The AAVS1 locus was originally defined in Genomics by Kotin, Menninger, Ward, and Berns.

Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter RM Kotin, JC Menninger, DC Ward, KI Berns Genomics 10 (3), 831-834

Disease relevance of AAVS1

• The nonpathogenic human virus adeno-associated virus type 2 (AAV) has evolved the potentially unique strategy to establish latency by site-specifically integrating its genome into human chromosome 19 (19q13.3-qter) at a locus designated AAVS1 [1] [2].
• DNA integration at the AAVS1 site also was demonstrated in hepatoma cells coinfected with the HD-expressing Rep78 and with the second HD vector carrying a transgene flanked by AAV-ITRs [3].
• Further, the levels of transgene expression appear to be independent of 19q allelic status or the number of endogenous AAVS1 sequences in the various glioma cell lines studied [4].
• The latter is of particular interest because the AAV integration site (AAVS1) is located on the long arm of chromosome 19 and 30-40% of human glioblastoma tumors are reported to have loss of heterozygosity in this region of chromosome 19q [4].
• In this study, we analyze the recombinant junctions generated after integration of the AAV genome into an Epstein-Barr virus shuttle vector carrying 8.2, 1.6, or 0.51 kb of the chromosome 19 preintegration sequence (AAVS1 locus) [5].

High impact information on AAVS1

• Direct repeats at a much greater than random occurrence were found distributed non-uniformly throughout the AAVS1 sequence [6].
• Adeno-associated virus (AAV) undergoes site-specific integration into human chromosome 19 through a deletion-substitution mechanism at the well characterized AAVS1 site [7].
• A p5 integration efficiency element mediates Rep-dependent integration into AAVS1 at chromosome 19 [7].
• RRS motifs are found within the cellular AAVS1 integration locus, the viral p5 promoter, and the inverted terminal repeats (ITRs) [8].
• Here, we show that AAVS1 is closely linked to the slow skeletal troponin T gene, TNNT1, which has been mapped previously to 19q13 [2].

Biological context of AAVS1

• The signals which direct recombination with the AAV genome were localized to a 510-nt region at the 5' end of the 8.2-kb AAVS1 DNA [9].
• An additional chromosome 19 element, which is responsible for DNA rearrangements in episomes propagating AAVS1 DNA, was identified and shown not to be required for AAV episomal integration, despite its location adjacent to the recombination signal [10].
• Two viral elements are necessary for the integration at AAVS1: Rep68/78 and the inverted terminal repeats (AAV-ITRs) [3].
• Use of a functional model system for AAV DNA integration into AAVS1 has allowed us to conclude that the recombination event is directed by cellular DNA sequences [11].
• The minimal DNA signals within AAVS1 required for targeted integration were identified and shown to contain functional motifs of the viral origin of replication [11].

Anatomical context of AAVS1

• Different regions of an 8.2-kb cloned DNA segment containing the target for adeno-associated virus (AAV) integration in human chromosome 19q13-3-qter (AAVS1 locus) were subcloned in an Epstein-Barr virus-based shuttle vector and propagated as episomes in a derivative of the 293 human embryonic kidney cell line [9].
• Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep(+) hybrid amplicon vector [12].
• Lastly, AAV genomic DNA integration into the AAVS1 site in vivo was assessed by virus injection into the quadriceps muscle of transgenic rats and mice [13].
• The finding that 55% of all analyzed integration sites were either within the AAVS1 or globin LCR region demonstrates that a high frequency of targeted integration of a large transgene cassette can be achieved in human hematopoietic stem cell lines [14].
• PCR amplification of the inverted terminal repeat (ITR)-AAVS1 junction revealed that the AAV genome integrated into the AAVS1 site in fibroblasts and hepatocytes [13].

Associations of AAVS1 with chemical compounds

• By comparing the migration of radiolabeled oligonucleotides containing wild-type or mutated sequences from the AAVS1 nicking site to appropriate standards, on native and denaturing polyacrylamide gels, we have found evidence that this region forms a stable secondary structure [15].
• Of 11 infected cell lines studied, nine (82%) showed integration of the neomycin resistance marker into AAVS1, which confirms the findings of previous investigations [16].
• The activity of this chimeric protein, named Rep1-491/P, is highly dependent on RU486 in various assays: in particular, it triggers site-specific integration at AAVS1 of an ITR-flanked cassette in a ligand-dependent manner, as efficiently as wild-type Rep68 but without generating unwanted genomic rearrangement at AAVS1 [17].

Regulatory relationships of AAVS1

• Our data suggest that TRP-185 suppresses AAV integration at the AAVS1 RBS and enhances AAV integration into a region downstream of the RBS [18].

Other interactions of AAVS1

• TAR RNA loop binding protein 185 (TRP-185), previously reported to associate with human immunodeficiency virus type 1 TAR RNA, binds to AAVS1 DNA [18].
• In a gel shift test, the 336-bp DNA did not bind an in vitro-prepared CCCTC-binding factor that binds to the chicken beta-globin insulator, suggesting that the AAVS1 insulator requires an as yet unidentified binding protein [19].

Analytical, diagnostic and therapeutic context of AAVS1

• In the presence of HeLa extract and the His-Tag-purified Rep68 protein, specific covalent junctions between AAV and AAVS1 were formed and detected by PCR [20].
• The localization of the GFP-Neo sequence in the AAVS1 region was determined by Southern blot and FISH analysis [13].
• Sequence analysis also confirmed that transient expression of Rep78 was sufficient for site-specific integration at the AAVS1 locus, as is observed with integration of wild-type AAV [21].
• Mutants were analyzed for their abilities to bind the GAGC motif, nick at the trs homolog, and integrate an ITR-containing plasmid into AAVS1 by electrophoretic mobility shift assay, trs endonuclease assay, and PCR-based integration assay [22].
• Since AAVS1 sequences are not conserved in a rodent's genome, no animal model is currently available to study AAV-mediated site-specific integration [13].

References