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Updated: Oct 14, 2025

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins
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A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

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Functional roles of the membrane-associated AAV protein MAAP

Lionel Galibert1, Amira Hyvönen2,3, Reetta A E Eriksson2,4

  • 1Kuopio Center for Gene and Cell Therapy, Kuopio, Finland. lionel.galibert@kct.fi.

Scientific Reports
|November 5, 2021
PubMed

Insights

Adeno-associated virus (AAV) uses overlapping genes, like the membrane-associated accessory protein (MAAP), to maximize its genome. MAAP is crucial for AAV replication, capsid assembly, and controlling adenovirus infections, impacting therapeutic vector production.

Area of Science:

  • * Virology
  • * Molecular Biology
  • * Gene Expression

Background:

  • * Adeno-associated virus (AAV) has a limited coding capacity (4.7 kb), necessitating genome optimization through overlapping genes.
  • * A novel open reading frame (ORF) within the AAV cap gene encodes the membrane-associated accessory protein (MAAP), distinct from VP1/2 but in the same region.
  • * MAAP is a 13 kDa protein unique to the dependovirus genus, with no known homologies.

Purpose of the Study:

  • * To confirm the translation initiation site and cellular localization of MAAP.
  • * To investigate the role of MAAP in AAV replication, capsid protein dynamics, and adenovirus (Ad) infection control.
  • * To evaluate the impact of MAAP modifications on AAV production and vector quality.

Main Methods:

  • * Co-transfection of plasmids encoding wild-type AAV (wt-AAV) and adenovirus (Ad) helper genes.
  • * Site-directed mutagenesis to inactivate or truncate MAAP translation.
  • * Confocal microscopy to determine MAAP localization.
  • * Analysis of AAV capsid protein expression, intracellular distribution, and virus production.

Main Results:

  • * MAAP translation initiates from the first CTG codon in the VP1 ORF2.
  • * MAAP localizes to the plasma membrane and perinuclear structures.
  • * MAAP inactivation or truncation affects AAV capsid protein emergence and distribution.
  • * MAAP facilitates AAV replication and influences Ad infection.
  • * C-terminal MAAP truncation improved virus production and capsid integrity, while other modifications increased non-viral DNA packaging.

Conclusions:

  • * MAAP is a significant factor in AAV infection biology.
  • * MAAP plays a critical role in regulating AAV replication and capsid assembly.
  • * MAAP modulation offers potential for enhancing therapeutic AAV vector production and quality.

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