, Bourdoncle (IMAG'IC facility of Institut Cochin) for their technical support, and Pr, Eric, vol.604
, Solary for the pRLL-EF1-PGK-GFP lentiviral vector plasmid. This work was supported by 605 funds from Agence Nationale de la Recherche
, Fondation Gustave, p.607
Institut National du Cancer (INCA 9414), NATIXIS, SIDACTION and the French 608 ,
, National Agency for Research on AIDS and viral Hepatitis (ANRSH) (to
HIV-1 induces 620 the formation of stable microtubules to enhance early infection, Cell Host Microbe, vol.621, pp.535-546, 2013. ,
Distinct functions of diaphanous-624 related formins regulate HIV-1 uncoating and transport, Proc Natl Acad Sci, vol.114, pp.6932-6941, 2017. ,
Role of Microtubules and Microtubule-Associated Proteins in 628 HIV-1 Infection, J Virol, vol.92, pp.85-103, 2018. ,
Sgt1 acts via an LKB1/AMPK 631 pathway to establish cortical polarity in larval neuroblasts, Dev Biol, vol.363, pp.258-265, 2012. ,
Hsp90-Sgt1 and Skp1 target human Mis12 complexes to ensure 634 efficient formation of kinetochore-microtubule binding sites, J Cell Biol, vol.189, pp.261-274, 2010. ,
SGT1 encodes an essential 637 component of the yeast kinetochore assembly pathway and a novel subunit of the SCF 638 ubiquitin ligase complex, Mol Cell, vol.4, pp.21-33, 1999. ,
Urinary bladder epithelium antigen induces CD8+ T 641 cell tolerance, activation, and autoimmune response, J Immunol, vol.178, pp.539-546, 2007. ,
Sgt1 is 644 required for human kinetochore assembly, EMBO Rep, vol.5, pp.626-631, 2004. ,
p21-651 mediated RNR2 repression restricts HIV-1 replication in macrophages by inhibiting dNTP 652 biosynthesis pathway, Proc Natl Acad Sci U S A, vol.8, pp.3997-4006, 2007. ,
Single-cell 655 imaging of HIV-1 provirus (SCIP), Proc Natl Acad Sci U S A, vol.110, pp.5636-5641, 2013. ,
Second 658 generation imaging of nuclear/cytoplasmic HIV-1 complexes, AIDS Res Hum Retroviruses, vol.659, pp.717-726, 2014. ,
The G140S 662 mutation in HIV integrases from raltegravir-resistant patients rescues catalytic defect due to 663 the resistance Q148H mutation, Nucleic Acids Res, vol.37, pp.1193-1201, 2009. ,
The 666 engagement of activating FcgammaRs inhibits primate lentivirus replication in human 667 macrophages, J Immunol, vol.177, pp.6291-6300, 2006. ,
,
, Visualization of the intracellular behavior of HIV in living cells, J Cell Biol, vol.159, pp.441-671, 2002.
Azido-677 containing diketo acid derivatives inhibit human immunodeficiency virus type 1 integrase in 678 vivo and influence the frequency of deletions at two-long-terminal-repeat-circle junctions, Am J Physiol Cell Physiol, vol.300, pp.3210-3222, 2004. ,
Intracellular transport of human immunodeficiency 682 virus type 1 integrase, J Cell Sci, vol.116, pp.4401-4408, 2003. ,
Control of microtubule organization and dynamics: two 685 ends in the limelight, Nat Rev Mol Cell Biol, vol.16, pp.711-726, 2015. ,
CLIP-170 tracks 688 growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites, 689 J Cell Biol, vol.183, pp.1223-1233, 2008. ,
Analysis of 692 microtubule dynamic instability using a plus-end growth marker, Nat Methods, vol.7, p.761 ,
Estimating the microtubule GTP 696 cap size in vivo, Curr Biol, vol.22, pp.1681-1687, 2012. ,
HIV-1 pre-integration complexes 699 selectively target decondensed chromatin in the nuclear periphery, PLoS One, vol.3, 2008. ,
Microtubule-702 associated proteins 1 (MAP1) promote human immunodeficiency virus type I (HIV-1) 703 intracytoplasmic routing to the nucleus, J Biol Chem, vol.290, pp.4631-4646, 2015. ,
Single HIV-1 Imaging Reveals Progression of Infection 706 through CA-Dependent Steps of Docking at the Nuclear Pore, Uncoating, and Nuclear 707 Transport, Cell Host Microbe, vol.23, pp.536-548, 2018. ,
HIV-1 integrase 710 trafficking in S. cerevisiae: a useful model to dissect the microtubule network involvement of 711 viral protein nuclear import, Yeast, vol.26, pp.39-54, 2009. ,
HIV-1 714 integrase interacts with yeast microtubule-associated proteins, Biochim Biophys Acta, vol.715, pp.40-48, 2002. ,
, Immunofluorescence of brain autopsies from uninfected persons (n=3) (a) and HIV-1 721 infected patients (n=3) (b) for SUGT1, CAp24 and nucleus
, Quantification of SUGT1 expression in CAp24 + (n=45) or CAp24 -(n=690) cells detected 723 in brain sections. Fluorescence intensities (FI) are shown. Means ± SEM are indicated. P 724 values were calculated using two-tailed unpaired t-test using Bonferroni correction
, monocytes/macrophages (f) WB of endogenous SUGT1 levels are shown (n=3). SUGT1 a 727 and b isoforms are indicated
, PBLs (h) and CD4 + CXCR4 + HeLa cells (i) are 729 shown (n=3), SUGT1 depletion in human MDMs (g)
, Effect of SUGT1 depletion on viral production obtained from MDMs (j, k), PBLs (l, m), p.731
, or CD4 + CXCR4 + HeLa cells (n=3) (n) infected with HIV-1 AD8 (j, k) or HIV-1 NL4-3 (l-n)
, CAp24 release for representative donor (j, l) and fold changes (n=7 for MDMs, p.733
, PBLs) (k, m) are shown
, Effect of SUGT1 depletion on viral production obtained at indicated times post-735 infection from MDMs (o) and PBLs (p) infected with HIV-1 AD8 (o) or HIV-1 NL4-3 (p)
, Figure 2. SUGT1 promotes early HIV-1 replication steps
, HIV-1 infectivity of control or SUGT1-depleted MDMs (a, b) and activated PBLs, p.740
, Luciferase activity from 741 representative donor (a, c) and fold changes (n=8 for MDMs and n=4 for PBLs) (b, d) are 742 shown, that were infected with HIV-1 ?EnvNL4-3-Luc
, PBLs (f) were transduced with lentiviral vectors expressing control 744 (shCo.), a pool of two shRNAs against SUGT1 (shSUGT1) and/ or SUGT1 resistant cDNA 745 (pSUGT1) for 72 hours prior infection with HIV-1 ?EnvNL4-3-Luc (VSV-G), MDMs (e) and
, i-q) Fold changes of HIV-1 early reverse transcripts (i, j), late reverse transcripts (k, l), pp.2-748
, LTRs circles (m, n, q) and integrated proviruses (o, p) were determined by qPCR in control 749 or SUGT1-depleted macrophages (i, k, m, o, q) or lymphocytes (j, l, n and p) that were 750 infected with HIV-1 ?EnvNL4-3-Luc (VSV-G) (i-p) or with HIV-1 ?EnvNL4-3-IND64E, p.751
, r, s) Representative confocal micrographs of HIV-1CMV-GFP-I-SCEI -infected HEK293T cells (r), p.753
, and percentages of HIV-1 infected (GFP + ) cells with ?H2AX + foci (s) are shown
, SEM are indicated (n=3). P values were calculated using two-tailed unpaired t test (**p 755, vol.01, p.0
, Figure 3. SUGT1 is associated with microtubules trafficking HIV-1
, VSV-G) infected U2OS 759 cells showing SUGT1 and ?-tubulin expression (aI). (aII) is a magnification of the dashed 760 region in (aI). (aII1-4) are the magnifications of the dashed regions in (aII). Fluorescence 761 overlap spectrums of, Representative SIM micrograph of 4 hour HIV-1 ?EnvNL4-3-GFP-Vpr
, HIV-1 IN and SUGT1 expression levels by WB in control and SUGT1-depleted U2OS 763 cells after 48 hours siRNA transfection and expression of exogenous HIV-1 IN for 24 hours. 764 (c, d) Representative confocal micrographs of HIV-1 HA-IN expression in control, p.765
, SUGT1-depleted U2OS cells (c) and percentages of cells showing nuclear or diffused HIV-1
, IN (d)
, Immunoprecipitation of HA-IN in control and HA-IN-overexpressing HEK293T cells and 768 expression of indicated proteins by WB
, WB and images are representative of three independent experiments. Means ± SEM are 770 indicated from at least three independent experiments. P values were calculated using two-771 way ANOVA test
, Representative confocal micrographs and magnifications showing ?-tubulin and nucleus 775 in control and SUGT1-depleted U2OS cells
, Fluorescence intensity of ?-tubulin
, Percentages of cells with +MTs parallel or perpendicular to the cell cortex. 778 (d-f) Representative confocal micrographs of ?-tubulin, AcK40 ?-tubulin and nucleus in 779 control and SUGT1-depleted U2OS cells (d)
, g-i) Representative confocal micrographs of ?-tubulin, EB1 and nucleus in control and 782
, Separate fluorescence images of (g) are shown in 783
, Quantification of EB1 comet length (n=559) 784 of cells (n=60) (i)
are indicated from at least three independent experiments. P values were 786 calculated using two-way ANOVA test for (c) and two-tailed unpaired t test for (f) and (i), p.787 ,
, , vol.0001
, HIV-1 132W (f) and HIV-1 DH12 815 (g). Representative WB revealing CAp24, HIV-1 CAp24 detected by WB in the cell supernatants (SN) of MDMs that were 814 depleted (or not) for SUGT1 and infected with HIV-1 BXO8 (e)
, Control and SUGT1-depleted primary human MDMs were infected with HIV-1 140/148 817 and evaluated for proviral integration and for CAp24 release at 72 h