Adeno-associated virus (AAV) is a Dependoparvovirus with a ssDNA ~4.7 kb genome in a ~25 nm icosahedral capsid structure. AAV genomes encode nine known functional proteins from two open reading frames between two inverted terminal repeats (ITRs). In recombinant AAV vectors for gene therapy use, the AAV genome is replaced with a transgene of interest flanked by ITRs and subsequently packaged within an AAV capsid made up of three viral structural proteins (VP1, VP2, and VP3) in an approximate 1:1:10 ratio, respectively. The AAV capsid, particularly VP3, has traditionally been ascribed to capsid-cellular receptor binding. However, AAV9 VP1/VP2 exhibits a capsid-promoter interaction that can alter neuronal cellular tropism in the rat and non-human primate central nervous system. This capsid-promoter interaction is altered by AAV9EU (AAV9 with six glutamates inserted at aa139) which exhibits a significant reduction in nuclear transgene DNA, a decrease in neuronal transduction, and a reduction in vivo relative transgene mRNA levels. AAV9EU has six amino acid insertions in VP1, VP2, and MAAP (membrane-associated accessory protein), but no combination of VP with MAAP recapitulated the AAV9EU in vivo phenotype. Surprisingly, AAV9 produced in the absence of MAAP9 exhibits an increase in relative transgene levels. While co-infusing two AAV9 vectors, differing only in transgene and MAAP9 presence during production, exhibit a significantly increased in vivo transgene fluorescence intensity by fivefold of both transgenes. Together, an MAAP9-related activity acts both in cis and in trans to increase AAV9 transgene mRNA levels and AAV9 transgene protein levels in vivo. IMPORTANCE: Recombinant adeno-associated viruses (AAVs) are used extensively in clinical gene therapy for treating a range of tissues and pathologies in humans. In particular, AAV9 occupies a prominent position in central nervous system (CNS) gene therapy given its central role in ongoing clinical trials and an FDA-approved therapeutic. Despite its widespread use, recent studies have identified unique roles for the AAV capsid in in vivo transgene expression; for example, interior-facing capsid residues of AAV VP1 and VP2 modulate cellular transgene expression in vivo. The following experiments identified that the AAV9 MAAP protein exerts a significant influence on in vivo transgene expression. This finding could further explain how AAV can remain latent after infection in vivo. Together, these studies provide novel functional insights that highlight the importance of further understanding basic AAV biology.