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Adeno-associated virus: from defective virus to effective vector.

Gonçalves MA - Virol. J. (2005)

Bottom Line: The initial discovery of adeno-associated virus (AAV) mixed with adenovirus particles was not a fortuitous one but rather an expression of AAV biology.In the present review, issues related to the development of recombinant AAV (rAAV) vectors, from the general principle to production methods, tropism modifications and other emerging technologies are discussed.In addition, the accumulating knowledge regarding the mechanisms of rAAV genome transduction and persistence is reviewed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Gene Therapy Section, Department of Molecular Cell Biology, Leiden University Medical Center, the Netherlands. m.goncalves@lumc.nl

ABSTRACT
The initial discovery of adeno-associated virus (AAV) mixed with adenovirus particles was not a fortuitous one but rather an expression of AAV biology. Indeed, as it came to be known, in addition to the unavoidable host cell, AAV typically needs a so-called helper virus such as adenovirus to replicate. Since the AAV life cycle revolves around another unrelated virus it was dubbed a satellite virus. However, the structural simplicity plus the defective and non-pathogenic character of this satellite virus caused recombinant forms to acquire centre-stage prominence in the current constellation of vectors for human gene therapy. In the present review, issues related to the development of recombinant AAV (rAAV) vectors, from the general principle to production methods, tropism modifications and other emerging technologies are discussed. In addition, the accumulating knowledge regarding the mechanisms of rAAV genome transduction and persistence is reviewed. The topics on rAAV vectorology are supplemented with information on the parental virus biology with an emphasis on aspects that directly impact on vector design and performance such as genome replication, genetic structure, and host cell entry.

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Schematic representation of the AAV DNA replication model. AAV DNA replication is thought to involve a self-priming single-strand displacement mechanism that is initiated by DNA polymerisation at the 3' hairpin primer of input single-stranded genomes. This leads to the formation of linear unit-length double-stranded molecules (duplex monomers, DMs) with one covalently closed end. These structures are resolved at the terminal resolution site (trs) by site-specific nicking of the parental strand opposite the original 3' end position (i.e., at nucleotide 125). The newly generated free 3' hydroxyl groups provide a substrate for DNA polymerases that unwind and copy the inverted terminal repeat (ITR). Finally, the palindromic linear duplex termini can renaturate into terminal hairpins putting the 3' hydroxyl groups in position for single-strand displacement synthesis. Next, single-stranded genomes and new DM replicative forms are made. When nicking does not occur, elongation proceeds through the covalently closed hairpin structure generating linear double-length double-stranded molecules (duplex dimers, DDs) with either a head-to-head or a tail-to-tail configuration. The DD replicative intermediates can be resolved to DMs through the AAV ITR sequences located at the axis of symmetry.
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Figure 3: Schematic representation of the AAV DNA replication model. AAV DNA replication is thought to involve a self-priming single-strand displacement mechanism that is initiated by DNA polymerisation at the 3' hairpin primer of input single-stranded genomes. This leads to the formation of linear unit-length double-stranded molecules (duplex monomers, DMs) with one covalently closed end. These structures are resolved at the terminal resolution site (trs) by site-specific nicking of the parental strand opposite the original 3' end position (i.e., at nucleotide 125). The newly generated free 3' hydroxyl groups provide a substrate for DNA polymerases that unwind and copy the inverted terminal repeat (ITR). Finally, the palindromic linear duplex termini can renaturate into terminal hairpins putting the 3' hydroxyl groups in position for single-strand displacement synthesis. Next, single-stranded genomes and new DM replicative forms are made. When nicking does not occur, elongation proceeds through the covalently closed hairpin structure generating linear double-length double-stranded molecules (duplex dimers, DDs) with either a head-to-head or a tail-to-tail configuration. The DD replicative intermediates can be resolved to DMs through the AAV ITR sequences located at the axis of symmetry.

Mentions: The human adeno-associated virus (AAV) was discovered in 1965 as a contaminant of adenovirus (Ad) preparations [1]. AAV is one of the smallest viruses with a non-enveloped icosahedral capsid of approximately 22 nm (Fig. 1), the crystal structure of which has been recently determined to a 3-angstrom resolution [2]. Because a co-infecting helper virus is usually required for a productive infection to occur, AAV serotypes are ascribed to a separate genus in the Parvoviridae family designated Dependovirus. Despite the high seroprevalence of AAV in the human population (approximately 80% of humans are seropositive for AAV2) the virus has not been linked to any human illness. The AAV has a linear single-stranded DNA genome of approximately 4.7-kilobases (kb). The AAV2 DNA termini consist of a 145 nucleotide-long inverted terminal repeat (ITR) that, due to the multipalindromic nature of its terminal 125 bases, can fold on itself via complementary Watson-Crick base pairing and form a characteristic T-shaped hairpin structure (Fig. 2) [3]. According to the AAV DNA replication model [4] this secondary structure provides a free 3' hydroxyl group for the initiation of viral DNA replication via a self-priming strand-displacement mechanism involving leading-strand synthesis and double-stranded replicative intermediates (Fig. 3). The virus does not encode a polymerase relying instead on cellular polymerase activities to replicate its DNA [5]. The ITRs flank the two viral genes rep (replication) and cap (capsid) encoding nonstructural and structural proteins, respectively. The rep gene, through the use of two promoters located at map positions 5 (p5) and 19 (p19), and an internal splice donor and acceptor site, encode four regulatory proteins that are dubbed Rep78, Rep68, Rep52 and Rep40 on basis of their apparent molecular weights. The Rep78 and Rep68 proteins participate in the AAV DNA replication process via their interaction with Rep-binding element (RBE) and terminal resolution site (trs) sequences located within the ITRs (Fig. 2). In addition, in response to environmental cues such as presence or absence of a helper virus these proteins either positively or negatively regulate AAV gene expression, respectively [6]. The Rep52 and Rep40 proteins are involved in the generation and accumulation of single-stranded viral genomes from double-stranded replicative intermediates [7]. The resulting single-stranded genomes with plus and minus polarities are packaged with equal efficiency [8]. The economy displayed by AAV is staggering and derives not only from its overlapping genetic organization but also from the integration of various biochemical activities in each of its few gene products. For instance, Rep78 and Rep68 are site-specific DNA binding proteins, as well as strand- and site-specific endonucleases [9]. They also exhibit helicase and ATPase activities [10], which are shared by Rep52 [11] and by Rep40 [12].


Adeno-associated virus: from defective virus to effective vector.

Gonçalves MA - Virol. J. (2005)

Schematic representation of the AAV DNA replication model. AAV DNA replication is thought to involve a self-priming single-strand displacement mechanism that is initiated by DNA polymerisation at the 3' hairpin primer of input single-stranded genomes. This leads to the formation of linear unit-length double-stranded molecules (duplex monomers, DMs) with one covalently closed end. These structures are resolved at the terminal resolution site (trs) by site-specific nicking of the parental strand opposite the original 3' end position (i.e., at nucleotide 125). The newly generated free 3' hydroxyl groups provide a substrate for DNA polymerases that unwind and copy the inverted terminal repeat (ITR). Finally, the palindromic linear duplex termini can renaturate into terminal hairpins putting the 3' hydroxyl groups in position for single-strand displacement synthesis. Next, single-stranded genomes and new DM replicative forms are made. When nicking does not occur, elongation proceeds through the covalently closed hairpin structure generating linear double-length double-stranded molecules (duplex dimers, DDs) with either a head-to-head or a tail-to-tail configuration. The DD replicative intermediates can be resolved to DMs through the AAV ITR sequences located at the axis of symmetry.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1131931&req=5

Figure 3: Schematic representation of the AAV DNA replication model. AAV DNA replication is thought to involve a self-priming single-strand displacement mechanism that is initiated by DNA polymerisation at the 3' hairpin primer of input single-stranded genomes. This leads to the formation of linear unit-length double-stranded molecules (duplex monomers, DMs) with one covalently closed end. These structures are resolved at the terminal resolution site (trs) by site-specific nicking of the parental strand opposite the original 3' end position (i.e., at nucleotide 125). The newly generated free 3' hydroxyl groups provide a substrate for DNA polymerases that unwind and copy the inverted terminal repeat (ITR). Finally, the palindromic linear duplex termini can renaturate into terminal hairpins putting the 3' hydroxyl groups in position for single-strand displacement synthesis. Next, single-stranded genomes and new DM replicative forms are made. When nicking does not occur, elongation proceeds through the covalently closed hairpin structure generating linear double-length double-stranded molecules (duplex dimers, DDs) with either a head-to-head or a tail-to-tail configuration. The DD replicative intermediates can be resolved to DMs through the AAV ITR sequences located at the axis of symmetry.
Mentions: The human adeno-associated virus (AAV) was discovered in 1965 as a contaminant of adenovirus (Ad) preparations [1]. AAV is one of the smallest viruses with a non-enveloped icosahedral capsid of approximately 22 nm (Fig. 1), the crystal structure of which has been recently determined to a 3-angstrom resolution [2]. Because a co-infecting helper virus is usually required for a productive infection to occur, AAV serotypes are ascribed to a separate genus in the Parvoviridae family designated Dependovirus. Despite the high seroprevalence of AAV in the human population (approximately 80% of humans are seropositive for AAV2) the virus has not been linked to any human illness. The AAV has a linear single-stranded DNA genome of approximately 4.7-kilobases (kb). The AAV2 DNA termini consist of a 145 nucleotide-long inverted terminal repeat (ITR) that, due to the multipalindromic nature of its terminal 125 bases, can fold on itself via complementary Watson-Crick base pairing and form a characteristic T-shaped hairpin structure (Fig. 2) [3]. According to the AAV DNA replication model [4] this secondary structure provides a free 3' hydroxyl group for the initiation of viral DNA replication via a self-priming strand-displacement mechanism involving leading-strand synthesis and double-stranded replicative intermediates (Fig. 3). The virus does not encode a polymerase relying instead on cellular polymerase activities to replicate its DNA [5]. The ITRs flank the two viral genes rep (replication) and cap (capsid) encoding nonstructural and structural proteins, respectively. The rep gene, through the use of two promoters located at map positions 5 (p5) and 19 (p19), and an internal splice donor and acceptor site, encode four regulatory proteins that are dubbed Rep78, Rep68, Rep52 and Rep40 on basis of their apparent molecular weights. The Rep78 and Rep68 proteins participate in the AAV DNA replication process via their interaction with Rep-binding element (RBE) and terminal resolution site (trs) sequences located within the ITRs (Fig. 2). In addition, in response to environmental cues such as presence or absence of a helper virus these proteins either positively or negatively regulate AAV gene expression, respectively [6]. The Rep52 and Rep40 proteins are involved in the generation and accumulation of single-stranded viral genomes from double-stranded replicative intermediates [7]. The resulting single-stranded genomes with plus and minus polarities are packaged with equal efficiency [8]. The economy displayed by AAV is staggering and derives not only from its overlapping genetic organization but also from the integration of various biochemical activities in each of its few gene products. For instance, Rep78 and Rep68 are site-specific DNA binding proteins, as well as strand- and site-specific endonucleases [9]. They also exhibit helicase and ATPase activities [10], which are shared by Rep52 [11] and by Rep40 [12].

Bottom Line: The initial discovery of adeno-associated virus (AAV) mixed with adenovirus particles was not a fortuitous one but rather an expression of AAV biology.In the present review, issues related to the development of recombinant AAV (rAAV) vectors, from the general principle to production methods, tropism modifications and other emerging technologies are discussed.In addition, the accumulating knowledge regarding the mechanisms of rAAV genome transduction and persistence is reviewed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Gene Therapy Section, Department of Molecular Cell Biology, Leiden University Medical Center, the Netherlands. m.goncalves@lumc.nl

ABSTRACT
The initial discovery of adeno-associated virus (AAV) mixed with adenovirus particles was not a fortuitous one but rather an expression of AAV biology. Indeed, as it came to be known, in addition to the unavoidable host cell, AAV typically needs a so-called helper virus such as adenovirus to replicate. Since the AAV life cycle revolves around another unrelated virus it was dubbed a satellite virus. However, the structural simplicity plus the defective and non-pathogenic character of this satellite virus caused recombinant forms to acquire centre-stage prominence in the current constellation of vectors for human gene therapy. In the present review, issues related to the development of recombinant AAV (rAAV) vectors, from the general principle to production methods, tropism modifications and other emerging technologies are discussed. In addition, the accumulating knowledge regarding the mechanisms of rAAV genome transduction and persistence is reviewed. The topics on rAAV vectorology are supplemented with information on the parental virus biology with an emphasis on aspects that directly impact on vector design and performance such as genome replication, genetic structure, and host cell entry.

Show MeSH
Related in: MedlinePlus