Adeno-associated virus (AAV) is a non-pathogenic parvovirus. First reported in 1965 as a contaminant of adenovirus, it has since been characterized as naturally replication deficient, requiring helper viruses such as adenovirus for propagation.
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 ( 7) , all transcripts of AAV2 contain a single intron. Unspliced RNAs encode Rep78 and Rep52, while Rep68 and Rep40 are encoded by spliced messages.
ITR sequences comprise 145 nucleotides each. 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.
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.
In the recent years, several novel AAV serotypes, including AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and over 100 AAV variants have been isolated from Ad stocks, or from human and nonhuman primate tissues. These serotypes differ in their tropism, or the types of cells they infect, making AAV a very useful system for preferentially transducing specific cell types. The chart below gives a summary of the tropism of AAV serotypes, indicating the optimal serotype(s) for transduction of a given organ.
* The genome sequence is not complete.
# AAV3B and AAV3 has only six amino acids difference in sequence.
Serology of AAVs is an important functional characteristic for cell specific transduction efficiency. The cellular entry of AAV vectors is often initiated by interaction of the capsid with cell surface glycosaminoglycan receptors. Subsequent secondary interactions of the viral capsid with coreceptors appear to dictate the intracellular trafficking pathway and biological fate of the virus.
The primary attachment receptor for AAV2 is heparan sulfate proteoglycan (HSPG). The coreceptors for AAV2 include 37/67 kd laminin receptor (LamR), fibroblast growth factor receptor-1(FGFR-1), hepatocyte growth factor receptor (HGFR), ανβ5 integrin, α5β1 integrin coreceptors, and CD9. Receptors and coreceptors have been identified for other serotypes as well, such as 2,3-O-linked sialic acid, 2,3-N-linked sialic acids, 2,6-N-linked sialic acids, N-linked galactose, epidermal growth factor receptor(EGFR), platelet-derived growth factor receptor (PDGFR). The high transduction efficiency of AAV3 in human liver cancer cells may be due to its specific coreceptor. Besides, HSPG, LamR, and FGFR1, it uses hepatocyte growth factor receptor (HGFR) as a cellular coreceptor for viral entry. The rapidly increasing knowledge about AAV receptors/coreceptors should provide important insight when determining optimum use of these reagents for vectors in human gene transfer studies. Re-expression or over-expression of receptors/coreceptors may be a choice to enhance transduction efficiency of AAV in the cells with lower level or lack of receptors/coreceptors.
More recently, a universal receptor, KIAA0319 or AAVR, has been identified that mediates rapid endocytosis after cell binding and attachment for all AAV serotypes.
The first step in viral tropism is the attachment to the target cell. AAV is recognized by glycosylated cell surface receptors of the host. For the best-studied serotype (AAV2), heparan sulfate was first identified as receptor. In addition to heparan sulfate proteoglycans, fibroblast growth factor receptor 1 (FGFR1) has been shown to be bound by AAV2 and and, in the context of recombinant viruses, its presence has been associated with enhanced transduction.
Subsequent to receptor binding AAV is thought to enter via clathrin-coated vescicles. AAV then traffics through the cytosol mediated by the cytoskeletal network. Owing to the somewhat low pH environment of the endosome, the VP1/VP2 region undergoes a conformational change. Following endosomal escape, AAV is transported into the nucleus and uncoated. AAV can also undergo proteolysis by the proteasome.
AAV replication initiates in the nucleus. However, it remains somewhat controversial how, and where, the DNA is released from the capsids. Nevertheless, it has been recognized that one of the rate-limiting steps in viral infection and vector transduction is the uncoating of the extraordinary stable AAV virion.
There are currently two classes of recombinant AAVs (rAAVs) in use: single-stranded AAV (ssAAV) and self-complementary AAV (scAAV). ssAAV are packaged as either sense (plus-stranded) or anti-sense (minus-stranded) genomes. These single-stranded forms are still transcriptionally inert when they reach the nucleus and must be converted to double-stranded DNA as a prerequisite of transcription. This conversion can be achieved by second strand synthesis via host cell DNA polymerases or by strand annealing of the plus and minus strands that may coexist in the nucleus.
Single-stranded virion DNA enters the host-cell nucleus and the 3′-inverted terminal repeat (ITR) acts as a primer for host DNA polymerase. (1) The 3′-ITR primer is elongated, displacing and replicating the ITR at the 5′ end. (2) The duplex ITR is re-folded into a double-hairpin configuration by host or viral DNA helicase, forming a new primer for DNA synthesis. (3) While the 3′-ITR is elongated and the complementary strand displaced, AAV Rep protein recognizes and binds to the ITR at the downstream end. (4) To generate complete monomeric genomes, Rep endonuclease nicks the terminal resolution site (trs) of the downstream ITR, initiating a second DNA replication complex, to copy the ITR before being reached by the complex initiated at the other end. (5m) The original replication complex displaces the daughter strand, including the newly synthesized ITR, and completes replication to the end of the genome, recreating the template for isomerization in step 3. (6m) The displaced single-stranded genome is packaged into the AAV capsid. (7m) Dimeric genomes are generated when Rep fails to nick the trs before being reached by the replication complex from the other end. (5d) Replication continues through the ITR, and the displaced strand, to generate a dimeric dsDNA template (6d) which can initiate a new round of DNA synthesis either by isomerizing the open end (as in step 4) or by terminal resolution of the hairpin end. (7d) Isomerization allows priming of DNA synthesis from the resolved end (8d), and replication of the dimeric template displaces a single-strand dimeric inverted repeat genome (9d), which can then be packaged into the AAV virion (10 d). dsDNA; double-stranded DNA; ssDNA, single-stranded DNA.