title: Substitution of adeno-associated virus Rep protein binding and nicking sites with human chromosome 19 sequences.
abstract: BACKGROUND
  Adeno-associated virus type 2 (AAV2) preferentially integrates its DNA at a ~2 kb region of human chromosome 19, designated AAVS1 (also known as MBS85). Integration at AAVS1 requires the AAV2 replication (Rep) proteins and a DNA sequence within AAVS1 containing a 16 bp Rep recognition sequence (RRS) and closely spaced Rep nicking site (also referred to as a terminal resolution site, or trs). The AAV2 genome is flanked by inverted terminal repeats (ITRs). Each ITR contains an RRS and closely spaced trs, but the sequences differ from those in AAVS1. These ITR sequences are required for replication and packaging.
  
  
  RESULTS
  In this study we demonstrate that the AAVS1 RRS and trs can function in AAV2 replication, packaging and integration by replacing a 61 bp region of the AAV2 ITR with a 49 bp segment of AAVS1 DNA. Modifying one or both ITRs did not have a large effect on the overall virus titers. These modifications did not detectably affect integration at AAVS1, as measured by semi-quantitative nested PCR assays. Sequencing of integration junctions shows the joining of the modified ITRs to AAVS1 sequences.
  
  
  CONCLUSIONS
  The ability of these AAVS1 sequences to substitute for the AAV2 RRS and trs provides indirect evidence that the stable secondary structure encompassing the trs is part of the AAV2 packaging signal.
authors.affiliation: Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, USA.
authors.forename: Victor J
authors.initials: VJ
authors.lastname: McAlister
authors.forename: Roland A
authors.initials: RA
authors.lastname: Owens
mesh_terms: D002888:Chromosomes, Human, Pair 19; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D011995:Recombination, Genetic; D020079:Terminal Repeat Sequences; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D019065:Virus Assembly; D016662:Virus Integration
chemical_list: D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D004247:DNA
references.citation: J Virol. 1999 Nov;73(11):9325-36
references.pmid: 10516041
references.citation: Mol Ther. 2008 Mar;16(3):494-9
references.pmid: 18180769
references.citation: J Virol. 1995 Apr;69(4):2038-46
references.pmid: 7884849
references.citation: Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7966-72
references.pmid: 8755586
references.citation: J Mol Biol. 1989 Mar 5;206(1):91-100
references.pmid: 2539485
references.citation: Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10039-43
references.pmid: 7937833
references.citation: J Virol. 1998 Jun;72(6):4874-81
references.pmid: 9573254
references.citation: EMBO J. 2001 Jun 15;20(12):3282-91
references.pmid: 11406604
references.citation: J Virol. 2001 Oct;75(20):9991-4
references.pmid: 11559833
references.citation: J Virol. 2001 Jan;75(1):375-83
references.pmid: 11119606
references.citation: Virology. 1988 Oct;166(2):316-27
references.pmid: 2845646
references.citation: J Virol. 1995 Nov;69(11):6880-5
references.pmid: 7474103
references.citation: Gene Ther. 2008 Jun;15(11):817-22
references.pmid: 18401436
references.citation: Curr Gene Ther. 2002 May;2(2):145-59
references.pmid: 12109212
references.citation: J Virol. 1997 Apr;71(4):3299-306
references.pmid: 9060699
references.citation: J Virol. 1988 Sep;62(9):3356-63
references.pmid: 2841488
references.citation: J Virol. 1987 Oct;61(10):3096-101
references.pmid: 3041032
references.citation: J Virol. 2007 Sep;81(18):9718-26
references.pmid: 17626070
references.citation: J Virol. 2004 Aug;78(15):7874-82
references.pmid: 15254160
references.citation: J Gen Virol. 2007 Jun;88(Pt 6):1722-1732
references.pmid: 17485532
references.citation: J Virol. 1995 Nov;69(11):6917-24
references.pmid: 7474109
references.citation: J Virol. 1986 Dec;60(3):823-32
references.pmid: 3023672
references.citation: Annu Rev Genet. 2004;38:819-45
references.pmid: 15568995
references.citation: J Virol. 2003 Jan;77(1):776-81
references.pmid: 12477885
references.citation: Virology. 1989 Nov;173(1):120-8
references.pmid: 2554565
references.citation: J Virol. 2004 Aug;78(16):8917-21
references.pmid: 15280500
references.citation: EMBO J. 1991 Dec;10(12):3941-50
references.pmid: 1657596
references.citation: Cell. 1990 May 4;61(3):447-57
references.pmid: 2159383
references.citation: Nature. 1974 Aug 9;250(5466):467-70
references.pmid: 4469597
references.citation: J Virol. 2009 Nov;83(22):11655-64
references.pmid: 19759155
references.citation: J Virol. 2003 Feb;77(3):1904-15
references.pmid: 12525625
references.citation: Virology. 1987 Nov;161(1):18-28
references.pmid: 2823460
references.citation: J Virol. 1996 Apr;70(4):2440-8
references.pmid: 8642672
references.citation: J Virol. 2005 Dec;79(23):14793-803
references.pmid: 16282479
references.citation: Int J Mol Med. 2000 Jul;6(1):17-27
references.pmid: 10851261
references.citation: Proc Natl Acad Sci U S A. 2009 May 5;106(18):7571-6
references.pmid: 19372372
references.citation: J Virol. 1997 Oct;71(10):7951-9
references.pmid: 9311886
references.citation: Virology. 1980 Apr 15;102(1):71-82
references.pmid: 6245509
references.citation: Gene Ther. 1996 Sep;3(9):748-55
references.pmid: 8875221
references.citation: EMBO J. 1992 Dec;11(13):5071-8
references.pmid: 1334463
references.citation: J Virol. 1992 Feb;66(2):1119-28
references.pmid: 1309894
references.citation: J Virol. 1997 Mar;71(3):2528-34
references.pmid: 9032395
references.citation: Mol Ther. 2004 Dec;10(6):981-9
references.pmid: 15564130
references.citation: J Virol. 1988 Aug;62(8):2745-54
references.pmid: 2839699
references.citation: J Virol. 2003 Apr;77(8):4881-7
references.pmid: 12663794
references.citation: J Virol. 1999 Feb;73(2):1580-90
references.pmid: 9882364
references.citation: Proc Natl Acad Sci U S A. 1990 Mar;87(6):2211-5
references.pmid: 2156265
references.citation: J Virol. 1995 Jun;69(6):3542-8
references.pmid: 7538173
references.citation: J Virol. 1990 Dec;64(12):6204-13
references.pmid: 2173787
references.citation: Curr Gene Ther. 2005 Jun;5(3):273-84
references.pmid: 15975005
references.citation: Proc Natl Acad Sci U S A. 2000 Apr 25;97(9):4862-6
references.pmid: 10758163
references.citation: Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5808-12
references.pmid: 8016070
references.citation: J Virol. 1999 Nov;73(11):8989-98
references.pmid: 10516005
references.citation: J Virol. 1997 Jun;71(6):4461-71
references.pmid: 9151837
references.citation: J Virol. 1993 Feb;67(2):997-1005
references.pmid: 8380475
references.citation: J Virol. 1999 Oct;73(10):8235-44
references.pmid: 10482574
references.citation: J Virol. 2005 Mar;79(6):3544-56
references.pmid: 15731249
references.citation: Mol Ther. 2005 Oct;12(4):744-53
references.pmid: 16023415
references.citation: J Biol Chem. 1993 Feb 15;268(5):3781-90
references.pmid: 7679117