Functional roles of the membrane-associated AAV protein MAAP

Lionel Galibert,Hanna P Lesch,Salla Mattola,Saija Weman,Justin D Albers,Kari J Airenne,Reetta A E Eriksson,Seppo Ylä-Herttuala,Tiina Nieminen,Sanna K Peltola,Maija Vihinen-Ranta,Amira Hyvönen,Sami Salminen,Vesa Aho

doi:10.1038/s41598-021-01220-7

Abstract

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.

Highlights

Adeno-associated virus (AAV) serotype 2 is a dependoparvovirus with a ssDNA genome of 4679 bases
Studies of Hela cells co-infected with wt-Ad and wild-type AAV (wt-AAV) suggest that the well-known phenomenon of AAV subcellular compartmentalisation is the result of the capsid assembly being initiated in the nucleolus while the DNA packaging likely occurs separately, in the n ucleoplasm[18]
Using a quantitative analysis of confocal microscopy data, we further studied the emergence of membrane-associated accessory protein” (MAAP) in wt-AAV2 and recombinant MAAP expression plasmid transfected cells and changes in the amount of MAAP at 24 hpt

Summary

Introduction

Adeno-associated virus (AAV) serotype 2 is a dependoparvovirus with a ssDNA genome of 4679 bases. Space within the AAV genome is so efficiently used that alternative splicing and non-canonical start codons in the cap gene encode three capsid proteins, VP1, VP2 and VP3 (VPs), from the three overlapping reading frames[12,13], as well as the Assembly Activating Protein (AAP) which is transcribed as the result of a frame-shift in the VP2/3 reading frame[14]. Unless MAAP was supplied in trans, MAAP-mutated AAVs were outcompeted by viruses encoding wt-cap Based on these results, Ogden and collaborators proposed that MAAP may be involved in competitive exclusion between different serotype AAV genomes. Ogden and collaborators proposed that MAAP may be involved in competitive exclusion between different serotype AAV genomes This finding is parsimonious with capsid and genome couplings observed in the generation of engineered AAV capsid libraries[20]

Methods

Results

Conclusion