Adeno-Associated Virus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 26

herpes simplex virus to trigger its replication and to generate progeny
virions [ 13 , 14 ]. In the absence of helper virus, AAV-2 can estab-
lish latency by integrating its genome site specifically into a locus
on human chromosome 19 [ 15 - 18 ].
The 4.7-kb genome of AAV-2 contains two open reading
frames (ORF), rep and cap , that are flanked by two identical
inverted terminal repeats (ITRs) [ 19 ]. The 145-bp ITR sequence
forms a unique T-shaped hairpin structure [ 20 ], which is the only
cis -acting sequence required for AAV DNA replication, packaging,
site-specific integration and rescue of the AAV genome [ 21 ].
The rep gene encodes four overlapping regulatory proteins, which
are involved in every step of the viral life cycle [ 22 ]. The two large
Rep proteins, Rep78 and Rep68, are required for AAV DNA rep-
lication, site-specific integration, and regulation of AAV gene
expression, whereas the two small Rep proteins, Rep52 and Rep40,
are involved in the accumulation and encapsidation of single-
stranded DNA genome [ 23 ]. The cap gene encodes three overlap-
ping structural proteins (VP1, VP2, and VP3) that form the
icosahedral capsid and an assembly activating protein (AAP)
required for capsid assembly [ 24 ]. The AAV capsid is composed of
60 subunits of VP1, VP2, and VP3 in an approximate stoichiometric
ratio of 1:1:10.
The first step in AAV cellular entry is the binding of the viral
capsid to cell surface receptors. To date, a number of attachment
sites have been identified. The primary attachment receptor for
AAV-2 and AAV-3 is the ubiquitous heparan sulfate proteoglycan
(HSPG) [ 25 ], while AAV-1, -4, -5, and -6 use sialic acid with
different linkage forms [ 26 - 28 ]. Efficient AAV cell binding and
infection also require the presence of coreceptors, i.e., integrin
αVβ5, FGFR 1, and HGFR for AAV2 [ 29 - 31 ], PDGFR for AAV-5
[ 32 ], and laminin receptor for AAV-2, 3, 8, and 9 [ 33 ]. Subsequent to
receptor binding, AAV is rapidly internalized via clathrin-mediated
endocytosis [ 34 ]. The virus is finally translocated to the nucleus
where the viral DNA is released from the capsids.
Once inside the nucleus, the AAV single-stranded (ss) DNA
genome must be converted into a double-stranded (ds) template
to ensure rep gene expression. The AAV ITR can fold into a
T-shaped secondary structure that serves as a primer for full-length
extension. This step is performed by a cellular DNA polymerase
and occurs in the absence of Rep and helper functions [ 35 ]. Once
the AAV template has been replicated, a duplex replication inter-
mediate is generated in which one end is covalently closed.
Resolution of the covalently closed ITR is achieved by the large
Rep proteins that specifically bind to a sequence within the ITR
[ 36 , 37 ] and introduce a single-strand nick at the adjacent terminal
resolution site [ 38 ]. The nicking reaction generates a 3′OH end
that serves as a primer to restore the ITR. Upon viral capsids
assembly, the newly replicated plus and minus strands are packaged
with equal frequency into preassembled capsids [ 39 ] and progeny