Creating a Novel Origin of Replication through Modulating DNA-Protein Interfaces

Viral integrations are important in human biology, yet genome-wide integration profiles have not been determined for many viruses. Adeno-associated virus (AAV) infects most of the human population and is a prevalent gene therapy vector. AAV integrates into the human genome with preference for a single locus, termed AAVS1. However, the genome-wide integration of AAV has not been defined, and the principles underlying this recombination remain unclear. Using a novel high-throughput approach, integrant capture sequencing, nearly 12 million AAV junctions were recovered from a human cell line, providing five orders of magnitude more data than were previously available. Forty-five percent of integrations occurred near AAVS1, and several thousand novel integration hotspots were identified computationally. Most of these occurred in genes, with dozens of hotspots targeting known oncogenes. Viral replication protein binding sites (RBS) and transcriptional activity were major factors favoring integration. In a first for eukaryotic viruses, the data reveal a unique asymmetric integration profile with distinctive directional orientation of viral genomes. These studies provide a new understanding of AAV integration biology through the use of unbiased high-throughput data acquisition and bioinformatics.

Section: Hotspots and Rep Binding Sites (Rbs)supporting

confidence: 80%

High-Throughput Sequencing Reveals Principles of Adeno-Associated Virus Serotype 2 Integration

Janovitz

Klein

Oliveira

et al. 2013

“…Plasmid pdsEMBL-CMV-E4orf6 (pMH210) was constructed by BamHI excision of the adenovirus E4orf6-coding sequence from pCMVE4orf6 (a kind gift from P. Hearing), treatment with the Klenow fragment to blunt the ends, and ligation into blunt-ended AgeI and NotI sites of pdsEMBLgfp4 (42). Plasmids pDD2-eGFP, pDD5-eGFP, pDD5ϩ2SNS-eGFP, and pDD2ϩ5NS-eGFP have been described previously and consist of a single wild-type or mutant ITR in DD form inserted along with a CMV-eGFP expression cassette into the pUC18 backbone (43,44). Plasmid pTRSn-CMV-eGFP was generated by substitution of a CMV-eGFP expression cassette, flanked by PstI and PciI restriction sites, for the majority of the genome in the snake AAV construct pSAAV (a kind gift from Peter Tijssen) via compatible PstI and NcoI restriction sites (45).…”

Section: Methodsmentioning

confidence: 99%

Insight into the Mechanism of Inhibition of Adeno-Associated Virus by the Mre11/Rad50/Nbs1 Complex

Lentz

Samulski

2015

Self Cite

Adeno-associated virus (AAV) is a dependent virus of the family Parvoviridae. The gene expression and replication of AAV and derived recombinant AAV (rAAV) vectors are severely limited (>10-fold) by the cellular DNA damage-sensing complex made up of Mre11, Rad50, and Nbs1 (MRN). The AAV genome does not encode the means to circumvent this block to productive infection but relies on coinfecting helper virus to do so. Using adenovirus helper proteins E1B55k and E4orf6, which enhance the transduction of AAV via degradation of MRN, we investigated the mechanism through which this DNA damage complex inhibits gene expression from rAAV. We tested the substrate specificity of inhibition and the contribution of different functions of the MRN complex. Our results demonstrate that both single-and double-stranded rAAV vectors are inhibited by MRN, which is in contrast to the predominant model that inhibition is the result of a block to second-strand synthesis. Exploring the contribution of known functions of MRN, we found that inhibition of rAAV does not require downstream DNA damage response factors, including signaling kinases ATM and ATR. The nuclease domain of Mre11 appears to play only a minor role in inhibition, while the DNA binding domain makes a greater contribution. Additionally, mutation of the inverted terminal repeat of the rAAV genome, which has been proposed to be the signal for interaction with MRN, is tolerated by the mechanism of inhibition. These results articulate a model of inhibition of gene expression in which physical interaction is more important than enzymatic activity and several key downstream damage repair factors are dispensable. A deno-associated virus (AAV) is a member of the genus Dependovirus within the family Parvoviridae. The virion is small (diameter, ϳ20 nm) and consists of a protein nucleocapsid containing a single-stranded DNA (ssDNA) genome of ϳ4.7 kb. The genome carries inverted terminal repeat (ITR) sequences at both ends, which self-anneal to form a T-shaped secondary structure (1, 2). Formation of this structure provides the 3= OH for secondstrand DNA synthesis, as well as the signal for genome packaging. Flanking ITRs are the only feature required in cis for replication and production of the virus. This property is exploited in recombinant AAV (rAAV) vectors, in which the coding region of the genome is replaced with a transgene expression cassette (3). The AAV genome carries two genes encoding replication (Rep) and capsid (Cap) proteins (4). Both are required for replication and packaging of AAV and are provided in trans for production of rAAV vectors. However, these proteins are not sufficient. Wildtype (wt) AAV and rAAV are dependent upon helper virus and host cell proteins for creation of a permissive cellular environment and DNA synthesis machinery, respectively (5, 6).Establishment of a permissive cellular environment for expression and replication of AAV requires mediation of the cellular DNA damage response (DDR). DDR signaling and repair pathways normally function...

“…30% of the population is seropositive for AAV-5 specific antibodies (2,5,6). In addition, it is the most divergent AAV yet discovered (4, 7) and, uniquely, does not cross-complement the replication of other serotypes (3,26,27). AAV-2 is known to integrate at a high frequency in the human genome, with preference for a single locus, AAVS1 (15,18,21).…”

Section: Discussionmentioning

confidence: 99%

“…Importantly, these sequences were separated by an appropriate number of nucleotides and were present in the same orientation as in the AAV-5 ITR. Recent studies have indicated that the AAV-5 ITR spacer and the three-dimensional structure of the terminal resolution site (TRS), where endonuclease cleavage occurs, may be critical to its functionality (27). Therefore, a number of elements critical to AAV-5 ITR function are represented in the newly described consensus integration motif.…”

Section: Discussionmentioning

confidence: 99%

“…Although the DNA sequence motif which binds AAV-5 Rep is similar to other serotypes, the endonuclease cleavage site is completely distinct (3). Underscoring this divergence, AAV-5 Rep protein does not cross-complement the replication of other serotypes (3,26,27). Thus, the primary driver of targeted viral integration differs between AAV-5 and the other known AAVs.…”

mentioning

confidence: 99%

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Highly Divergent Integration Profile of Adeno-Associated Virus Serotype 5 Revealed by High-Throughput Sequencing

Janovitz

Oliveira

Sadelain

et al. 2014

Adeno-associated virus serotype 5 (AAV-5) is a human parvovirus that infects a high percentage of the population. It is the most divergent AAV, the DNA sequence cleaved by the viral endonuclease is distinct from all other described serotypes and, uniquely, AAV-5 does not cross-complement the replication of other serotypes. In contrast to the well-characterized integration of AAV-2, no published studies have investigated the genomic integration of AAV-5. In this study, we analyzed more than 660,000 AAV-5 integration junctions using high-throughput integrant capture sequencing of infected human cells. The integration activity of AAV-5 was 99.7% distinct from AAV-2 and favored intronic sequences. Genome-wide integration was highly correlated with viral replication protein binding and endonuclease sites, and a 39-bp consensus integration motif was revealed that included these features. Algorithmic scanning identified 126 AAV-5 hot spots, the largest of which encompassed 3.3% of all integration events. The unique aspects of AAV-5 integration may provide novel tools for biotechnology and gene therapy. IMPORTANCE Viral integration into the host genome is an important aspect of virus host cell biology. Genomic integration studies of the small single-stranded AAVs have largely focused on site preferential integration of AAV-2, which depends on the viral replication protein (Rep).We have now established the first genome wide integration profile of the highly divergent AAV-5 serotype. Using integrant capture sequencing, more than 600,000 AAV-5 integration junctions in human cells were analyzed. AAV-5 integration hot spots were 99.7% distinct from AAV-2. Integration favored intronic sequences, occurred on all chromosomes, and integration hot spot distribution was correlated with human genomic GAGC repeats and transcriptional activity. These features support expansion of AAV-5 based vectors for gene transfer considerations.