Site-Specific Integration of Adeno-Associated Virus Involves Partial Duplication of the Target Locus

Site-specific integration of adeno-associated virus involves partial duplication of the target locus Els Henckaertsa, Nathalie Dutheila, Nadja Zeltnerb, Steven Kattmanc, Erik Kohlbrennerb, Peter Wardd, Nathalie Cle´ mentb, Patricia Rebollob, Marion Kennedyc, Gordon M. Kellerc, and R. Michael Lindena,b,1 aDepartment of Infectious Diseases, King’s College London School of Medicine, London SE1 9RT, United Kingdom; Departments of bGene and Cell Medicine and dMedicine, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029; and cMcEwen Centre for Regenerative Medicine, University Health Network, Toronto, ON, Canada M5G 1L7 Edited by Kenneth I. Berns, University of Florida College of Medicine, Gainesville, FL, and approved March 9, 2009 (received for review July 21, 2008) A variety of viruses establish latency by integrating their genome identified within AAVS1 (14). The MBS85 protein is thought to be into the host genome. The integration event generally occurs in a involved in the regulation of actin–myosin fiber assembly, and its nonspecific manner, precluding the prediction of functional con- translation initiation start codon is located only 17 nt downstream sequences from resulting disruptions of affected host genes. The of the RBS (14, 15). The AAVS1 locus is also closely linked to the nonpathogenic adeno-associated virus (AAV) is unique in its ability muscle-specific genes TNNT1, encoding slow skeletal muscle tro- to stably integrate in a site-specific manner into the human MBS85 ponin T, and TNNI3, encoding cardiac troponin I (16). gene. To gain a better understanding of the integration mechanism The fact that AAVS1 is located in a highly gene-dense region and and the consequences of MBS85 disruption, we analyzed the that virtually all viral–cellular junctions are found within MBS85 molecular structure of AAV integrants in various latently infected highlights the potential complexity of the integration mechanism human cell lines. Our study led to the observation that AAV and raises the question about the possible consequences of AAV integration causes an extensive but partial duplication of the latency (i.e., MBS85 disruption). With the help of an extensive target gene. Intriguingly, the molecular organization of the inte- library of previously identified viral–cellular junctions, it has be- grant leaves the possibility that a functional copy of the disrupted come clear that most of the integration sites characterized to date target gene could potentially be preserved despite the resulting lie within the first exon and intron of MBS85, possibly leaving 1 rearrangements. A latently infected, Mbs85-targeted mouse ES cell allele undisrupted (summarized in ref. 17). Besides these observa- MICROBIOLOGY line was generated to study the functional consequences of the tions, many aspects of this unique viral strategy remain elusive. observed duplication-based integration mechanism. AAV-modified In this study, we investigated the AAV insertion profile in various ES cell lines continued to self-renew, maintained their multilineage latently infected human cell lines and observed that AAV integrates differentiation potential and contributed successfully to mouse via a mechanism that includes the partial duplication of the target development when injected into blastocysts. Thus, our study locus, potentially preserving a functional copy of the disrupted reveals a viral strategy for targeted genome addition with the target gene. We took advantage of our previous observation that apparent absence of functional consequences. the AAVS1 locus is conserved in the mouse (15) and generated a latently infected mouse ES cell line to study the functional conse- embryonic stem cells ͉ MBS85 ͉ gene targeting ͉ Rep ͉ AAVS1 quences of the observed duplication-based site-specific integration event. AAV-modified ES cell lines continued to self-renew, main- ild-type adeno-associated virus has adopted a lifestyle that is tained their multilineage differentiation potential, and contributed Wunique among eukaryotic viruses. This nonautonomous successfully to mouse development when injected into blastocysts. parvovirus has evolved to efficiently replicate in cells that have been Based on our findings, we propose a mechanism that could explain infected with helper viruses (e.g., adeno- or herpesviruses) (1). In how this nonpathogenic virus can integrate into one of the most the absence of helper virus infection, AAV can establish latency densely populated regions within the human genome in the absence through site-specific genome integration into human chromosome of apparent deleterious effects. 19 at 19q13.42 (2, 3). It is well established that AAV-mediated site-specific integration Results requires the AAV Rep78/68 proteins in trans (4, 5), a cis-acting viral Identification of the Viral–Cellular Junctions in Various AAV-Infected DNA sequence, which consists of a tetranucleotide repeat called Human Cell Lines. Latently infected HeLa cell lines were generated the Rep-binding site (RBS) (6) and a 33-nt cellular sequence by using standard procedures. The junctions between the left ITR present at the integration site, termed AAVS1 (7). This sequence of AAV and cellular DNA were identified by linker-mediated (LM) consists of a RBS and terminal resolution site (TRS), 2 motifs that PCR technology (18). Because the endonuclease activity of Rep within the ITRs of AAV (6, 8) together serve as the replication initiates AAV-mediated targeted integration (19), we have arbi- origin (9). The large Rep proteins can simultaneously bind the trarily designated the second T residue of the MBS85 TRS motif cellular and viral RBS, suggesting a mechanism of site-specific (GGTTGG), as the nucleotide number 1. The left junction in the integration that is based on Rep-mediated tethering of the AAV genome to the AAVS1 sequence (10). The next step in the integration process involves Rep-mediated site-specific nicking of the Author contributions: E.H., N.D., and R.M.L. designed research; E.H., N.D., N.Z., S.K., E.K., AAVS1 TRS, generating a free DNA 3Ј-OH and a covalent 5Ј and P.W. performed research; N.C., P.R., M.K., and G.M.K. contributed new reagents/ analytic tools; E.H., N.D., N.Z., S.K., E.K., P.W., M.K., G.M.K., and R.M.L. analyzed data; and DNA–Rep complex, similar to the initiation of AAV DNA repli- E.H. and R.M.L. wrote the paper. cation (11). The subsequent steps remain to be elucidated, although The authors declare no conflict of interest. the requirement of a functional AAV replication origin within This article is a PNAS Direct Submission. AAVS1 is indicative for the involvement of AAVS1 replication Freely available online through the PNAS open access option. (7). This Rep-induced replication is thought to be at the basis of 1To whom correspondence should be addressed at: Department of Infectious Diseases, the previously hypothesized amplification of the integration locus King’s College London School of Medicine, London SE1 9RT, United Kingdom. E-mail: (12, 13). [email protected]. Interestingly, a gene called protein phosphatase 1 regulatory in- This article contains supporting information online at www.pnas.org/cgi/content/full/ hibitor subunit 12C or MBS85 (myosin-binding subunit 85) was 0806821106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806821106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 23, 2021 Fig. 1. Molecular structure of site-specifically integrated wtAAV2 isolated from different latently infected human cell lines. Black boxes represent the first 3 exons of the MBS85 gene. The horizontal dashed line indicates that the sizes are not proportional. Vertical dashed lines indicate the junctions between MBS85 and wtAAV2. White boxes represent the left (AAV-L) and right (AAV-R) side of integrated wtAAV2. Numbers indicate the nucleotide positions of the junctions relative to the AAVS1 TRS motif. first cell line, HeLa-T1, is located in MBS85 at a significant distance from the cellular TRS (12.9 kb) and the viral ITR is missing the first 86 nt [Fig. 1 and supporting information (SI) Fig. S1]. In HeLa-T2 cells, the left junction is located in MBS85 at 14 kb from the TRS, and the viral ITR has been deleted by the first 109 nt (Fig. 1 and Fig. S1). The junctions between cellular DNA and right AAV ITR, identified by direct PCR, are located in MBS85 at 13 and 4 nt downstream from the TRS motif in, respectively, HeLa-T1 and -T2 cells (Fig. 1). The right ITRs are also partially deleted (Fig. S1). In HeLa-T1 cells, we isolated a second left and right junction, at, Fig. 2. Site-specific integration of wtAAV2 in HeLa cells occurs through respectively, 12 and 0.3 kb downstream from the TRS (Fig. 1 and partial duplication of the target site. (A) The duplication hypothesis. Sizes of Fig. S1). Based on the observed positions of the left viral–cellular the restriction fragments that hybridize to the MBS85-specific (pRVK, forward junctions, we designed a direct PCR technique using different hatched box) and/or wtAAV2 (backward hatched box) probes are shown for forward primers located at 2-kb intervals in MBS85 combined with the disrupted (Top) and undisrupted allele (Bottom). Black and gray boxes a fixed reverse primer in AAV. Using this method, we identified the respectively represent MBS85 exons and integrated wtAAV2. The dashed line left junction in a third cell line, HeLa-T3, at 9.3 kb downstream indicates that the sizes of the introns are not proportional. (Center) A sche- from the TRS (Fig. 1). The right junction in this cell line is located matic representation of the PCRs showing duplication of MBS85 sequences. (B) Southern blot analysis of EcoRI-digested genomic DNA from HeLa and in MBS85 at 24 nt downstream from the TRS (Fig. 1). Both left and HeLa-T3 cells using the MBS85 or wtAAV2 probe. Cohybridization is indicated right ITRs of the provirus are partially deleted (Fig.

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