Abstract
Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity1. They have been successfully used in clinical trials to treat hereditary diseases such as haemophilia B (ref. 2), and have been approved for treatment of lipoprotein lipase deficiency in Europe3. Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration1,4, yet basic aspects of AAV cellular entry are still poorly understood. In particular, the protein receptor(s) required for AAV entry after cell attachment remains unknown. Here we use an unbiased genetic screen to identify proteins essential for AAV serotype 2 (AAV2) infection in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor (AAVR)). We characterize AAVR as a protein capable of rapid endocytosis from the plasma membrane and trafficking to the trans-Golgi network. We show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently block AAV2 infection. Moreover, genetic ablation of AAVR renders a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR serves as a critical host factor for all tested AAV serotypes. The importance of AAVR for in vivo gene delivery is further highlighted by the robust resistance of Aavr−/− (also known as Au040320−/− and Kiaa0319l−/−) mice to AAV infection. Collectively, our data indicate that AAVR is a universal receptor involved in AAV infection.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Accession codes
References
Kotterman, M. A. & Schaffer, D. V. Engineering adeno-associated viruses for clinical gene therapy. Nature Rev. Genet. 15, 445–451 (2014)
Nathwani, A. C. et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N. Engl. J. Med . 365, 2357–2365 (2011)
Gaudet, D., Methot, J. & Kastelein, J. Gene therapy for lipoprotein lipase deficiency. Curr. Opin. Lipidol. 23, 310–320 (2012)
Lisowski, L. et al. Selection and evaluation of clinically relevant AAV variants in a xenograft liver model. Nature 506, 382–386 (2014)
Summerford, C. & Samulski, R. J. Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J. Virol. 72, 1438–1445 (1998)
Kashiwakura, Y. et al. Hepatocyte growth factor receptor is a coreceptor for adeno-associated virus type 2 infection. J. Virol. 79, 609–614 (2005)
Qing, K. et al. Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2. Nature Med. 5, 71–77 (1999)
Carette, J. E. et al. Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature 477, 340–343 (2011)
Bonifacino, J. S. & Hierro, A. Transport according to GARP: receiving retrograde cargo at the trans-Golgi network. Trends Cell Biol. 21, 159–167 (2011)
McGough, I. J. & Cullen, P. J. Recent advances in retromer biology. Traffic 12, 963–971 (2011)
Poelmans, G., Buitelaar, J. K., Pauls, D. L. & Franke, B. A theoretical molecular network for dyslexia: integrating available genetic findings. Mol. Psychiatry 16, 365–382 (2011)
Ellis, B. L. et al. A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with nine natural adeno-associated virus (AAV1–9) and one engineered adeno-associated virus serotype. Virol. J. 10, 74 (2013)
Hansen, J., Qing, K., Kwon, H. J., Mah, C. & Srivastava, A. Impaired intracellular trafficking of adeno-associated virus type 2 vectors limits efficient transduction of murine fibroblasts. J. Virol. 74, 992–996 (2000)
Ibraghimov-Beskrovnaya, O. et al. Strong homophilic interactions of the Ig-like domains of polycystin-1, the protein product of an autosomal dominant polycystic kidney disease gene, PKD1. Hum. Mol. Genet. 9, 1641–1649 (2000)
Bhella, D. The role of cellular adhesion molecules in virus attachment and entry. Phil. Trans. R. Soc. Lond. B 370, 20140035 (2015)
Maxfield, F. R. & McGraw, T. E. Endocytic recycling. Nature Rev. Mol. Cell Biol. 5, 121–132 (2004)
Kelly, B. T. & Owen, D. J. Endocytic sorting of transmembrane protein cargo. Curr. Opin. Cell Biol. 23, 404–412 (2011)
Nonnenmacher, M. & Weber, T. Adeno-associated virus 2 infection requires endocytosis through the CLIC/GEEC pathway. Cell Host Microbe 10, 563–576 (2011)
Ghosh, P., Dahms, N. M. & Kornfeld, S. Mannose 6-phosphate receptors: new twists in the tale. Nature Rev. Mol. Cell Biol. 4, 202–213 (2003)
Beglova, N. & Blacklow, S. C. The LDL receptor: how acid pulls the trigger. Trends Biochem. Sci. 30, 309–317 (2005)
Ohka, S. et al. Receptor (CD155)-dependent endocytosis of poliovirus and retrograde axonal transport of the endosome. J. Virol. 78, 7186–7198 (2004)
Zincarelli, C., Soltys, S., Rengo, G. & Rabinowitz, J. E. Analysis of AAV serotypes 1–9 mediated gene expression and tropism in mice after systemic injection. Mol. Ther. 16, 1073–1080, (2008)
Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015)
Balazs, A. B. et al. Antibody-based protection against HIV infection by vectored immunoprophylaxis. Nature 481, 81–84 (2012)
Nonnenmacher, M. & Weber, T. Intracellular transport of recombinant adeno-associated virus vectors. Gene Ther. 19, 649–658 (2012)
Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. A Stat. Soc . 57, 289–300 (1995)
Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nature Protocols 8, 2281–2308 (2013)
Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nature Protocols 7, 171–192 (2012)
Campeau, E. et al. A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS ONE 4, e6529 (2009)
Holster, S. et al. Expression of the dyslexia candidate gene Kiaa0319-like in insect cells J. Biochem. Mol. Biol. Post Gen. Era 2, 45–52 (2013)
Seaman, M. N. Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer. J. Cell Biol. 165, 111–122 (2004)
Jae, L. T. et al. Virus entry. Lassa virus entry requires a trigger-induced receptor switch. Science 344, 1506–1510 (2014)
Acknowledgements
The authors thank K. Kirkegaard, M. Kay and T. Brummelkamp for critical reading of the manuscript and valuable advice; T. Lerch, J. Tyner, D. Kabat and H. Nakai for assistance with preliminary experiments; H. P. Bächinger for advice and assistance with surface plasmon resonance experiments; Stanford Shared FACS facility and its staff; X. Ji (Stanford Functional Genomics Facility); Stanford mouse facility; T. Doyle for small animal imaging training; L. Popov for guidance in generating immunofluorescent images; G. Fuchs for technical assistance; and members of the Carette and Chapman laboratories for intellectual discussions and support. The work was funded in part by NIH R01 GM066875 (M.S.C.), DP2 AI104557 (J.E.C.) and U19 AI109662 (J.E.C.). J.E.C. is a David and Lucile Packard Foundation fellow.
Author information
Authors and Affiliations
Contributions
S.P., M.S.C. and J.E.C. were responsible for overall design of the study. S.P. performed the haploid genetic screen, generated isogenic knockout and AAVR complement cell lines, and performed antibody inhibition and AAVR tracking studies. N.L.M. designed soluble AAVR construct and performed binding and soluble AAVR inhibition studies. J.E.C. designed AAVR-generated deletion mutant constructs. A.S.P. performed the wild-type AAV2 infection assay and all in vivo studies, under the technical expertise of C.M.N. O.D. was responsible for heterologous overexpression and purification of soluble AAVR, J.D. assisted in the production of FGFR1KO and METKO cell lines, Y.I. performed surface plasmon resonance measurements, L.T.J. generated the B3GALT6KO cell line, and J.E.W. created the ΔC-tail construct. S.P., M.S.C. and J.E.C. wrote the manuscript.
Corresponding authors
Ethics declarations
Competing interests
Stanford University has filed a patent claim regarding AAVR applications in AAV vector technology (inventors: S.P., M.S.C., J.E.C., A.S.P., N.L.M. and O.D.).
Additional information
DNA sequencing data have been deposited in the NCBI sequencing read archive under NCBI BioProject PRJNA284536 with BioSample SAMN03703230 (gene-trap control data set) and SAMN04244346 (AAV screen).
Extended data figures and tables
Extended Data Figure 1 Surface molecules FGFR1 and MET are not essential for AAV2 infection.
a, Region of FGFR1, MET, or B3GALT6 genes (previously identified co-receptors/attachment factors5,6,7) targeted by CRISPR gRNA or TALENs in wild-type HAP1 cells, and the resulting genotypes of derived knockout cell lines (see Extended Data Table 1 for full sequences). All CRISPR- or TALEN-created mutations disrupt the open reading frame of the targeted gene. b, Surface staining for the respective receptors in respective cell lines. Isotype antibodies for the receptor antibodies (Ab) were used as controls. c, AAV2–RFP infection (MOI 5,000 vg per cell; measured after 24 h) of wild-type and knockout cell lines. Data depict the mean and s.d. for triplicate infections. *P < 0.05, ***P < 0.001; analysed using unpaired parametric two-sided Student’s t-test, with Welch post-correction. SSC, side scatter. Fluorescently labelled antibody conjugates, fluorescein isothiocyanate (FITC) and phycoerythrin (PE), were used to visualize surface receptors.
Extended Data Figure 2 Haploid unbiased genetic screen evaluating host factors important for AAV2 infection.
a, A schematic depicting the strategy for the AAV2 genetic screen. A library of mutagenized haploid HAP1 cells was created with a retroviral gene-trap vector, and subsequently infected with AAV2–RFP (MOI: 20,000 vg per cell) for 24 h. RFP-negative cells were sorted using FACS to isolate cells with mutations in genes essential for AAV2 infection. These cells were re-infected for a second iteration of selection. DNA was then extracted from this enriched population and sequenced to map specifically where the gene-trap insertions occurred that resulted in the mutation. b, The gating strategy for the FACS-based AAV2 screen.
Extended Data Figure 3 AAVR is a critical host factor for AAV2 infection.
a, Effect of AAVR isogenic knockout (AAVRKO) upon AAV2–luciferase infection, evaluated in HAP1 and HeLa cell background from a MOI of 100 to 100,000 vg per cell. b, RT–qPCR to detect wild-type AAV2 infection in wild-type HeLa or AAVRKO cells. Cells were infected with wild-type AAV2 and adenovirus (helper virus required for AAV2 replication), and AAV2 Rep68 mRNA levels were measured to assess AAV2 infection. c, Immunoblot analysis evaluating AAVR expression in wild-type, AAVRKO and AAVRKO overexpressing AAVR (AAVR Comp.) cell lines of HAP1 and HeLa origin. GAPDH was immunoblotted as a control. AAVR (predicted 115 kilodaltons (kDa)) appears at 150 kDa owing to six glycosylation sites. d, AAV2–luciferase infection (MOI 20,000 vg per cell; measured after 24 h) in AAVRKO cells stably complemented with AAVR or control lentiviral vector, evaluated in several AAV2-susceptible human and mouse cell lines. e, Comparison of AAV2–RFP infection (MOI: 20,000 vg per cell; measured after 24 h) in wild-type, AAVRKO, METKO and FGFR1KO cells, evaluated in several AAV2-susceptible human cell lines. Data depict the mean and s.d. for triplicate infections.
Extended Data Figure 4 AAVR specifically binds to AAV2.
a, ELISA measurement of the binding to AAV2 particles of MBP at concentrations of 0.05–2,000 nM. This serves as a control to the ELISA data depicted in Fig. 2c. b, Representative surface plasmon resonance sensograms (collected in triplicate), with a ligand (AAVR) concentration of 4 nM and an analyte (AAV2) concentration as indicated, to measure binding of AAV2 particles to AAVR. c, Simultaneous addition to cells of AAV2–GFP particles with soluble AAVR or MBP (both at 0.1 μM) to evaluate binding effect of AAVR on AAV2 infection. Fluorescence was imaged 24 h after infection. These data complement Fig. 2d. Data in a depict the mean and s.d. for triplicate infections. Scale bars, 50 μm. RU, response units.
Extended Data Figure 5 AAVR ΔC-tail is detected at the cell surface and does not endocytose to the TGN.
AAVRKO cells (a) or ΔC-tail-expressing cells (c) were incubated with anti-AAVR antibodies for 1 h at 4 °C, washed and then transferred to 37 °C. At respective time points, cells were fixed and antibody-bound AAVR was visualized. These data complement Fig. 3b. b, Permeabilized and unpermeabilized immunostaining of full-length AAVR and ΔC-tail when expressed in AAVRKO cells. These data complements Fig. 3c. Scale bars, 10 μm.
Extended Data Figure 6 AAVR endocytosis is crucial for AAV2 infection.
a, Schematic of the miniAAVR and domain-swapped derivatives probing the localization of AAVR through the swapping of the AAVR C-tail with that of well-characterized recycling receptors: Ci-MPR (traffics from plasma membrane through endosomes to the TGN), LDLR and PVR (both traffic from plasma membrane to endosomal compartments but are not reported to traffic to TGN). b, Corresponding permeabilized and unpermeabilized immunofluorescence images of constructs depicted in a when expressed in AAVRKO cells. c, AAV2–RFP infection (MOI: 20,000 vg per cell; measured after 24 h) in AAVRKO cells stably expressing constructs depicted in a. Data depict the mean and s.d. for triplicate infections. Scale bars, 10 μm.
Extended Data Figure 7 AAVR is essential for AAV infection in vivo.
a, Genotypes of FVB mice littermates used to perform in vivo studies. AAVR knockout mice (Aavr−/−) were bred from heterozygous (Aavr+/−) parent mice; Aavr+/− and Aavr−/− mice display frameshift mutations in targeted genes in 1 and 2 alleles, respectively. Sequences recognized by the TALENs are displayed in yellow. b, AAV9–luciferase infection for all infected mice depicted for day 3, 10 and 14 (day 7 is shown in Fig. 4d). c, Bioluminescence in all wild-type (Aavr+/+), Aavr+/− and Aavr−/− FVB mice 7 days after AAV9–luciferase infection (does not include those shown in Fig. 4b). Radiance range of 2 × 105 to 1 × 107 photons s−1 cm−2 sr−1. Significance was determined using unpaired two-sided Mann–Whitney t-test; **P < 0.01.
Supplementary information
Supplementary Information
This file contains Supplementary Table 1 and Supplementary Figure 1, which contains the raw source data for Extended Data Figure 3c. (PDF 432 kb)
Rights and permissions
About this article
Cite this article
Pillay, S., Meyer, N., Puschnik, A. et al. An essential receptor for adeno-associated virus infection. Nature 530, 108–112 (2016). https://doi.org/10.1038/nature16465
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature16465
This article is cited by
-
A humanized mouse model for adeno-associated viral gene therapy
Nature Communications (2024)
-
Enhancing transduction efficiency of adeno-associated virus 9 by cell line engineering: implication for gene therapy potency assay
Biotechnology and Bioprocess Engineering (2024)
-
A Novel Retrograde AAV Variant for Functional Manipulation of Cortical Projection Neurons in Mice and Monkeys
Neuroscience Bulletin (2024)
-
Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters
Gene Therapy (2023)
-
A flexible system for tissue-specific gene expression in mice using adeno-associated virus
Nature Methods (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
