J. Kleinschmidt, 49]) or B1 (detecting free capsid proteins of all AAV serotypes used here except AAV4)

A. Antibody, GeneTex) was diluted 1:500 in the hybridoma supernatants Cells were then washed twice for 10 min each with IF blocking buffer and then incubated for 1 h at 37°C with secondary antibodies diluted 1:700 in blocking buffer. To quantify B1 and A20/ADK stainings, an anti-mouse AF488-labeled secondary antibody (Life Technologies) was used. The anti-HA antibody was detected with an anti-rabbit AF647- labeled secondary antibody (Life Technologies) To stain nuclei, Hoechst stain (Molecular Probes, Thermo Fisher Scientific) diluted 1:3,000 was added to all samples. Next, cells were washed with 1 PBS and stored at 4°C in the dark in 200 l 1 PBS per well, HeLa cells were treated the same way, except that cells were analyzed at 42 h posttransfection and stained with the anti-HA antibody (and Hoechst stain, 29110.

. Cdna, 75 l H 2 O. Reactions were run in a StepOnePlus real-time PCR system (Thermo Fisher Scientific) using the following program: 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min, and a final melting step from 60°C to 95°C in increments of 0.3°C. Samples were measured in duplicates and normalized to a GAPDH housekeeper (primer pair 1117 For the final analysis, 1118.

. Polyclonal and . Production, Thermo Fisher Scientific, and produced by immunization of rabbits with peptide GKDSST TTGDSDPRDSTS (AAP2 residues 121 to 138) (17) Affinity purification yielded the final polyclonal anti-AAP2 antibody (preserved in 1% BSA), which was used for Western blot analyses of insect cell samples and for immunostainings. Dot blot analysis. Crude cell lysates containing AAV particles were produced as stated above. A nitrocellulose membrane (Thermo Fisher Scientific) was cut to size, soaked in PBS for 10 min, and assembled into a Bio-Dot microfiltration apparatus (Bio-Rad) according to the manufacturer's protocol. Next, 15 l of the indicated crude lysates was loaded. To promote capsid disassembly for detection of free, unassembled viral capsid proteins, 100 l of each sample was heated to 95°C for 5 min, and 15 l REFERENCES 1 Adenovirus-associated defective virus particles, The anti-AAP2 antibody was ordered from the company Perbio, pp.754-756, 1965.

D. Grimm and S. Zolotukhin, E Pluribus Unum: 50 Years of Research, Millions of Viruses, and One Goal???Tailored Acceleration of AAV Evolution, Molecular Therapy, vol.23, issue.12, pp.1819-1831, 2015.
DOI : 10.1038/mt.2015.173

M. Agbandje-mckenna and J. Kleinschmidt, AAV Capsid Structure and Cell Interactions, Methods Mol Biol, vol.807, pp.47-92, 2011.
DOI : 10.1007/978-1-61779-370-7_3

M. Kotterman and D. Schaffer, Engineering adeno-associated viruses for clinical gene therapy, Nature Reviews Genetics, vol.434, issue.7, pp.445-451, 2014.
DOI : 10.1016/j.ymthe.2006.05.009

URL : http://europepmc.org/articles/pmc4393649?pdf=render

D. Grimm, J. Lee, L. Wang, T. Desai, B. Akache et al., In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses, Journal of Virology, vol.82, issue.12, pp.5887-5911, 2008.
DOI : 10.1128/JVI.00254-08

J. Koerber, J. Jang, and D. Schaffer, DNA Shuffling of Adeno-associated Virus Yields Functionally Diverse Viral Progeny, Molecular Therapy, vol.16, issue.10, pp.1703-1709, 2008.
DOI : 10.1038/mt.2008.167

W. Li, A. Asokan, Z. Wu, T. Van-dyke, N. Diprimio et al., Engineering and Selection of Shuffled AAV Genomes: A New Strategy for Producing Targeted Biological Nanoparticles, Molecular Therapy, vol.16, issue.7, pp.1252-1260, 2008.
DOI : 10.1038/mt.2008.100

S. Gray, B. Blake, H. Criswell, S. Nicolson, R. Samulski et al., Directed Evolution of a Novel Adeno-associated Virus (AAV) Vector That Crosses the Seizure-compromised Blood???Brain Barrier (BBB), Molecular Therapy, vol.18, issue.3, pp.570-578, 2010.
DOI : 10.1038/mt.2009.292

S. Powell, N. Khan, C. Parker, R. Samulski, G. Matsushima et al., Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism, Gene Therapy, vol.807, issue.11, pp.807-814, 2016.
DOI : 10.1083/jcb.85.3.890

L. Yang, J. Jiang, L. Drouin, M. Agbandje-mckenna, C. Chen et al., A myocardium tropic adenoassociated virus (AAV) evolved by DNA shuffling and in vivo selection, 2009.

P. Asuri, M. Bartel, T. Vazin, J. Jang, T. Wong et al., Directed Evolution of Adeno-associated Virus for Enhanced Gene Delivery and Gene Targeting in Human Pluripotent Stem Cells, Molecular Therapy, vol.20, issue.2, pp.329-338, 2012.
DOI : 10.1038/mt.2011.255

D. Dalkara, L. Byrne, R. Klimczak, M. Visel, L. Yin et al., In vitro and in vivo models for the study of human polyomavirus infection, Viruses, vol.8, 2013.

K. Excoffon, J. Koerber, D. Dickey, M. Murtha, S. Keshavjee et al., Directed evolution of adeno-associated virus to an infectious respiratory virus, Proceedings of the National Academy of Sciences, vol.3, issue.5, pp.3865-3870, 2009.
DOI : 10.1073/pnas.0601433103

J. Jang, J. Koerber, J. Kim, P. Asuri, T. Vazin et al., An Evolved Adeno-associated Viral Variant Enhances Gene Delivery and Gene Targeting in Neural Stem Cells, Molecular Therapy, vol.19, issue.4, pp.667-675, 2011.
DOI : 10.1038/mt.2010.287

URL : https://doi.org/10.1038/mt.2010.287

J. Koerber, R. Klimczak, J. Jang, D. Dalkara, J. Flannery et al., Molecular Evolution of Adeno-associated Virus for Enhanced Glial Gene Delivery, Molecular Therapy, vol.17, issue.12, pp.2088-2095, 2009.
DOI : 10.1038/mt.2009.184

P. Ward and C. Walsh, Chimeric AAV Cap sequences alter gene transduction, Virology, vol.386, issue.2, pp.237-248, 2009.
DOI : 10.1016/j.virol.2009.01.012

URL : https://doi.org/10.1016/j.virol.2009.01.012

F. Sonntag, K. Schmidt, and J. Kleinschmidt, A viral assembly factor promotes AAV2 capsid formation in the nucleolus, Proceedings of the National Academy of Sciences, vol.140, issue.1-2, pp.10220-10225, 2010.
DOI : 10.1016/j.jviromet.2006.10.005

M. Naumer, F. Sonntag, K. Schmidt, K. Nieto, C. Panke et al., Properties of the Adeno-Associated Virus Assembly-Activating Protein, Journal of Virology, vol.86, issue.23, pp.13038-1304801675, 2012.
DOI : 10.1128/JVI.01675-12

F. Sonntag, K. Kother, K. Schmidt, M. Weghofer, C. Raupp et al., The Assembly-Activating Protein Promotes Capsid Assembly of Different Adeno-Associated Virus Serotypes, Journal of Virology, vol.85, issue.23, pp.12686-1269705359, 2011.
DOI : 10.1128/JVI.05359-11

L. Earley, Y. Kawano, K. Adachi, X. Sun, M. Dai et al., ABSTRACT, Journal of Virology, vol.89, issue.6, pp.3038-304803125, 2015.
DOI : 10.1128/JVI.03125-14

L. Earley, J. Powers, K. Adachi, J. Baumgart, N. Meyer et al., Adeno-associated virus assemblyactivating protein is not an essential requirement for capsid assembly of AAV serotypes 4, 5, and 11, J Virol, pp.1980-1996, 2017.

M. Urabe, C. Ding, and R. Kotin, Insect Cells as a Factory to Produce Adeno-Associated Virus Type 2 Vectors, Human Gene Therapy, vol.13, issue.16, pp.1935-1943, 2002.
DOI : 10.1089/10430340260355347

R. Smith, J. Levy, and R. Kotin, A Simplified Baculovirus-AAV Expression Vector System Coupled With One-step Affinity Purification Yields High-titer rAAV Stocks From Insect Cells, Molecular Therapy, vol.17, issue.11, pp.1888-1896, 2009.
DOI : 10.1038/mt.2009.128

G. Aslanidi, K. Lamb, and S. Zolotukhin, An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells, Proceedings of the National Academy of Sciences, vol.6, issue.6, pp.5059-5064, 2009.
DOI : 10.1038/sj.gt.3300938

M. Mietzsch, V. Casteleyn, S. Weger, S. Zolotukhin, and R. Heilbronn, 9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA, Human Gene Therapy, vol.26, issue.10, pp.688-697, 2015.
DOI : 10.1089/hum.2015.050

W. Xiao, N. Chirmule, S. Berta, B. Mccullough, G. Gao et al., Gene therapy vectors based on adeno-associated virus type 1, J Virol, vol.73, pp.3994-4003, 1999.

A. Douar, K. Poulard, D. Stockholm, and O. Danos, Intracellular Trafficking of Adeno-Associated Virus Vectors: Routing to the Late Endosomal Compartment and Proteasome Degradation, Journal of Virology, vol.75, issue.4, pp.1824-1833, 2001.
DOI : 10.1128/JVI.75.4.1824-1833.2001

D. Duan, Y. Yue, Z. Yan, J. Yang, and J. Engelhardt, Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus, Journal of Clinical Investigation, vol.105, issue.11, pp.1573-1587, 2000.
DOI : 10.1172/JCI8317

URL : http://www.jci.org/articles/view/8317/files/pdf

Z. Yan, R. Zak, G. Luxton, T. Ritchie, U. Bantel-schaal et al., Ubiquitination of both Adeno-Associated Virus Type 2 and 5 Capsid Proteins Affects the Transduction Efficiency of Recombinant Vectors, Journal of Virology, vol.76, issue.5, pp.2043-2053, 2002.
DOI : 10.1128/jvi.76.5.2043-2053.2002

A. Wistuba, A. Kern, S. Weger, D. Grimm, and J. Kleinschmidt, Subcellular compartmentalization of adeno-associated virus type 2 assembly, J Virol, vol.71, pp.1341-1352, 1997.

D. Grimm, K. Pandey, H. Nakai, T. Storm, and M. Kay, Liver Transduction with Recombinant Adeno-Associated Virus Is Primarily Restricted by Capsid Serotype Not Vector Genotype, Journal of Virology, vol.80, issue.1, pp.426-439, 2006.
DOI : 10.1128/JVI.80.1.426-439.2006

URL : http://jvi.asm.org/content/80/1/426.full.pdf

J. Rabinowitz, X. W. Samulski, and R. , Insertional Mutagenesis of AAV2 Capsid and the Production of Recombinant Virus, Virology, vol.265, issue.2, pp.274-285, 1999.
DOI : 10.1006/viro.1999.0045

K. Warrington, . Jr, O. Gorbatyuk, J. Harrison, S. Opie et al., Adeno-Associated Virus Type 2 VP2 Capsid Protein Is Nonessential and Can Tolerate Large Peptide Insertions at Its N Terminus, Journal of Virology, vol.78, issue.12, 2004.
DOI : 10.1128/JVI.78.12.6595-6609.2004

URL : http://jvi.asm.org/content/78/12/6595.full.pdf

H. Petrs-silva, A. Dinculescu, Q. Li, S. Min, V. Chiodo et al., High-efficiency Transduction of the Mouse Retina by Tyrosine-mutant AAV Serotype Vectors, Molecular Therapy, vol.17, issue.3, pp.463-471, 2009.
DOI : 10.1038/mt.2008.269

C. Qiao, W. Zhang, Z. Yuan, J. Shin, J. Li et al., Adeno-Associated Virus Serotype 6 Capsid Tyrosine-to-Phenylalanine Mutations Improve Gene Transfer to Skeletal Muscle, Human Gene Therapy, vol.21, issue.10, pp.1343-1348, 2010.
DOI : 10.1089/hum.2010.003

URL : http://europepmc.org/articles/pmc2957235?pdf=render

L. Zhong, B. Li, C. Mah, L. Govindasamy, M. Agbandje-mckenna et al., Next generation of adeno-associated virus 2 vectors: Point mutations in tyrosines lead to high-efficiency transduction at lower doses, Proceedings of the National Academy of Sciences, vol.111, issue.9, pp.7827-7832, 2008.
DOI : 10.1172/JCI200316887

D. Grimm, M. Kay, and J. Kleinschmidt, Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6, Molecular Therapy, vol.7, issue.6, pp.839-850, 2003.
DOI : 10.1016/S1525-0016(03)00095-9

Y. Kawano, S. Neeley, K. Adachi, and H. Nakai, An experimental and computational evolution-based method to study a mode of coevolution of overlapping open reading frames in the AAV2 viral genome, PLoS One, vol.8, issue.e66211, 2013.

L. Gallo-ramirez, O. Ramirez, and L. Palomares, Intracellular localization of adeno-associated viral proteins expressed in insect cells, Biotechnology Progress, vol.67, issue.2, pp.483-493, 2011.
DOI : 10.1002/rmv.614

G. Rohrmann, Baculovirus molecular biology, National Center for Biotechnology Information, 2013.

E. Lombardo, J. Ramirez, J. Garcia, and J. Almendral, Complementary Roles of Multiple Nuclear Targeting Signals in the Capsid Proteins of the Parvovirus Minute Virus of Mice during Assembly and Onset of Infection, Journal of Virology, vol.76, issue.14, 2002.
DOI : 10.1128/JVI.76.14.7049-7059.2002

L. Riolobos, J. Reguera, M. Mateu, and J. Almendral, Nuclear Transport of Trimeric Assembly Intermediates Exerts a Morphogenetic Control on the Icosahedral Parvovirus Capsid, Journal of Molecular Biology, vol.357, issue.3, pp.1026-1038, 2006.
DOI : 10.1016/j.jmb.2006.01.019

J. Gil-ranedo, E. Hernando, L. Riolobos, C. Dominguez, M. Kann et al., The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly, PLOS Pathogens, vol.90, issue.6, 2015.
DOI : 10.1371/journal.ppat.1004920.s004

URL : https://doi.org/10.1371/journal.ppat.1004920

N. Schurmann, L. Trabuco, C. Bender, R. Russell, and D. Grimm, Molecular dissection of human Argonaute proteins by DNA shuffling, Nature Structural & Molecular Biology, vol.57, issue.7, pp.818-826, 2013.
DOI : 10.1016/0263-7855(96)00018-5

R. Samulski, L. Chang, and T. Shenk, A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication, J Virol, vol.61, pp.3096-3101, 1987.

T. Matsushita, S. Elliger, C. Elliger, G. Podsakoff, L. Villarreal et al., Adeno-associated virus vectors can be efficiently produced without helper virus, Gene Therapy, vol.5, issue.7, pp.938-945, 1998.
DOI : 10.1038/sj.gt.3300680

URL : http://www.nature.com/gt/journal/v5/n7/pdf/3300680a.pdf

D. Grimm, K. Streetz, C. Jopling, T. Storm, K. Pandey et al., Fatality in mice due to oversaturation of Role of AAP in AAV Vectors and VP Stability Journal of Virology, pp.1198-1215, 2006.

D. Costa, S. Blouin, V. Broucque, F. Penaud-budloo, M. Francois et al., Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR, Mol Ther Methods Clin Dev, vol.5, p.16019, 2016.

A. Wistuba, S. Weger, A. Kern, and J. Kleinschmidt, Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins, J Virol, vol.69, pp.5311-5319, 1995.

K. Borner, D. Niopek, G. Cotugno, M. Kaldenbach, T. Pankert et al., Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines, Nucleic Acids Research, vol.17, issue.21, p.199, 2013.
DOI : 10.3851/IMP2073

K. Borner, J. Hermle, C. Sommer, N. Brown, B. Knapp et al., From experimental setup to bioinformatics: an RNAi screening platform to identify host factors involved in HIV-1 replication, 2010.
URL : https://hal.archives-ouvertes.fr/hal-00545273

K. Livak and T. Schmittgen, Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2???????CT Method, Methods, vol.25, issue.4, pp.402-408, 2001.
DOI : 10.1006/meth.2001.1262