AAV2 Virus production were prepared as follows. 293 T cells (European Collection of Cell Cultures 293 T Number: 12022001) were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, supplemented with 2 mM L-glutamine, and penicillin–streptomycin. Polyethylenimine (PEI) transfections of AAV2 plasmid and adenovirus helper plasmid (1:1 ratio, total 350 ng/cm2) were performed on 293 T cells in T25 flasks (60,000 cells/cm2). The cell density reached 70–90% confluence at the time of transfection. The PEIpro (Polyplus Transfection, ref# 115-100)/DNA weight ratio was maintained at 1:1 in serum-free DMEM medium. Virus was harvested 24 h and 72 h after transfection. For viral genome titer determination and AAV2 capsid ELISA samples, virus was harvested using Triton-X-100 buffer (0.5% Triton-X-100 and 2 mM MgCl2 in 1 × PBS) and Denarase (50 U/ml). Lysis buffer was added to the media and cells were incubated for 2 h at 37 ˚C before cell lysate was collected. Crude lysates were used in the experiments without further purification.

With a limited coding capacity of 4.7 kb, adeno-associated virus (AAV) genome has evolved over-lapping genes to maximise the usage of its genome. An example is the recently found ORF in the cap gene, encoding membrane-associated accessory protein (MAAP), located in the same genomic region as the VP1/2 unique domain, but in a different reading frame. This 13 KDa protein, unique to the dependovirus genus, is not homologous to any known protein. Our studies confirm that MAAP translation initiates from the first CTG codon found in the VP1 ORF2. We have further observed MAAP localised in the plasma membrane, in the membranous structures in close proximity to the nucleus and to the nuclear envelope by co-transfecting with plasmids encoding the wild-type AAV (wt-AAV) genome and adenovirus (Ad) helper genes. While keeping VP1/2 protein sequence identical, both inactivation and truncation of MAAP translation affected the emergence and intracellular distribution of the AAV capsid proteins. We have demonstrated that MAAP facilitates AAV replication and has a role in controlling Ad infection. Additionally, we were able to improve virus production and capsid integrity through a C-terminal truncation of MAAP while other modifications led to increased packaging of contaminating, non-viral DNA. Our results show that MAAP plays a significant role in AAV infection, with profound implications for the production of therapeutic AAV vectors.