GPR108 Is a Highly Conserved AAV Entry Factor

Abstract

Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells in vitro. These findings are further validated in vivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. In vitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both in vitro and in vivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.

Keywords: AAV; CRISPR screen; GPR108; adeno-associated virus; endosomal escape; entry; in vivo; receptor.

Graphical abstract

Figure 1

rh32.33 Entry Screen (A) Huh7 AAVR KO cells undergo lentiCRISPR mutagenesis using a two-vector lentiviral GeCKO system. sgRNA library contains six sgRNAs per gene, targeting the entire human genome and 1,000 miRNAs to generate a cell line knockout library. Cells are subjected to multiple rounds of high MOI transduction and sgRNA deep sequencing to determine gene deletions enriched in GFP-negative cells to identify genes required for rh32.33 entry. (B) Entry screen hits from second round of GFP transduction. The x axis shows individual genes within the GeCKO library, grouped by gene ontology analysis. The y axis shows significance of hit based on RRA analysis. The bubble diameter indicates the number of individual sgRNAs per gene enriched in the selected population, relative to unselected control.

Figure 2

GPR108 Is a Highly Conserved AAV Entry Factor (A) Phylogenetic tree of extant and putative evolutionary intermediate AAV serotypes, color-coded for AAVR and GPR108 dependence. Red, AAVR and GPR108; green, GPR108 only; blue, AAVR only. (B) Transduction of indicated AAV serotypes in WT or GPR108 KO Huh7 cells at 10,000 VG/cell with hAd5 helper virus. CMV.Luciferase.SVPA (AAVrh10, AAV8, AAVAnc82, AAV9, AAVAnc81, AAVAnc80, AAV3, AAV6.2, AAV1, AAVrh32.33, AAV4, AAV5) or CMV.eGFP.T2A.Luciferase.SVPA (AAVAnc83, AAVAnc110, AAV2) transgene. (C) Huh7 WT or GPR108 KO cells transfected with FLAG-tagged human GPR107 or GPR108 followed by transduction of the indicated serotype in the presence of hAd5 helper virus. 10,000 VG/cell CMV.Luciferase.SVPA transgene. (D) H1 HeLa cells deleted for GPR108, then stably rescued with GPR108 lentivirus, followed by transduction of the indicated serotypes at 10,000 VG/cell with hAd5 helper virus. CMV.Luciferase.SVPA transgene. All in vitro transductions are shown as SE measurement from minimum of three independent experiments, done in duplicate. Background is maximum observed value from untransduced control cells.

Figure 3

GPR108 Dependence Is Dictated by the VP1 Unique Region of Capsid and Is Transferrable to Other Serotypes (A) Design of chimeric capsids used to determine GPR108 usage domain with amino acid number labeled above. vPLA, viral phospholipase domain. (B) Transduction of Huh7 WT, AAVR KO, GPR108 KO, or double-KO cells with the indicated WT or chimeric capsids (100 μL of crude vector preparation, plus hAd5 helper virus, CMV.eGFP.T2A.Luciferase transgene). (C) Transduction of MEF cells derived from WT or GPR108 KO C57BL/6 mice transduced with 10,000 VG/cell iodixanol-purified WT or chimeric capsids in the presence of hAd5 helper virus, CMV.Luciferase.SVPA transgene. (D) Bioluminescence quantification of iodixanol-purified CMV.Luciferase.SVPA packaged WT or chimeric capsids injected in WT C57BL/6 mice 7 days post-injection. n = 5 mice per group; ROI for quantification is the whole mouse.

Figure 4

Loss of AAV Entry Factors Causes an Entry Defect Upstream of Nuclear Import (A) qPCR binding assay for cell-bound viral genomes in Huh7 WT, AAVR KO, GPR108 KO, or double-KO cells, assessed for the indicated capsid serotype after 1 h of binding on ice. Average of three independent experiments; error bars = SEM. (B) Western blot of cytoplasmic, membrane, or nuclear subcellular fractions from AAV entry/nuclear import assay (tubulin [α-tubulin], AIF [apoptosis-inducing factor 1], or H3 [histone H3]). (C) Percentage of total Anc80 (CMV.eGFP.WPRE) genomes quantified by ddPCR (CMV primer/probe) on subcellular fractions isolated from WT, AAVR KO, GPR108 KO, or AAVR KO GPR108 KO cells 48 h post-transduction at 10 VG/cell in 1 million cells. One representative experiment; error bars = SEM of three technical replicates. (D) Total number of recovered genomes from entry assay represented in (C). (E) Indirect immunofluorescence of FLAG-tagged GPR107 or GPR108 with anti-FLAG M2 primary antibody (green) or anti-TGN46 antibody as a trans-Golgi network marker (red) and DAPI staining of cell nuclei (blue).

Figure 5

GPR108 N- and C-Terminal Domains Are Required for AAV Entry (A) Predicted membrane topology of nonfunctional GPR107, with indicated disulfide bond location, furin cleavage site, alanine-glycine linker, and functional GPR108. (B) Rescue of Anc80 transduction by transient transfection with FLAG-tagged GPR107/GPR108 chimeric proteins or transfected empty vector (EV). Cells transduced with 10,000 VG/cell, CMV.Luciferase.SVPA transgene after hAd5 helper virus co-infection. (C) Corresponding anti-FLAG western blot from cellular membrane fractions demonstrating protein expression in cells transduced in (B). Black arrows point to expected full-length protein; gray arrows point to expected glycosylation states; red arrows point to protein aggregates; and yellow arrows point to expected N-terminal domain cleavage product. AIF (apoptosis-inducing factor 1) was used as a membrane protein loading control. (D) Indirect immunofluorescence of chimeric proteins using anti-FLAG antibody as described in Figure 4E.

Figure 6

GPR108 Usage Is Conserved in Mice (A) Transduction of Anc80, AAV9, AAV9.PHP-B, rh32.33, AAV4, and AAV5 in Huh7 WT or GPR108 KO cells transfected with FLAG-tagged mouse GPR107 or GPR108 (10,000 VG/cell CMV.Luciferase.SVPA transgene plus hAd5 helper virus). (B) Anti-FLAG western blot of cell membrane fraction of human or mouse GPR107- or GPR108-transfected or empty vector (EV)-transfected Huh7 cells. (C) WT and GPR108 KO Hepa cells transduced with AAV9, AAV9.PHP-B, rh32.33, AAV4, and AAV5 (10,000 VG/cell CMV.Luciferase.SVPA transgene).

Figure 7

GPR108 Is Required for Transduction in Primary Cells and In Vivo (A) Mouse embryonic fibroblast (MEF) cells derived from WT or GPR108 KO mice transduced with the indicated serotype. The same transduction protocol was used as described in Figure 2B. (B) In vivo bioluminescence of WT or GPR108 KO mice injected retro-orbitally with 10¹¹ VG/mouse of CMV.Luciferase.SVPA packaged AAV8, rh32.33, or AAV5 at 14 days post-injection. (C) Quantification of bioluminescence at 7, 14, 28, or 56 days post-injection from mice in (B). n = 3 mice per group; uninjected is maximum observed luminescence from uninjected mice across all time points. Error bars represent SEM.

Figure 8

Summary of Capsid Variant Entry Factor Requirements and Model (A) Three different entry pathway classes exist in which capsids require both AAVR and GPR108 (red), AAVR only (blue), or GPR108 only (green). (B) Green capsids use an as-of-yet unidentified receptor or receptor complex in place of AAVR, dictated by the VP3 outer surface of intact capsids, while blue and red capsids both require AAVR (purple). (C) Blue capsids use an as-of-yet unidentified entry factor in place of GPR108, dictated by the internalized VP1-unique N terminus of capsid, while red and green capsids require GPR108 (brown).