43. Distinct Intracellular Localization of AAPs Derived from AAV Serotypes 1-11 and Their Ability To Cross-Complement in Heterologous Capsid Assembly
Authors: John Powers, Lauriel Earley, Kei Adachi, Hiroyuki Nakai
Journal: Molecular Therapy (2015)

AAV expresses a unique non-structural viral protein that exhibits no homology to any known protein, called assembly-activating protein (AAP). It has been shown that AAP derived from AAV serotype 2 (AAP2) is a nucleolar localizing protein that transports VP proteins to the nucleolus and promotes capsid assembly. Although understanding the AAV capsid assembly process is key to developing novel AAV vectors for improved and targeted gene delivery, little is known about the roles and functions of AAPs, in particular AAPs derived from alternative serotypes. Here, by analyzing AAPs derived from 11 serotypes (AAP1 to 11), we show that strong nucleolar enrichment, the hallmark of the prototype AAP2, is not the case in other serotype AAPs, and AAP5 exhibits an unambiguous nucleolar exclusion pattern. We also show that, although all AAPs can promote assembly of many heterologous VP3 proteins into a capsid, particular VP3-AAP combinations do not effectively cross-complement. To elucidate intracellular localization of AAP1 to 11 and their functional roles, we constructed plasmids expressing VP3s or FLAG-tagged codon-optimized AAPs derived from AAV1 to 11 under the control of the CMV promoter. Confocal immunofluorescence (IF) microscopy revealed that all AAPs translocate exclusively in the nucleus with strong nucleolar enrichment (e.g., AAP2), nucleolar and nucleoplasmic retention (e.g., AAP9), or clear nucleolar exclusion (e.g., AAP5). To investigate the cross-complementation abilities of AAP1 to 11 with AAV1 to 11 VP3s (11 × 11=121 combinations) in a biologically triplicated experiment, we utilized our lab's novel AAV Barcode-Seq approach. In this approach, HEK 293 cells were transfected with a mixture of VP3, AAP, AAV2-Rep and adenovirus helper plasmids together with a unique barcode plasmid for each combination. An empty plasmid backbone was used as a no AAP control. Five days after transfection, cells and culture media were harvested and pooled for each replicate, viral DNA was extracted, and barcodes were PCR amplified and sequenced by multiplexed Illumina sequencing. This approach allowed for titering AAV vector production in ~400 different cell culture wells in a simple manner. The data demonstrated that, although the AAPs from the 11 serotypes are capable of assembling and packaging most heterologous serotype VP3 capsids, some combinations showed a large reduction in the ability to assemble capsids compared to the native AAP + VP3 pairings. Some of the incompatible combinations, including AAP11 + AAV8 VP3, were confirmed by IF. We also applied the same Illumina-based approach to investigate whether overexpression of heterologous AAPs can enhance production of infectious AAV serotype vector particles, and identified combinations showing >1.5 × enhancement, although the majority of combinations did not exhibit such an enhancing effect. Thus, this study provides the first comprehensive dataset on the biological properties of AAPs derived from 11 serotypes and significantly helps understand the functional roles of AAP in AAV vector production.