Adeno-associated viruses(AAVs) are small, non-enveloped viruses that belong to the family Parvoviridae[1,2]. They are widely used as gene delivery vectors for various research and therapeutic applications due to their non-pathogenic nature, ability to transduce a wide range of cell types, and capacity to mediate long-term gene expression in target cells.
AAV Genome Structure
The most extensively studied serotype of AAV is type 2 (AAV2), which serves as a prototype for the AAV family. The AAV genome is a molecule of single-stranded DNA of approximately 4.7 kb. The plus and minus strands are packaged with equal efficiency into separate preformed particles. At either end of the genome are inverted terminal repeats (ITRs) that form T-shaped, base-paired hairpin structures, and contain cis-elements required for replication and packaging.
Two genes (rep and cap) encode for four nonstructural proteins required for replication (Rep78, Rep68, Rep52, and Rep40) and three structural proteins that make up the capsid (VP1, VP2, and VP3). The Cap ORF may also express AAP and X-gene, which may help genome replication.
There are three viral promoters that are identified by their relative map position within the viral genome: p5, p19, and p40. Although the transcription profiles vary for different AAV serotypes , all transcripts of AAV2 contain a single intron. Unspliced RNAs encode Rep78 and Rep52, while Rep68 and Rep40 are encoded by spliced messages.
Inverted Terminal Repeat (ITR)
ITR sequences comprise 145 nucleotides each. They are located at both ends of the AAV genome, flanking the coding region. The ITRs is required for both integration of the AAV DNA into the host cell genome and rescue from it as well as for efficient AAV DNA replication and encapsidation. With regard to AAV vectors, ITRs is the only cis element required to AAV packaging.
AAV Virion Structure
The AAV virion is an icosahedral nonenveloped particle with an encapsidated single-stranded DNA genome. The AAV2 virion is roughly 25 nm in diameter and is composed of 60 copies of the three capsid proteins VP1, VP2, and VP3 in a 1:1:10 ratio. The VP1 and VP2 proteins share the VP3 sequence and have additional residues at their N-termini. The N-terminus of VP1 has a conserved phospholipase A2 sequence that has been implicated in virus escape from endosomes and is crucial for infectivity. The VP2 protein is not essential for assembly or infection.
- VP1: The largest capsid protein, VP1 contributes to the structural stability and assembly of the capsid.
- VP2: VP2 is an intermediate-sized protein that plays a role in forming the capsid shell and stabilizing the virion.
- VP3: VP3 is the smallest capsid protein and forms the majority of the capsid structure. It contributes to the outer surface of the virion.
- Tropism Determinants: The specific serotype of AAV determines its tropism, or the types of cells it can infect. Variations in capsid protein sequences among different serotypes contribute to differences in receptor binding and cellular entry.
- Receptor Binding: AAVs attach to target cells by interacting with specific cell surface receptors. Different AAV serotypes may have different receptor preferences, influencing their ability to transduce various cell types.
AAV Life Circle
AAVs are helper-dependent members of the Dependovirus genus of the parvoviruses that have evolved to replicate under a diverse set of conditions. The AAVs are small viruses with limited coding capacity, and they are therefore highly reliant on the cellular environment and machinery. Productive AAV infection requires helper functions that can be supplied by co-infecting helper viruses. Helper viruses shown to promote AAV replication include Adenovirus (Ad) and herpes simplex virus (HSV) and vaccinia virus (VV). Specific adenovirus genes such as E1a, E1b55k, E2a, E4orf6 and associated viral protein have been identified to provide known helper functions for AAV. The helper induces changes to the cellular environment that can serve to facilitate AAV gene expression and replication. In the absence of helper virus, AAV can establish a latent infection in many cell types, from which it can be rescued by subsequent helper virus infection. In the case of AAV2, latency is associated with targeted integration at a specific locus on human chromosome 19 and this requires the viral Rep protein.
