Highly divergent integration profile of adeno-associated virus serotype 5 revealed by high-throughput sequencing

Abstract

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.

FIG 1

AAV-5 genome organization, experimental design, and structure of integrant junctions. (A) AAV-5 genome features. Inverted terminal repeats (green) form the ends of the single-strand 4.6-kb viral genome. Three promoters (P7, P19, and P41) drive expression of two genes, Rep (red) and Cap (blue). Viral replication protein binding sites (gray arrows) are located in each ITR. P1 and P2 (black arrows) are approximate locations for sequencing primer 1 and 2 binding; SP1 is biotinylated (gray circle). (B) Experimental design, including IC-Seq outline. HeLa cells infected with wtAAV-5 were grown for 3 weeks prior to DNA extraction. Genomic DNA was sonicated, blunted, A-tailed, and ligated to T-tailed asymmetric linkers. Integrations were amplified by seminested ligation-mediated PCR, incorporating bead pull-down target enrichment, followed by linker cleavage and Illumina linker ligation and paired-end high-throughput sequencing. (C) Structure of captured AAV-5 junctions demonstrating viral genome sequence (green), host DNA (red), and linker (blue). Three representative junction sequences, including 20 nucleotides on either side, are displayed. (D) Genomic location and features of representative AAV-5 junctions. The base pair length of the viral genome sequence (v) and chromosomal fragment (c) are displayed; the E value describes number of matches expected by chance in the human genome as determined by NCBI blastn. Breakpoints in the viral genome occurred in the inverted terminal repeats (ITRs).

FIG 2

Genome-wide distribution of AAV-5 integration. (A) Summary of high-throughput AAV-5 integration data. (B) Unique AAV-5 integration events per mappable megabase of each human chromosome, adjusted for copy number. (C) Genome-wide view of unique AAV-5 integration events (blue bars) and computationally identified AAV-5 integration hot spots (red). Darkness, size, and proximity to the center correspond with increasing integrations per hot spot. Chromosomal size and banding pattern is displayed in the external ring. (D) Density profile, at 10-bp intervals, of unique integrations in an 800-bp region around the largest AAV-5 hot spot.

FIG 3

AAV-5 integration is associated with specific gene regions and activity markers. (A) Fold enhancement compared to a random distribution, or relative frequency, of AAV-5 integration events (black) and AAV-5 integration hot spots (gray) in genes and specific gene regions. Dashed line indicates frequency expected in a random model. **, P < 0.001 (determined using a permutation test). (B) Percentage of AAV-5 hot spots (black) in transcription level gene groups, compared to a distribution based on a random model (gray). (C) Enrichment of AAV-5 integrations (black) and hot spots (gray) in several activity-related markers, displayed as relative frequency. Dashed line indicates expected frequency based on a random model. **, P < 0.001 (determined using a permutation test); +, enrichment that approaches infinite, given an estimated real value for graphical display (see Materials and Methods).

FIG 4

AAV-5 integration is associated with viral replication protein binding and endonuclease sites. (A) Integrations per chromosome as a function of Rep binding sites per chromosome, as defined by GAGC dimers, adjusted for locus copy number. Simple linear regression and R² value results are shown. (B) Enrichment of AAV-5 integrations (black) and hot spots (gray) in Rep binding sites (GAGCx2) and endonuclease sites, displayed as relative frequency. Dashed line indicates expected frequency based on a random model. **, P < 0.001 (determined using a permutation test); +, enrichment that approaches infinite, given an estimated real value for graphical display (see Materials and Methods).

FIG 5

Discovery of a large AAV-5 consensus integration motif. (A) Sequence LOGO diagram of the 39-bp motif associated with AAV-5 integration hot spots. Stack height indicates information content, in bits, while letter size denotes the probability of the letter occurring in that position. Labeled bars display apparent AAV-5 functional correlation. (B) The sequences of the top five supported AAV-5 integration motifs associated with hot spots. Sites are ranked by P value, as determined by the probability of a random sequence generating the same or higher match score. Labeled bars indicate apparent AAV-5 functional correlation. (C) Arrangement of AAV-5 integrations around consensus motif. All supporting loci are arranged with the beginning, nuclease cleavage site, of the consensus (gray arrow) at position zero, and associated integrations are displayed. The mean position of integrations is portrayed (gray circle) and the standard error of the mean (SEM) span is indicated (black bars). (D) Proposed Rep-mediated model of AAV-5 integration. (Diagram 1) Rep binding sites (gray arrows) and endonuclease sites (gray box) are present in the human genome. (Diagram 2) AAV-5 Rep proteins oligomerize, possibly into opposing ring structures, on the Rep binding sites. The helicase domains, linker domains, and DNA binding/endonuclease domains are depicted by red, orange, and yellow, respectively. (Diagram 3) The AAV-5 Rep complexes nick endonuclease-cleavable sites. Helicase activity is low, and complexes are unable to traverse substantial distances. Thus, the stalled complexes deliver AAV-5 genomes only to the immediate vicinity of endonuclease sites.