Abstract
Adeno associated virus serotype 2 (AAV2), a non-pathogenic parvovirus reliant on helper viruses, is studied extensively as a potential gene delivery vector. A +1 open reading frame within the cap gene encodes a nonstructural protein of 204 amino acids termed assembly activating protein (AAP), which has been attributed a critical role in transporting the viral capsid protein VP3 into the nucleolus for assembly. However, AAP remains poorly characterized because of its relatively late discovery and lack of commercial antibodies. In the absence of other virus proteins, AAP localizes in the nucleolus due to five redundant nuclear and nucleolar localization signals. Additionally, AAP, a predicted intrinsically disordered protein, forms spontaneous dose dependent nuclear globular condensates, a trait of liquid-liquid phase separated inclusions. Consistent with LLPS biophysical properties, the AAP condensates recovered rapidly from photobleaching and are sensitive to aliphatic diol treatment; moreover, AAP self-oligomerizes. We produced an AAP specific antibody to analyze the role of this protein during productive AAV2 replication. In this context, we observed that AAP also forms nuclear globular condensates with LLPS biophysical properties in cells co-infected with AAV2 and either herpes simplex virus type 1 (HSV 1) or adenovirus type 5 (AdV 5) as the helper viruses. The screening of AAP deletion mutants revealed that the N terminal region (amino acids 1 to 61) is necessary for condensate formation and self oligomerization. Interestingly, this AAP region contains a predicted alpha helix spanning amino acids 16 to 45. The substitution in this region of the hydrophobic residues by alanines drastically impaired AAP LLPS biophysical properties and its ability to trigger AAV2 capsid assembly. Identifying the amino acids involved in assembly and LLPS may improve AAV vector production.
Competing Interest Statement
The authors have declared no competing interest.
