This study has revealed that AAV4, 5, and 11 VP3 monomers can assemble without AAP. Recently, through approaches that include the use of quantitative dot blot assays, we investigated the ability of AAV1 to 12 VP3 monomers to assemble into capsids in the absence of AAP expression and the ability of AAP1 to 12 to promote assembly of VP3 monomers from heterologous serotypes. Subsequent studies on AAV serotypes 1, 8, and 9 corroborated the critical role of AAPs in capsid assembly, as VP3 monomer proteins of AAV1, 8, and 9 were unable to form a fully assembled capsid in the absence of co-expression of AAP 2. The inability of the AAV2 capsids to assemble in the absence of AAP expression has been independently confirmed by multiple groups, including ours 1, 2, 3, 4, 5. Functional studies of this novel protein were initially focused on the AAP from the prototype AAV2 (AAP2), which has established the essential role of AAP2 in targeting unassembled VP proteins to the nucleolus for their accumulation and formation into fully assembled capsids 1, 3, 4. The ORFs for AAP have been identified bioinformatically in the genomes of all parvoviruses within the genus Dependoparvovirus, but not within the genomes of viruses of different genera of the parvovirus family 1, 2. This novel protein has been named assembly-activating protein (AAP) after the role it plays in promoting capsid assembly 1. and found to play a critical role in the assembly of AAV2 capsid VP monomer proteins into a viral capsid 1. In 2010, a previously unidentified nonstructural protein encoded by a +1 frame-shifted ORF within the AAV2 cap gene was discovered by Sonntag et al. The AAV genome contains open-reading frames (ORFs) for the rep and cap genes. In addition, we show that the choice of nuclease significantly affects the readout of the dot blot assay, and thus, choosing an optimal enzyme is critical for successful assessment of AAV titers.Īdeno-associated virus (AAV) is a small, non-enveloped, single-stranded DNA virus with a genome of approximately 4.7 kilobases (kb). The assay also showed that, unlike many of the common serotype AAPs that promote heterologous capsid assembly by cross-complementation, Snake AAP does not promote assembly of AAV9 capsids. The assay revealed that Snake AAV capsid assembly requires Snake AAP and cannot be promoted by AAPs from AAV5 and AAV9. To demonstrate the utility of this dot blot assay, we set out to characterize capsid assembly and AAP dependency of Snake AAV, a previously uncharacterized reptile AAV, as well as AAV5 and AAV9, which have previously been shown to be AAP-independent and AAP-dependent serotypes, respectively. This manuscript details a straightforward dot blot assay for AAV quantitation and its application to assess AAP dependency and serotype specificity in capsid assembly. This unexpected heterogeneity in the biological properties and functional roles of AAPs among different AAV serotypes underscores the importance of studies on AAPs derived from diverse serotypes. Furthermore, we demonstrated that AAPs and assembled capsids of different serotypes localize to different subcellular compartments. Although many AAV serotypes require AAP for assembly, we have recently reported that AAV4, 5, and 11 are exceptions to this rule. In the latter respect, the recent discovery of a non-structural AAV protein, termed assembly-activating protein (AAP), has shed new light on the processes involved in assembly of the viral capsid VP proteins into a capsid. While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus biology.
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