, 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

. Roussy, Institut National du Cancer (INCA 9414), NATIXIS, SIDACTION and the French 608

, National Agency for Research on AIDS and viral Hepatitis (ANRSH) (to

Y. Sabo, D. Walsh, D. S. Barry, S. Tinaztepe, K. De-los-santos et al., HIV-1 induces 620 the formation of stable microtubules to enhance early infection, Cell Host Microbe, vol.621, pp.535-546, 2013.

M. K. Delaney, V. Malikov, Q. Chai, G. Zhao, and M. H. Naghavi, Distinct functions of diaphanous-624 related formins regulate HIV-1 uncoating and transport, Proc Natl Acad Sci, vol.114, pp.6932-6941, 2017.

A. Dharan and E. M. Campbell, Role of Microtubules and Microtubule-Associated Proteins in 628 HIV-1 Infection, J Virol, vol.92, pp.85-103, 2018.

R. O. Andersen, D. W. Turnbull, E. A. Johnson, and C. Q. Doe, Sgt1 acts via an LKB1/AMPK 631 pathway to establish cortical polarity in larval neuroblasts, Dev Biol, vol.363, pp.258-265, 2012.

A. E. Davies and K. B. Kaplan, 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.

K. Kitagawa, D. Skowyra, S. J. Elledge, J. W. Harper, and P. Hieter, 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.

W. Liu, D. P. Evanoff, X. Chen, and Y. Luo, Urinary bladder epithelium antigen induces CD8+ T 641 cell tolerance, activation, and autoimmune response, J Immunol, vol.178, pp.539-546, 2007.

P. Steensgaard, M. Garre, I. Muradore, P. Transidico, E. A. Nigg et al., Sgt1 is 644 required for human kinetochore assembly, EMBO Rep, vol.5, pp.626-631, 2004.

A. Mayor, F. Martinon, D. Smedt, T. Petrilli, V. Tschopp et al., 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.

D. Primio, C. Quercioli, V. Allouch, A. Gijsbers, R. Christ et al., Single-cell 655 imaging of HIV-1 provirus (SCIP), Proc Natl Acad Sci U S A, vol.110, pp.5636-5641, 2013.

A. C. Francis, D. Primio, C. Quercioli, V. Valentini, P. Boll et al., Second 658 generation imaging of nuclear/cytoplasmic HIV-1 complexes, AIDS Res Hum Retroviruses, vol.659, pp.717-726, 2014.

O. Delelis, I. Malet, L. Na, L. Tchertanov, V. Calvez et al., 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.

A. David, A. Saez-cirion, P. Versmisse, O. Malbec, B. Iannascoli et al., The 666 engagement of activating FcgammaRs inhibits primate lentivirus replication in human 667 macrophages, J Immunol, vol.177, pp.6291-6300, 2006.

D. Mcdonald, M. A. Vodicka, G. Lucero, T. M. Svitkina, G. G. Borisy et al.,

, Visualization of the intracellular behavior of HIV in living cells, J Cell Biol, vol.159, pp.441-671, 2002.

K. W. Dunn, M. M. Kamocka, J. H. Mcdonald, E. S. Svarovskaia, R. Barr et al., 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.

E. Devroe, A. Engelman, and P. A. Silver, Intracellular transport of human immunodeficiency 682 virus type 1 integrase, J Cell Sci, vol.116, pp.4401-4408, 2003.

A. Akhmanova and M. O. Steinmetz, Control of microtubule organization and dynamics: two 685 ends in the limelight, Nat Rev Mol Cell Biol, vol.16, pp.711-726, 2015.

P. Bieling, S. Kandels-lewis, I. A. Telley, J. Van-dijk, C. Janke et al., 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.

A. Matov, K. Applegate, P. Kumar, C. Thoma, W. Krek et al., Analysis of 692 microtubule dynamic instability using a plus-end growth marker, Nat Methods, vol.7, p.761

D. Seetapun, B. T. Castle, A. J. Mcintyre, P. T. Tran, and D. J. Odde, Estimating the microtubule GTP 696 cap size in vivo, Curr Biol, vol.22, pp.1681-1687, 2012.

A. Albanese, D. Arosio, M. Terreni, and A. Cereseto, HIV-1 pre-integration complexes 699 selectively target decondensed chromatin in the nuclear periphery, PLoS One, vol.3, 2008.

J. Fernandez, D. M. Portilho, A. Danckaert, S. Munier, A. Becker et al., 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.

A. C. Francis and G. B. Melikyan, 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.

S. Desfarges, B. Salin, C. Calmels, M. L. Andreola, V. Parissi et al., 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.

V. R. De-soultrait, A. Caumont, P. Durrens, C. Calmels, V. Parissi et al., 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)

±. Means and . Sem, 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

. (****p<0, , 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