cells, and various cellular transport proteins provide crucial functions for lentiviral nuclear entry and integration. We previously showed that the viral capsid (CA) protein mediated the dependency on cellular nucleoporin (NUP) 153 during HIV-1 infection, and now demonstrate a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG)-repeat enriched NUP153 C-terminal domain (NUP153C). NUP153C fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153C) potently restricted HIV-1, providing an intracellular readout for the NUP153C-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV) bound NUP153C under these conditions, results that correlated with direct binding between purified proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 protein during virus infection. Mutagenesis experiments concordantly identified NUP153C and CA residues important for binding and lentiviral infectivity. Different FG motifs within NUP153C mediated binding to HIV-1 versus EIAV capsids. HIV-1 CA binding mapped to residues that line the common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74) inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6) protein, with Asn57 (Asp58 in EIAV) playing a particularly important role. PF74 and CPSF6 accordingly each competed with NUP153C for binding to the HIV-1 CA pocket, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153C expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicates that the NUP153C-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. Our results highlight similar mechanisms of binding for disparate host factors to the same region of HIV-1 CA during viral ingress. We conclude that a subset of lentiviral CA proteins directly engage FG-motifs present on NUP153 to affect viral nuclear import

Impaired IL-23-dependent induction of IFN-gamma underlies mycobacterial disease in patients with inherited TYK2 deficiency

Human cells homozygous for rare loss-of-expression (LOE) TYK2 alleles have impaired, but not abolished, cellular responses to IFN-alpha/beta (underlying viral diseases in the patients) and to IL-12 and IL-23 (underlying mycobacterial diseases). Cells homozygous for the common P1104A TYK2 allele have selectively impaired responses to IL-23 (underlying isolated mycobacterial disease). We report three new forms of TYK2 deficiency in six patients from five families homozygous for rare TYK2 alleles (R864C, G996R, G634E, or G1010D) or compound heterozygous for P1104A and a rare allele (A928V). All these missense alleles encode detectable proteins. The R864C and G1010D alleles are hypomorphic and loss-of-function (LOF), respectively, across signaling pathways. By contrast, hypomorphic G996R, G634E, and A928V mutations selectively impair responses to IL-23, like P1104A. Impairment of the IL-23-dependent induction of IFN-gamma is the only mechanism of mycobacterial disease common to patients with complete TYK2 deficiency with or without TYK2 expression, partial TYK2 deficiency across signaling pathways, or rare or common partial TYK2 deficiency specific for IL-23 signaling.ANRS Nord-Sud ; CIBSS ; CODI ; Comité para el Desarrollo de la Investigación ; Fulbright Future Scholarshi

Search for stop and higgsino production using diphoton Higgs boson decays

Results are presented of a search for a "natural" supersymmetry scenario with gauge mediated symmetry breaking. It is assumed that only the supersymmetric partners of the top-quark (stop) and the Higgs boson (higgsino) are accessible. Events are examined in which there are two photons forming a Higgs boson candidate, and at least two b-quark jets. In 19.7 inverse femtobarns of proton-proton collision data at sqrt(s) = 8 TeV, recorded in the CMS experiment, no evidence of a signal is found and lower limits at the 95% confidence level are set, excluding the stop mass below 360 to 410 GeV, depending on the higgsino mass

Severe early onset preeclampsia: short and long term clinical, psychosocial and biochemical aspects

Preeclampsia is a pregnancy specific disorder commonly defined as de novo hypertension and proteinuria after 20 weeks gestational age. It occurs in approximately 3-5% of pregnancies and it is still a major cause of both foetal and maternal morbidity and mortality worldwide1. As extensive research has not yet elucidated the aetiology of preeclampsia, there are no rational preventive or therapeutic interventions available. The only rational treatment is delivery, which benefits the mother but is not in the interest of the foetus, if remote from term. Early onset preeclampsia (<32 weeks’ gestational age) occurs in less than 1% of pregnancies. It is, however often associated with maternal morbidity as the risk of progression to severe maternal disease is inversely related with gestational age at onset2. Resulting prematurity is therefore the main cause of neonatal mortality and morbidity in patients with severe preeclampsia3. Although the discussion is ongoing, perinatal survival is suggested to be increased in patients with preterm preeclampsia by expectant, non-interventional management. This temporising treatment option to lengthen pregnancy includes the use of antihypertensive medication to control hypertension, magnesium sulphate to prevent eclampsia and corticosteroids to enhance foetal lung maturity4. With optimal maternal haemodynamic status and reassuring foetal condition this results on average in an extension of 2 weeks. Prolongation of these pregnancies is a great challenge for clinicians to balance between potential maternal risks on one the eve hand and possible foetal benefits on the other. Clinical controversies regarding prolongation of preterm preeclamptic pregnancies still exist – also taking into account that preeclampsia is the leading cause of maternal mortality in the Netherlands5 - a debate which is even more pronounced in very preterm pregnancies with questionable foetal viability6-9. Do maternal risks of prolongation of these very early pregnancies outweigh the chances of neonatal survival? Counselling of women with very early onset preeclampsia not only comprises of knowledge of the outcome of those particular pregnancies, but also knowledge of outcomes of future pregnancies of these women is of major clinical importance. This thesis opens with a review of the literature on identifiable risk factors of preeclampsia

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

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

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

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