An essential receptor for adeno-associated virus infection

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 immunogenicity. 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 Europe. Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration, 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.

Extended Data Figure 1. Surface molecules, FGFR1 and c-MET, are not essential for AAV2 infection

a, Region of FGFR1, c-MET, or B3GALT6 genes (previously-identified co-receptors/attachment factors^–) targeted by CRISPR guide RNA or TALENs in wild-type HAP1 cells, and the resulting genotypes of derived knock-out cell lines. (see full sequence in Extended Data Table 1). 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 were used as controls. c, AAV2-RFP infection (MOI 5,000 viral genomes (vg)/cell; measured after 24 hrs) of wild-type and knock-out cell lines. Data depicts the mean with s.d. for triplicate infections. * - p <0.05, *** - p <0.001; analyzed using an unpaired, parametric, two-sided student t-test, with a Welch post-correction. c-MET: hepatocyte growth factor receptor; FGFR1: fibroblast growth factor receptor-1. FITC or PE refer to fluorescently-labeled antibody conjugates used to visualize surface receptors. MOI: multiplicity of infection, RFP: red-fluorescent protein, SSC: side scatter.

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/cell) for 24 hrs. RFP-negative cells were sorted using FACS to isolate those 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 specifically map where the gene trap insertions occurred that resulted in the mutation. b, The gating strategy for the FACS-based AAV2 screen. FACS: fluorescence-activated cell sorting, RFP: red-fluorescent protein, SSC: side scatter.

Extended Data Figure 3. AAVR is a critical host factor for AAV2 infection

a, Effect of AAVR isogenic knock-out (AAVR^KO) upon AAV2-luciferase infection, evaluated in HAP1 and HeLa cell background from MOI of 100 to 100,000 vg/cell. b, Quantitative RT-PCR to detect wild-type AAV2 infection in wild-type (WT) HeLa or AAVR^KO 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 WT, AAVR^KO and AAVR^KO overexpressing AAVR (AAVR Comp.) cell lines of HAP1 and HeLa origin. GAPDH was immunoblotted as a control. AAVR (predicted 115 kDa) appears at 150 kDa due to 6 glycosylation sites. d, AAV2-luciferase infection (MOI 20,000 vg/cell; measured after 24 hrs) in AAVR^KO 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/cell; measured after 24 hrs) in WT, AAVR^KO, c-MET^KO and FGFR1^KO cells, evaluated in several AAV2-susceptible human cell lines. RLU: relative light units. Data depicts the mean with s.d. error bars 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 Figure 2c. b, Representative surface plasmon resonance sensograms (collected in triplicate), with a ligand (AAVR) concentration of 4nM and an analyte (AAV-2) concentration as indicated, to measure binding of AAV-2 particles to AAVR. c, Simultaneous addition to cells of AAV2-GFP particles with soluble AAVR or MBP (both at 0.1 μM) to evaluate AAVR’s binding effect on AAV2 infection. Fluorescence was imaged 24 hrs post infection. This data complements Figure 2d. Data in a depicts the mean with s.d. error bars for triplicate infections. Scale bars represent 50 μm.

Extended Data Figure 5. AAVR ΔC-tail is detected at the cell surface and does not endocytose to the TGN

AAVR^KO cells (a) or ΔC-tail-expressing cells (c) were incubated with anti-AAVR antibodies for 1 hr at 4°C, washed and then transferred to 37°C. At respective time points, cells were fixed and antibody-bound AAVR was visualized. This data complements Figure 3b. b, Permeabilized and unpermeabilized immunostaining of full-length AAVR and ΔC-tail when expressed in AAVR^KO cells. This data complements Figure 3c. Scale bars represent 10 μm.

Extended Data Figure 6. AAVR endocytosis is crucial for AAV2 infection

a, Schematic of the AAVR minimal construct (miniAAVR) and domain-swapped derivatives probing the localization of AAVR through the swapping of AAVR’s C-tail with that of well-characterized recycling receptors: cation-independent mannose-6-phosphate receptor (Ci-MPR) (traffics from plasma membrane (PM) through endosomes to the TGN), low density lipoprotein receptor (LDLR) and poliovirus receptor (PVR) (both traffic from PM 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 AAVR^KO cells. c, AAV2-RFP infection (MOI 20,000 vg/cell; measured after 24 hrs) in AAVR^KO cells stably expressing constructs depicted in a. Data depicts the mean with s.d. for triplicate infections. Scale bars represent 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 KO (AAVR^−/−) were bred from heterozygous (AAVR^+/−) parent mice; AAVR^+/− and AAVR^−/− mice display frameshift mutations in targeted genes in 1 or 2 alleles respectively. Sequences recognized by the TALENs are displayed in yellow. b, AAV9-luciferase infection (as measured by average radiance) for all infected mice depicted for Day 3, 10 and 14 (Day 7 is shown in Figure 4d). c, Bioluminescence in all wild-type (AAVR^+/+), AAVR^+/− and AAVR^−/− FVB mice 7 days post AAV9-luciferase infection (does not include those displayed in Figure 4b). Radiance range of 2×10⁵ – 1×10⁷ p/s/cm²/sr. The P value was determined using an unpaired, two-sided Mann-Whitney t-test where ** - P <0.01, NS – not significant.

Fig. 1. An unbiased, haploid genetic screen identifies KIAA0319L (AAVR), an essential host factor for AAV2 infection

a, Bubble plot illustrating significance of enrichment of gene-trap insertions within identified genes (relative to unselected control population). Bubbles represent genes with width proportional to number of independent gene trap insertions. Top forty significant genes (p ≤ 0.001) are colored and grouped by function. b, AAV2-RFP infection in wild-type (WT) cells and AAVR knock-out (AAVR^KO) cells, evaluated in AAV2-susceptible human and mouse cell lines. c, AAV2-RFP infection of poorly permissive human and murine cell lines with and without AAVR overexpression. Data depicts mean with s.d. error bars for triplicate infections. Infections were performed using MOI 20,000 vg/cell for 24 hrs. The P value was determined using an unpaired, parametric, two-sided student t-test, with a Welch post-correction, where * - P <0.05, ** - P <0.01, *** - P <0.001.

Fig. 2. AAVR binds specifically to AAV2 via its Ig-like PKD domains

a, Schematic of AAVR domains and deletion mutants; dotted line represents deletions. b, AAV2-RFP infection of HAP1 AAVR^KO cells expressing AAVR deletion mutants (MOI 20,000 vg/cell). c, ELISA showing binding to AAV2 particles of soluble AAVR (fusion protein between MBP and AAVR PKD 1–5). d, AAV2 neutralization assay incubating cells with soluble AAVR or MBP during AAV2-GFP infection, (MOI 7,500 vg/cell). e, Antibody inhibition assay incubating wild-type HeLa cells with anti-AAVR or IgG isotype control antibodies (at respective concentrations) at 4°C before AAV2-luciferase infection (MOI 1,000 vg/cell). Data depicts mean with s.d. error bars for triplicate infections; transgene expression measured after 24 hrs. SP: signal peptide, MANEC: motif at N-terminus with eight cysteines, PKD: polycystic kidney disease, TM: transmembrane, C-tail: C-terminal cytoplasmic tail, MBP: maltose binding protein, RLU: relative light units.

Fig. 3. AAVR traffics from the plasma membrane to the trans-Golgi network, and its endocytosis is necessary for AAV2 infection

a, Endogenous AAVR localization in wild-type HeLa cells shown with markers for cis-medial Golgi (giantin) and trans-Golgi network (TGN46). b,Tracking AAVR endocytosis using anti-AAVR antibodies. AAVR-complement cells were incubated with anti-AAVR antibodies for 1 hr at 4°C, washed and then transferred to 37°C. At respective time points, cells were fixed and anti-AAVR antibodies were visualized to depict the trafficking of surface AAVR. c, AAVR surface expression on AAVR^KO cells with and without overexpression of full-length AAVR and ΔC-tail (depicted in schematic). d, AAV2-RFP infection (MOI 20,000 vg/cell; measured after 24 hrs) in AAVR^KO cells stably expressing constructs depicted in c. Data depicts the mean with s.d. error bars for triplicate infections. Scale bars represent 10 μm.

Fig. 4. AAVR is a critical host factor for the infection of a wide array of naturally-occurring AAV serotypes, and is essential for AAV infection in vivo

a, Infection of wild-type HeLa cells, AAVR knock-out (AAVR^KO) cells, and AAVR^KO cells overexpressing AAVR (AAVR complement), using AAV vectors of different serotypes (MOI 10⁵ vg/cell; RFP/GFP expression measured at 24 hrs). b, Bioluminescence of AAV9-infected wild-type (AAVR^+/+), heterozygous (AAVR^+/−) and AAVR^KO (AAVR^−/−) FVB mice over 14 days; representative mice from each group are shown with a radiance range of 5×10⁵ – 1×10⁷ p/s/cm²/sr. c, AAV9-luciferase infection for AAVR^+/+, AAVR^+/−, and AAVR^−/− groups (measured as average radiance) at the respective days post infection. d, AAV9-luciferase infection of mice at Day 7. Data depicts the mean (with s.d. error bars in a and c). The P value was determined using an unpaired, two-sided Mann-Whitney t-test where ** - P <0.01, NS – not significant.