85. The Joint Nuclear-Nucleolar Signal in the AAV2 Assembly-Activating Protein (AAP) Contains a Secondary Signal Important for Yielding High AAV Titer

Abstract

Assembly-activating protein (AAP) is a small protein expressed from an overlapping reading frame in the VP2/VP3 section of the cap gene in the AAV genome and is known to promote translocation of VP proteins to the nucleolus and enable capsid assembly. In the AAP derived from AAV serotype 2 (AAP2), there is a region rich in basic amino acids near the C terminus (position 144-184, or AAP2-144-184) that contains a joint nuclear-nucleolar localization signal (NLS-NoLS). This joint NLS-NoLS is composed of 5 basic amino acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4) and RRIK (AAP2BR5) from the N terminus to the C terminus. Interestingly, there reside at least 3 overlapping NLSs and redundant NoLSs within this signal, indicating that the NLS-NoLS region has a role important for the AAV life cycle other than transporting AAP2 to the correct intracellular location. Here we show that the NLS-NoLS region contains a signal that maintains AAP2 protein expression levels but is not essential for its intracellular localization, and the titer in AAV2 production is proportionate to the amount of correctly localized AAP2 protein. In the study, we transiently transfected HeLa cells with a series of plasmids expressing a CMV promoter-driven FLAG-tagged, codon-optimized AAP2 (the wild type and 30 arginine/lysine-to-alanine mutants) and analyzed the cells by immunofluorescence microscopy. To determine the protein expression levels, we extracted proteins from the wild-type or mutant AAP2 plasmid-transfected HeLa cells, and performed a quantitative western blot analysis in duplicate using FluorChem M system. The titer of assembled AAV2 VP3 capsids in HEK 293 cells transfected with pAAV2-RepVP3 (pAAV-ITR-vector plasmid expressing all the Rep proteins and VP3 but no AAP2), pHelper (adenovirus helper plasmid), and the wild-type or mutant AAP2-expressing plasmid, was quantified by an A20 antibody-based AAV2 capsid ELISA. There were 16 AAP2 mutants that showed nucleolar enrichment similar to the wild type. They were all single AAP2BR mutants but showed various degrees of impairment of capsid assembly. Mutations involving the AAP2BR1 and BR2 were most influential to localization and the protein expression levels strongly correlated to capsid production with a Pearson's correlation coefficient of 0.94. A previous directed evolution study from our lab showed an evolutionary pressure to maintain strong positive charge and hydrophilicity in the AAP2BR1 in AAP2 mutants with a complete NLS-NoLS. Since not all of the clusters of basic-rich amino acids are required for nuclear-nucleolar localization, it is likely that these regions have a secondary function involved in maintaining the stability of AAP2. These results also indicate that, since levels of AAP directly correlate with the amount of virus produced, this protein does not act as a catalyst, but is directly involved in the capsid assembly process without being recycled.