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

Abstract

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.

Adeno-Associated Virus Type 2 Wild-Type and Vector-Mediated Genomic Integration Profiles of Human Diploid Fibroblasts Analyzed by Third-Generation PacBio DNA Sequencing

“…Expression of rep in the presence of rep-deficient rAAV vectors shifted targeting preferences from random integration back to hot spots in the neighborhood of consensus RBS in genomic open chromatin regions (19). Recent high-throughput DNA sequence analysis of AAVwt integration retrieved several orders of magnitude higher numbers of AAV2 integration sites, confirming and further extending our findings (20).…”

“…The latter had rarely been detected in HeLa cells before (19,20). In addition, a series of novel hot spots was detected, e.g., on chromosome 7q32.3 and on chromosome 5q31.2 (Table 1), only some of which have been described before at reduced frequencies in AAVwt-infected HeLa cells (20).…”

“…A number of hot spots from the previous HeLa data sets were not detected in human diploid fibroblasts. The top hot spots in HeLa cells on chromosome 5p13.3 (AAVS2) and on chromosome 3p25.1 (AAVS3) (19,20) displayed only three and one AAVwt integrations, respectively, in human fibroblasts, despite over 10-fold-higher total junction numbers.…”

Intégration des vecteurs AAV et mutagenèse insertionnelle

“…En effet, l'intégration de l'AAV sauvage peut avoir lieu dans une zone très étendue, en amont et en aval du site AAVS1, et entraîner des modifications profondes du génome cellulaire (délétions, duplications) ainsi que des délétions des extrémi-tés du génome viral. Il est ainsi impossible de connaître, a priori, les séquences virales présentes au niveau des jonctions avec l'ADN Figure 3A) [20][21][22]. Cela s'explique par la présence dans le génome cellulaire de nombreux sites RBS reconnus par les protéines Rep de l'AAV.…”

Proportion of transcriptionally active DNA virus integrants: a meta-analysis

“…In addition, other DNA viruses with transformation potential can integrate: Merkel cell polyomavirus (MCPV), human herpes 6 virus (HHV-6) and, in a lesser extent, adenovirus (AdV) [4][5][6]. The infection with these viruses is associated with a wide range of cancer types: HBV with hepatocarcinoma; HPV with virtually all cases of cervical cancer and in a significant proportion of other tumors, for instance in the head and neck; HHV-4 with a number of malignancies, in particular Burkitt and Hodgkin lymphomas, in both immunologically deficient and competent patients; HHV-8 DNA has been retrieved in all Kaposi sarcoma lesions even in immunocompetent hosts; MCPV is recognized as the causative agent of the Merkel cell carcinoma; HHV-6 has been involved in lymphoproliferative diseases; and AdV has been shown to transform rodent cells [1,[7][8][9].…”