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Chapter 38 : Adeno-Associated Virus

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Abstract:

The ability of adeno-associated virus (AAV) to establish a latent infection prompted an investigation of the nature of the viral genome within latently infected cells and led to the discovery that AAV stably integrates with high efficiency into host cell chromatin, with a preponderance of integration events occurring within a specific locus of human chromosome 19 known as AAV integration site 1 (AAVS1). The major open reading frame of the rep gene gives rise to a family of four overlapping nonstructural proteins, known as Rep78, Rep68, Rep52, and Rep40, via a combination of alternative promoter usage and differential utilization of a splicing event. Parallels between the potential chromosomal integration mechanisms of AAV and T-DNA sequences are discussed in detail. Researchers reported the sequence organization of Rep-mediated viral-cellular junctions produced in an in vitro recombination system. Recombination between AAV and cellular sequences at stretches of DNA with significant homology is rarely observed among junctions generated in vivo, and, based on the relatively large copy number of PCR substrates in the in vitro assay, these junctions may have potentially arisen from PCR priming events at Rep binding site (RBS) sequences common to the substrates. The major rescued products were linear duplex molecules with covalently closed ends. Resolution of the integration intermediates described above would use cellular DNA repair mechanisms. Exploration of the phylogenetic relationships among various mobile genetic elements may aid in identifying common mechanisms used in the mobilization and integration of numerous genetic elements of both prokaryotes and eukaryotes.

Citation: Smith, Jr. R, Kotin R. 2002. Adeno-Associated Virus, p 905-923. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch38
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Figures

Image of Figure 1.
Figure 1.

Map of the AAV genome. (A) The AAV genome contains two large open reading frames encoding the Rep and Cap proteins. Three AAV promoters, P5, P19, and P40, regulate expression of a series of 3′-coterminal transcripts that use a common polyadenylation signal (vertical arrow). The ITRs are represented by hatched boxes. An expansion of the terminal repeat shows one arrangement of the internal palindromes within the ITR. (B) Transcripts encoding the nonstructural (Rep) and structural proteins (VP-1, VP-2, and VP-3) of AAV are shown. Coding sequences are represented by open boxes; untranslated regions are represented by horizontal lines. The V-shaped lines indicate a spliced section of RNA. The protein products and transcript sizes are indicated. The molecular masses for the structural proteins are listed. See text for more details.

Citation: Smith, Jr. R, Kotin R. 2002. Adeno-Associated Virus, p 905-923. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch38
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Image of Figure 2.
Figure 2.

AAV ITR. The 3′-ITRis shown in its maximally basepaired conformation. The dodecameric Rep binding site (RBS) core consists of a GCTC repeat (horizontal arrows). The vertical arrow indicates the Rep nicking site (RNS). The hairpin conformation of the 3′-terminal palindrome functions as a primer for AAV DNA synthesis.

Citation: Smith, Jr. R, Kotin R. 2002. Adeno-Associated Virus, p 905-923. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch38
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Image of Figure 3.
Figure 3.

Modular domain structure of Rep. The Rep proteins can be depicted as an assortment of three functional domains conferring (i) sequence-specific DNA binding and nicking activities, (ii) nuclear localization and helicase activity, and (iii) protein kinase A inhibition. The relative locations of the RCR motifs (black boxes labeled 2 and 3) are indicated.

Citation: Smith, Jr. R, Kotin R. 2002. Adeno-Associated Virus, p 905-923. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch38
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Image of Figure 4.
Figure 4.

Rep-mediated cleavage-joining reaction. A theoretical spatial arrangement of amino acid side chains in the catalytic pocket of Rep78/68 is shown. Tyr-156, within RCR motif 3, and Tyr-152 are shown on the same face of an α-helix. Histidine residues 90 and 92, of RCR motif 2, are also depicted. (A) Binding of a single-stranded DNA substrate aligns the scissile phosphodiester bond of the RNS within the catalytic pocket. (B) Nucleophilic attack on the phosphodiester backbone of the substrate by Tyr-156 is facilitated by the RCR motif 2 histidine pair. (C) DNA cleavage results in covalent attachment of Rep to the 5′ end of the cleavage site DNA (adapted from reference ).

Citation: Smith, Jr. R, Kotin R. 2002. Adeno-Associated Virus, p 905-923. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch38
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Image of Figure 5.
Figure 5.

AAV replication model. (Column I) Formation of monomer AAV genomes. (A) The single-stranded virion genome is shown with both terminal palindromes in the hairpin conformation. (B) The 3″-ITRis extended by an RNA primer-independent, cellular polymerase complex (filled circle) producing a closed-ended duplex molecule (the replicative-form monomer, RF) that may express viral genes. The line thickness increases with each round of DNA initiation. (C) The large Rep proteins, Rep78 and Rep68, bind to a specific sequence within the A stem of the duplex ITR. (D and E) In a process referred to as terminal resolution, Rep nicks the template strand at the RNS (indicated by an open arrowhead in line B) forming a covalent nucleoprotein complex. Rep helicase activity unwinds the terminal palindrome allowing cellular polymerase activity to repair the end of the genome. (F) Re-formation of the terminal hairpin allows strand-displacement synthesis to occur, resulting in a progeny viral genome and an RF. These replication products are equivalent to those shown on lines A and B, and may function as templates for the next round of DNA replication. (Column II) Formation of duplex-dimer and higher-order concatemeric structures. (G) The newly synthesized ITR sequence of the RFM intermediate adopts a hairpin conformation, thus allowing continuation or reinitiation of DNA synthesis. (H and I) The polymerase complex displaces the complementary strand of the RFM, producing a replicative-form dimer (RF) that is covalently closed at one end. Rep nicks the internal ITR at the RNS (open arrowhead), thus becoming covalently attached to the cleaved DNA. (J) Rep unwinds the ITR sequence and encounters an RNS on the opposite strand. (K) Nicking of the second RNS initiates a strand-exchange reaction that resolves the dimer. The dashed arrow indicates an iteration of the process, resulting in longer tandem repeats of the genome.

Citation: Smith, Jr. R, Kotin R. 2002. Adeno-Associated Virus, p 905-923. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch38
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Image of Figure 6.
Figure 6.

Map of AAVS1. A 4-kb region of AAVS1 is shown schematically (GenBank accession number S5139).A CpG island occurs within the first 1 kb of the sequence. A partial cDNA has been mapped to nucleotide positions 1616 to 2315 ( ). A chromosome 19 q-arm-specific minisatellite repeat (parallel vertical lines) occurs at the 3′ end of the sequence between nt 3655 and 4022. Brackets labeled I and II indicate frequently reported locations of viral-cellular junctions ( ). Region I is centered around nt 400 and represents integration junctions reported for AAVS1 sequences maintained episomally ( ). Viral-cellular junctions within intact, chromosomal AAVS1 sequences are frequently detected at location II (centered around nt 1050). An AAV--like element, occurring within the CpG island, is indicated by an arrow.

Citation: Smith, Jr. R, Kotin R. 2002. Adeno-Associated Virus, p 905-923. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch38
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Image of Figure 7.
Figure 7.

Alignment of AAVS1 and AAV ITR sequences. Nucleotides 366 to 435 of AAVS1 are shown. This region contains sequences homologous to the AAV RNS and RBS and can function as a minimal Rep-dependent origin of replication.

Citation: Smith, Jr. R, Kotin R. 2002. Adeno-Associated Virus, p 905-923. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch38
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Image of Figure 8.
Figure 8.

AAV-AAVS1 junctions. (A) Junctions between wild-type AAV and AAVS1 DNA are indicated. Common characteristics include sequence overlap (or microhomology) between the viral and cellular sequences (underlined), limited truncation of the viral ITR, and, in some instances, short stretches of heterologous DNA insertion (lowercase letters). (B) Junctions between viral and cellular DNA sequences in which the cross-over point within AAVS1 occurs at the Rep nicking site of the AAVS1 DNA. Junctions are as reported in the following references: junctions 1 to 3, reference 77; junction 4, reference 47; junctions 5 to 10, reference 86; junctions 11 and 12, reference 26; junctions 13 and 14, reference 20. Nucleotide coordinates shown are as indicated in each respective reference. An asterisk indicates a nonprototypic orientation of the ITR.

Citation: Smith, Jr. R, Kotin R. 2002. Adeno-Associated Virus, p 905-923. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch38
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Image of Figure 9.
Figure 9.

Resolution of AAV DNA. A plasmid containing AAV genomic DNA is represented as a circle. An expanded view of one virus-plasmid junction is shown. The linear conformation of the duplex ITR palindromes exists in equilibrium with the cruciform conformation of the ITR. Rep-mediated cleaving-joining activity at the base of the A stems resolves the ITR sequences, resulting in two separate, covalently closed DNA molecules.

Citation: Smith, Jr. R, Kotin R. 2002. Adeno-Associated Virus, p 905-923. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch38
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Image of Figure 10.
Figure 10.

Recombination models. (Column I) (A) Rep (shaded disk) recognizes its cognate binding site within the -like element of AAVS1 and nicks the RNS (open arrow), resulting in covalent attachment of Rep to the chromosomal DNA. (B) Rep unwinds the DNA in a 3′-to-5″ direction relative to the helicase-bound strand. (C) AAV genomic DNA hybridizes to the newly created gap within AAVS1. Microhomologies between the AAV ITRs and AAVS1 position and stabilize the ends of the viral DNA within the gap. (D) Cellular exonuclease activity trims the exposed ends of the viral DNA, and the viral strand is joined to the genomic DNA by cellular ligase activity. Nicking and extension of the bottom strand would complete the integration process. (Columns II, III, and IV) Rep is covalently attached to the input viral genome (wavy lines). The DNA-binding activity of Rep directs the nucleoprotein complex to the AAVS1 locus of chromosome 19 (parallel lines). (Column II) (A) In a cleavage-joining reaction, Rep donates viral sequences to cellular DNA. (B) The helicase activity of Rep unwinds the nicked strand. (C) Microhomology between the AAV ITR and the chromosomal DNA stabilizes a heteroduplex structure. The cleavage-joining activity of Rep ligates the free 3″-OH of the AAV genome to chromosomal DNA. (D) Degradation of the displaced, single-stranded chromosomal DNA would produce a deletion of cellular sequences. Alternatively, nicking of the chromosomal DNA opposite the viral strand and extension from the 3′-OH by using the viral DNA as a template would result in an integration event that would appear as a simple insertion. (Column III) (A) Rep nicks the cellular DNA and unwinds AAVS1 without transferring viral sequences. (B) The 3″-OH generated by the nick is extended by leading-strand DNA synthesis (filled circle, thicker line). Upon encountering an RNS-like element or, possibly, an unusual DNA secondary structure, Rep nicks the opposite strand, resulting in DNA strand-exchange and attachment of viral sequences to chromosomal DNA. (C) The single-stranded viral DNA is converted to duplex DNA by cellular DNA polymerase activity by using the hairpin 3′-ITR as a primer. Rep transfers the covalently attached cellular DNA to the AAV ITR. (D) Gap-repair activity completes the integration process. (Column IV) (A) Rep unwinds AAVS1 DNA before nicking. (B) On encountering an RNSlike element, the cleaving-joining activity of Rep transfers AAV sequences to the cellular DNA. The AAV DNA is converted to duplex by a cellular polymerase complex (filled circle). (C) Rep-mediated cleavage-joining activity seals the bottom strand. (D) Cellular endonuclease activity degrades the displaced cellular DNA. The upper strand nicks are then sealed by cellular ligase activity. In all cases, Rep may be noncovalently bound to the RBS elements within the viral ITR and/or AAVS1. This would allow for protein-protein interactions to occur between Rep complexes on two or more DNA substrates. Thus, the double-strand break in column III, line C, may be held together by Rep-Rep interactions.

Citation: Smith, Jr. R, Kotin R. 2002. Adeno-Associated Virus, p 905-923. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch38
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