44. Unique Properties of AAV5 Assembly-Activating Protein (AAP) and Its Role in Capsid Assembly

Adeno-associated virus (AAV) has now been used successfully as a vector for in vivo gene therapy and continues to hold great promise for future AAV-based therapies, in part because if its numerous, naturally occurring variants including the widely appreciated AAV serotypes 1 through 9. Each variant exhibits distinct biological properties; and therefore, their mechanistic basis should be thoroughly understood in each variant for successful vector development and gene therapy. In this respect, assembly-activating protein (AAP), a non-structural AAV protein that was discovered in 2010 and remains not well characterized, has gained increasing attention for its pivotal role in the AAV life cycle, particularly in promoting the assembly of VP proteins into a full capsid. AAV2 AAP (AAP2) has a joint nuclear-nucleolar localization signals (NLS-NoLS) near its C terminus and the nucleolar localization of AAP2 mediated by the NLS-NoLS is a prerequisite for effective capsid assembly, which takes place predominantly in the nucleolus and nucleostemin-positive nuclear bodies. Here we report previously unknown biological properties and functions of AAV5 AAP (AAP5) that are distinct from those of AAP2. To examine the role of AAP5 in AAV5 capsid assembly, we transiently transfected HeLa cells with a plasmid expressing a CMV-driven AAV5 VP3 and a plasmid expressing a FLAG-tagged, codon-optimized AAP5. The cells were fixed and immunostained for FLAG, VP3, and assembled AAV5 capsids using anti-FLAG, B1, and ADK5a monoclonal antibodies, respectively. Unlike AAP2, AAP5 and AAV5 capsids were nucleolar excluded, demonstrating that the AAP5 does not have an NoLS and the nucleolus is not the site for AAV5 capsid assembly. Surprisingly, regardless of the presence of AAP5, ADK5a-positive signals indicative of assembled AAV5 capsids could be easily detected at 48 hours post transfection. To support this observation, vector genome-packaged AAV5 particles could be produced at a high titer using an AAV5 helper plasmid that expresses AAV2 Rep and AAV5 VP1, 2 and 3 proteins but does not express fully functional AAP5 due to introduction of a premature stop codon, S133X, resulting in truncation of the C terminus containing the functionally important basic-rich region. We also find that, in a cross-complementation study using AAV2VP3, AAP2 and AAP5, the localization of capsids is determined by AAV serotype, not by AAP localization, indicating that host protein-capsid interactions both determine the site of assembly and are different for each serotype. Indeed, we have observed a strong colocalization of AAV2 capsids with nucleostemin that is absent with AAV5 capsids. These observations, together with the fact that AAV5 is phylogenetically the most divergent serotype, indicate that the role of AAP and host-proteins in the AAV5 life cycle may be different from that for AAV2 and potentially other serotypes. Our study provides new insights into the functional roles of AAP derived from AAV5 and shows that intracellular host-virus interactions are not necessarily the same for all AAV serotypes.