2 AAV biology
The AAV belongs to the parvoviridae family: a family of single-stranded
DNA viruses that have evolved over the years, expanding a spectrum of
hosts[20]. The viruses of this family share some common structural
and functional features such as conserved phospholipase
A2 (PLA2)-like enzymatic domain and
identical replication components and pathways [20], [21]. Based
on the host specificity, the parvoviridae family is divided into two
subfamilies: Parvovirinae and Densovirinae, which infect mammalian and
invertebrate hosts, respectively (Figure 1A). The Parvovirinae subfamily
is further divided into eight genera, including Dependovirus, into which
AAV is classified. The insect cells are a natural host of Densovirinae
viruses, and from a taxonomical standpoint, they should provide an
adequate cellular environment for replication of viruses from closely
related subfamily Parvovirinae, including AAV. This relation was first
recognized and later found to be applicable by Urabe et al., where the
group demonstrated the first IC-BEVS based rAAV production platform
[22].
AAV virus particle comprises a vector genome: the functional element of
the virus, and the capsid, a metastable entity that preserves it. The
vector genome is a linear single-stranded DNA with a size of about
~4.7kb and a major coding region consisting of the
sequences for non-structural replicase proteins (Rep) and structural
capsid proteins (Cap or VP). This coding region is flanked by an
inverted terminal repeat sequences (ITR). The ITR is a segment of 145bp
with a T-shaped hairpin-like structure formed via complementary base
pairing of palindromic sequence of 125 bp. The remaining 20 bases stay
unpaired. The ITR region also encompasses the origin of replication
formed by the terminal resolution site and rep-binding element (RBE).
The free 3‘ hydroxyl region acts as an initiation sequence for DNA
replication.
AAV contains four non-structural Rep proteins (Rep 78, Rep 68, Rep 52,
and Rep40). Rep78 and Rep52 are expressed under the transcriptional
control of p5 and p19 promoters, respectively. The larger Rep78 and its
spliced variant Rep68 are crucial for- and participate in vector genome
integration, excision, rescue, and replication at various stages of AAV
life cycle[23]–[25]. The smaller Rep proteins Rep52 and its
spliced variant Rep40 play a critical role in the packaging of vector
DNA in preformed capsids and regulation of replication by repressing p5
in the absence of a helper virus[26], [27]. The three structural
proteins (VP1, VP2, and VP3) expressed from the right open reading frame
(ORF) under p40 promoter form an icosahedron capsid that carries the
viral genome and delivers it to the host cell traversing through post
cell surface attachment. A representative genomic map of wtAAV2 [28]
and its transcriptional profile are shown in Figure 1B.