With a limited coding capacity of 4.7 kb, adeno-associated virus (AAV) genome has evolved overlapping 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 diferent reading frame. This 13 KDa protein, unique to the dependovirus genus, is not homologous to any known protein. Our studies confrm that MAAP translation initiates from the frst 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 afected 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 modifcations led to increased packaging of contaminating, non-viral DNA. Our results show that MAAP plays a signifcant role in AAV infection, with profound implications for the production of therapeutic AAV vectors.