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.

Parvovirus B19 integration into human CD36+ erythroid progenitor cells

“…NS1 possesses structural and enzymatic functions that are similar to the AAV Rep proteins, with a high degree of amino acid homology (Figure 1A). Activity of the replication proteins appears to be the major determinate for integration of AAV (Chiorini et al, 1996; Huser et al, 2014; Huser et al, 2010; Janovitz et al, 2013; Janovitz et al, 2014; Kotin et al, 1990). We hypothesized that B19V infection can result in DNA damage and vDNA integration into human host DNA, where genomic insertion sites are largely driven by the sequence affinity and activity of the NS1 replication protein.…”

“…Large-scale infections of 1.5×10 7 cells at 2000 ge/cell were carried out, and total DNA was harvested 24 hours post inoculation. Purified DNA from two samples was processed through the IC-Seq protocol (Figure 1E (Janovitz et al, 2013; Janovitz et al, 2014) described in the materials and methods) using B19V specific primers flanking the P6 promoter (illustrated in Figure 1D). The rigorous fragmentation, purification, and amplification steps of the IC-Seq protocol are designed to eliminate PCR artifact (see materials and methods).…”

“…For each replicate, three aliquots of 6 million CD36+EPCs, containing ~250 ug of genomic DNA in total were processed using the TC-Seq and IC-Seq protocols as previously described (Janovitz et al, 2013; Janovitz et al, 2014; Klein et al, 2011; Oliveira et al, 2012) (illustrated in Figure 1E). Briefly, aliquots were lysed in Proteinase K buffer (100 mM Tris (pH 8), 0.2% SDS, 200 mM NaCl, 5 mM EDTA) with 200 ug/mL Proteinase K. Genomic DNA was purified by phenol-chloroform extraction and ethanol precipitation.…”

“…Sequences of the pLinker primer and asymmetric linker oligos were described previously (Janovitz et al, 2013; Janovitz et al, 2014; Klein et al, 2011). The B19V primer sequences utilized were: P1: 5’-[Bio-TEG]CAGAGCAAAGAATGGCGGCGG-3’ and P2: 5’-GCGGGCTCGTTCACTCGGTC-3’ (approximate location illustrated in Figure 1D).…”

Use of Adeno-Associated Virus to Enrich Cardiomyocytes Derived from Human Stem Cells

“…Compared to genetic selection, 24 mitochondria labeling, 25 or surface marker-based cell sorting, 28-30 methods described here have several advantages. First, though incidences of human genome integration have been reported, [31][32][33][34] FACS analysis of AAV1-pTR-UF11-transduced iPSC derivatives with and without G418 selection. Cells were probed for cardiac-specific cTnT (n = 3 for each conditions; *p = 0.0007).…”