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Stable secondary structure near the nicking site for adeno-associated virus type 2 Rep proteins on human chromosome 19.

ABSTRACT: Adeno-associated virus serotype 2 (AAV-2) can preferentially integrate its DNA into a 4-kb region of human chromosome 19, designated AAVS1. The nicking activity of AAV-2's Rep68 or Rep78 proteins is essential for preferential integration. These proteins nick at the viral origin of DNA replication and at a similar site within AAVS1. The current nicking model suggests that the strand containing the nicking site is separated from its complementary strand prior to nicking. In AAV serotypes 1 through 6, the nicking site is flanked by a sequence that is predicted to form a stem-loop with standard Watson-Crick base pairing. The region flanking the nicking site in AAVS1 (5'-GGCGGCGGT/TGGGGCTCG-3' [the slash indicates the nicking site]) lacks extensive potential for Watson-Crick base pairing. We therefore performed an empirical search for a stable secondary structure. 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. Further confirmation was provided by circular dichroism analyses. We identified six bases that appear to be important in forming this putative secondary structure. Mutation of five of these bases, within the context of a double-stranded nicking substrate, reduces the ability of the substrate to be nicked by Rep78 in vitro. Four of these five bases are outside the previously recognized GTTGG nicking site motif and include parts of the CTC motif that has been demonstrated to be important for integration targeting.

SUBMITTER: Jang MY

PROVIDER: S-EPMC1075745 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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Stable secondary structure near the nicking site for adeno-associated virus type 2 Rep proteins on human chromosome 19.

Jang Ming Y MY Yarborough Orlando H OH Conyers Gary B GB McPhie Peter P Owens Roland A RA

Journal of virology 20050301 6

Adeno-associated virus serotype 2 (AAV-2) can preferentially integrate its DNA into a 4-kb region of human chromosome 19, designated AAVS1. The nicking activity of AAV-2's Rep68 or Rep78 proteins is essential for preferential integration. These proteins nick at the viral origin of DNA replication and at a similar site within AAVS1. The current nicking model suggests that the strand containing the nicking site is separated from its complementary strand prior to nicking. In AAV serotypes 1 through ...[more]

PMID: 15731249

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Project description:The Rep proteins of adeno-associated virus type 2 (AAV) are known to bind to Rep recognition sequences (RRSs) in the AAV inverted terminal repeats (ITRs), the AAV p5 promoter, and the preferred AAV integration site in human chromosome 19, called AAVS1. Integration of the AAV genome into AAVS1 appears to be mediated by an interaction between the Rep proteins of AAV and Rep binding sites within the viral genome and the integration locus. In an attempt to identify potential alternate integration sites, we looked for recognition sites for AAV Rep proteins in the human genome by performing a BLASTN computerized homology search. We used the 16-mer core sequences of the RRSs in the AAV ITRs and AAVS1 separately as query sequences and identified 18 new RRSs in or flanking the genes coding for the following: tyrosine kinase activator protein 1 (TKA-1); colony stimulating factor-1; insulin-like growth factor binding protein 2 (IGFBP-2); histone H2B.1; basement membrane heparan sulfate proteoglycan, also known as perlecan; the AF-9 gene product, which is involved in the chromosomal translocation t (9:11)(p22:q23); the betaB subunit of the hormone known as inhibin; interleukin-2 enhancer binding factor; an endoplasmic reticulum-Golgi intermediate compartment resident protein called p63; a global transcription activator (hSNF2L); the beta-actin repair domain; a retinoic acid-inducible factor, also known as midkine; a breast tumor autoantigen; a growth-arrest- and DNA-damage-inducible protein called gadd45; the cyclin-dependent kinase inhibitor called KIP2, which inhibits several G1 cyclin-cyclin-dependent kinase complexes; and the hereditary breast and ovarian cancer gene (BRCA1). RRSs were also identified in a newly discovered open reading frame on chromosome 10 and in the ERCC1 locus on human chromosome 19. The ability of a maltose binding protein-Rep68 fusion protein to bind to these sequences was confirmed by electrophoretic mobility shift assays. These sites may serve as alternate integration sites for AAV or play a role in Rep-mediated effects on human cells.

Project description:Recombinant adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last 2 decades, research focused primarily on the characterization and isolation of new cap, genes resulting in hundreds of natural and engineered AAV capsid variants, while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the single-stranded DNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major by-product of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2-rep system. In further experiments the 3' end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids approximately 2- to -4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production. IMPORTANCE A major by-product of all adeno-associated virus (AAV) vector production systems are "empty" capsids, void of the desired therapeutic gene, and thus do not provide any curative benefit for the treatment of the targeted disease. In fact, empty capsids can potentially elicit additional immune responses in vivo gene therapies if not removed by additional purification steps. Thus, there is a need to increase the genome packaging efficiency and reduce the number of empty capsids from AAV biologics. The novel Rep hybrids from different AAV serotypes described in this study are capable of reducing the percentage of empty capsids in all tested AAV serotypes and improve overall yields of genome-containing AAV capsids at the same time. They can likely be integrated easily into existing AAV manufacturing protocols to optimize the production of the generated AAV gene therapy products.

Dataset Information

Stable secondary structure near the nicking site for adeno-associated virus type 2 Rep proteins on human chromosome 19.

Publications

Stable secondary structure near the nicking site for adeno-associated virus type 2 Rep proteins on human chromosome 19.

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