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Substitution of adeno-associated virus Rep protein binding and nicking sites with human Chromosome 19 sequences

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 se...

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Autores principales: McAlister, Victor J, Owens, Roland A
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944168/
https://www.ncbi.nlm.nih.gov/pubmed/20825662
http://dx.doi.org/10.1186/1743-422X-7-218
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author McAlister, Victor J
Owens, Roland A
author_facet McAlister, Victor J
Owens, Roland A
author_sort McAlister, Victor J
collection PubMed
description 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.
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spelling pubmed-29441682010-09-24 Substitution of adeno-associated virus Rep protein binding and nicking sites with human Chromosome 19 sequences McAlister, Victor J Owens, Roland A Virol J Research 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. BioMed Central 2010-09-08 /pmc/articles/PMC2944168/ /pubmed/20825662 http://dx.doi.org/10.1186/1743-422X-7-218 Text en Copyright ©2010 McAlister and Owens; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
McAlister, Victor J
Owens, Roland A
Substitution of adeno-associated virus Rep protein binding and nicking sites with human Chromosome 19 sequences
title Substitution of adeno-associated virus Rep protein binding and nicking sites with human Chromosome 19 sequences
title_full Substitution of adeno-associated virus Rep protein binding and nicking sites with human Chromosome 19 sequences
title_fullStr Substitution of adeno-associated virus Rep protein binding and nicking sites with human Chromosome 19 sequences
title_full_unstemmed Substitution of adeno-associated virus Rep protein binding and nicking sites with human Chromosome 19 sequences
title_short Substitution of adeno-associated virus Rep protein binding and nicking sites with human Chromosome 19 sequences
title_sort substitution of adeno-associated virus rep protein binding and nicking sites with human chromosome 19 sequences
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944168/
https://www.ncbi.nlm.nih.gov/pubmed/20825662
http://dx.doi.org/10.1186/1743-422X-7-218
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