AAV5-CAG-Luc

Adeno-associated virus (AAV) is a member of the Parvoviridae family that primarily infects mammalian cells and is reportedly non-pathogenic to humans. It was first reported in 1965 as an adenoviral contaminant and has since been described as naturally replication-defective, requiring a helper virus such as adenovirus for propagation. The linear, single-stranded DNA genome of AAV (~4.7 kb) encodes two genes, rep and cap, flanked by inverted terminal repeats (ITRs), which are required for packaging of the viral genome within the capsid. The ITRs act as primers for second-strand synthesis and are the only cis-elements in the genome required for viral production. For recombinant AAV (rAAV) vector production, the remainder of the viral genome can be removed, provided in trans on a separate plasmid, and replaced with the desired transgene.

The rep gene encodes four overlapping nonstructural proteins required for replication, integration, and packaging. Rep78 and Rep68 bind to the ITRs and exhibit helicase and endonuclease activities necessary for AAV genome replication. Rep52 and Rep40 exhibit 3’ to 5’ helicase activity and package the viral genome into capsids during virus production. The cap gene encodes three structural proteins, VP1, VP2, and VP3, which self-assemble into a 60-mer icosahedral capsid in a ratio of approximately 1:1:10. These three proteins are transcribed from the same open reading frame and share a C-terminal domain but have different N-termini due to alternative start codons and alternative splicing. cap also encodes for a non-structural protein, assembly-activating protein (AAP), in an alternate open reading frame from the VPs initially shown to be required for AAV2 capsid assembly. Capsid assembly dependence on AAP is serotype-specific, as AAV4, -5, and -11 do not require AAP to assemble.