Abstract
Adeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection. Some AAV serotypes are secreted in a pre-lytic manner as free or extracellular vesicle (EV)-associated particles, although mechanisms underlying such are unknown. Here, we discover that the membrane-associated accessory protein (MAAP), expressed from a frameshifted open reading frame in the AAV cap gene, is a novel viral egress factor. MAAP contains a highly conserved, cationic amphipathic domain critical for AAV secretion. Wild type or recombinant AAV with a mutated MAAP start site (MAAPΔ) show markedly attenuated secretion and correspondingly, increased intracellular retention. Trans-complementation with MAAP restored secretion of multiple AAV/MAAPΔ serotypes. Further, multiple processing and analytical methods corroborate that one plausible mechanism by which MAAP promotes viral egress is through AAV/EV association. In addition to characterizing a novel viral egress factor, we highlight a prospective engineering platform to modulate secretion of AAV vectors or other EV-associated cargo.
Highlights
Adeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection
Amino acid sequence alignment of membrane-associated accessory protein (MAAP) derived from different AAV serotypes revealed conserved N- and C-terminal regions containing hydrophobic and basic amino acid residues interconnected by a threonine/serine (T/S) rich region (Fig. 1B)
Our lab and others previously dissected the functional attributes of the Assembly-Activating Protein (AAP), which is encoded from an alternative open reading frames (ORFs) in the AAV cap gene[4,5,30]
Summary
Adeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection. We discover that the membrane-associated accessory protein (MAAP), expressed from a frameshifted open reading frame in the AAV cap gene, is a novel viral egress factor. Multiple studies have demonstrated that a significant fraction of recombinant AAV associate with extracellular vesicles (EVs) and are released as free particles into the supernatant fraction of the cell culture media[13]. It is well known that cells shed a variety of membrane-bound vesicles varying in size from 20 nm to 1 μm in diameter, which have been termed exosomes, microparticles or microvesicles (collectively referred to here as extracellular vesicles or EVs) Such EVs can package different macromolecules including proteins, nucleic acids and viruses, thereby making them an attractive therapeutic platform[14]. Our findings highlight a viral egress mechanism evolved by AAV as well as highlight the potential to exploit this novel protein for loading therapeutic cargo including AAV vectors into EVs and AAV vector production in general
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