Gene Delivery Using Viral Vectors - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 226

Properties of ITR sequences are as follows:
1. They are required for efficient multiplication of the AAV genome.
2. These sequences have the ability to form a hairpin, which contributes to so-called self-
priming that allows primase-independent synthesis of the second DNA strand.
3. The ITRs sequences require both integration of the AAV DNA into the host cell genome
(the 19th chromosome in humans) and rescue from it.
4. They are also useful for efficient encapsidation of the AAV DNA combined with the gen-
eration of fully assembled, deoxyribonuclease-resistant AAV particles [93-96] .
The rep gene, through the use of two promoters and alternative splicing, encodes our
regulatory proteins, dubbed Rep78, Rep68, Rep52, and Rep40. Capsid proteins assem-
ble into a near-spherical protein shell of 60 subunits, and these proteins are required in
AAV genome replication. The cap gene, through alternative splicing and initiation of
translation, gives rise to three capsid proteins: VP1 (virion protein 1), VP2, and VP3,
with molecular weight of 87, 72, and 62 kDa, respectively. Immunofluorescence results
suggest that capsid assembly is confined to the nucleoli of infected cells, and these
fully assembled AAV capsids enter the nucleoplasm in an AAV rep-dependent manner.
Selective AAV-DNA encapsidation is presumably directed by protein-protein interac-
tions between two complexes; the first complex is the preformed empty capsids and the
second complex is the complex of Rep78 or Rep68 with the virus genome. Next, the
Rep52 and Rep40 proteins are responsible for transferring the AAV genome DNA into
empty particles through the pores. It should be noted that this is just one of the many
working hypotheses for AAV replication.
5.3.3.1.2 Adeno-Associated Virus Life Cycle
Propagation of AAV can be either in a latent state as an integrated provirus that can
be rescued by superinfection with adenovirus, or by lytic infection in the presence of
a coinfecting adenovirus.
1. Attachment of AAV2 is primarily mediated by heparan sulfate proteoglycans, while internal-
ization is aided by the coreceptors, such as avb5 and FGFR1 as well as others. The use of
ubiquitous heparan sulfate proteoglycans as docking sites explains in part the well-known
broad tropism of this virus. However, the mechanisms behind the trafficking of AAV particles
into the nucleus are still not fully understood.
2. After entry into the host cell nucleus, AAV can follow either one of two distinct and inter-
changeable pathways of its life cycle:
a. The lytic pathway
b. The lysogenic pathway
The former develops in cells infected with a helper virus, such as adenovirus or HSV,
whereas the latter is established in host cells in the absence of a helper virus.
3. When AAV infects a human cell alone, its gene expression program is autorepressed, and
latency ensues by preferential site-specific integration of the virus genome into a region
of roughly 2 kb on the long arm (19q13.3 qter) of human chromosome 19, designated
AAVS1. This site-specific integration involves the AAV ITRs and rep proteins (Rep78,
Rep68).