Adeno-associated virus site-specifically integrates into a muscle-specific DNA region

Abstract

The nonpathogenic human virus adeno-associated virus type 2 (AAV) has evolved the potentially unique strategy to establish latency by site-specifically integrating its genome into human chromosome 19 (19q13.3-qter) at a locus designated AAVS1. This nonhomologous, site-specific recombination of viral DNA with the human genome provides a basis for developing targeted gene therapy vectors. To assess whether the region surrounding AAVS1 might have contributed to the selection of the specific integration site, we have investigated this locus. Here, we show that AAVS1 is closely linked to the slow skeletal troponin T gene, TNNT1, which has been mapped previously to 19q13.4. In support of this idea, we demonstrate that site-specific AAV DNA integration can result in the formation of TNNT1-AAV junctions. The question now arises whether muscle represents a natural target tissue for latent AAV infection. This possibility is of additional interest in view of recent observations that muscle tissue is particularly well suited for AAV-mediated gene transfer. The question also occurs whether latent infection by AAV can lead to phenotypic changes of the multinucleated muscle fiber cells.

Figure 1

AAVS1 and TNNT1 are linked. (A) EcoRI maps of two cosmids (31925 and 31855), a plasmid isolated from a genomic library (pS1), and a bacterial artificial chromosome (BC788605) are shown. The cosmids were preselected for containing AAVS1. pS1 was selected based on hybridization to AAVS1 probes. Gray boxes indicate EcoRI fragments for which independent DNA sequences are available [AAVS1, accession no. S51329, Kotin et al. (13); TNNT1, accession nos. AJ011712 and AJ011713]. The dark gray box indicates cosmid 31855. The DNA sequence of this cosmid has been made available by the human genome center at LLNL (AC005782) and is in agreement with the published AAVS1 sequence (S51329). The first 8 kb of this cosmid are graphically displayed. The sequence starts in AAVS1 at nucleotide 991 and extends for 35 kb into the centromeric region. The cosmids were digested with EcoRI and hybridized to probes indicated in the bottom part. The EcoRI fragments were aligned with help of the map of BC788605 (URL: http://www-bio.llnl.gov). In BC788605, the EcoRI sites are indicated by vertical lines, and the distances between them are shown in kb. In addition, HindIII (H) sites are shown. The positions of the HindIII sites are deduced from published sequences (for TNNT1 and AAVS1) and from sequence data generated for this study (for AAVS1). H*: this HindIII site was deduced from the published cardiac troponin I gene (TNNI3) sequences (30, 34). The HindIII site within TNNI3 was included based on the recent observation by Barton et al. (30) that the TNNT1 gene is closely linked to the TNNI3 gene. (B) Hybridization probes used for this study. AAVS1 probes: AAVS1pRVK: EcoRI-KpnI (nucleotides 1–3536 in S51329) and AAVS1pPD: DraI-PvuII (nucleotides −1723 to −1970 relative to S51329). TNNT1 probes: TNNT1 5′, nucleotides 56–663 (27); TNNT1 3′, nucleotides 663–896 (27); TNNT1ex11–13, nucleotides 562–754 (27). (C and D) Genomic DNA isolated from blood and from HEK293 cells (293) and cosmid DNA (cosmids 31925, 31855) as well as plasmid pS1 DNA was digested with either EcoRI (Fig. 1C) or HindIII (D) and separated by gel electrophoresis. Southern analysis was performed by using AAVS1 (pRVK and pPD) and TNNT1 (TNNT1 5′) probes. Ethidium bromide (EtBr) stainings of the gels before hybridization are shown where indicated. Molecular weights (M_r) are indicated in kb. Note, the genomic and cosmid DNA samples were run on separate gels to avoid cross-contaminations. The sizes of the genomic bands were determined from these independent Southern blots and are indicated in kb. The hybridization patterns were consistent with the EcoRI and HindIII maps shown in A.

Figure 2

wtAAV2 DNA integration can lead to the disruption of both AAVS1 and TNNT1. Southern blots of cellular DNA (ca. 10 μg) isolated from blood (negative control) and from cells latently infected with AAV (M19, M21, 7374, HA16) are shown. The genomic DNA was digested with either EcoRI (A) or HindIII (B). The blot was first hybridized to the 3′TNNT1 cDNA probe followed by hybridization to an AAV probe and subsequently to AAVS1pRVK. Between hybridizations, the blot was stripped (50% formamide, 2 h, 65°C) and the membrane was exposed to a PhosphorImager screen for 12 h to quantify remaining label. The exposure times are indicated for each blot. Cohybridization of AAV and TNNT1 is indicated by arrows (A, M19; B, M19, HA16) and of AAV, AAVS1, and TNNT1 by ▴ (A, HA16). Potential cohybridization of AAV and TNNT1 in 7374 cells was seen after EcoRI digestion (A); however, no TNNT1 disruption was apparent after HindIII digestion (B). Note, the variations in hybridization intensities with AAV as well as with AAVS1 probes are consistent with previous observations (see for example ref. 29)

Figure 3

The plasmid pHA16–3 contains viral–cellular junctions involving both AAVS1 and TNNT1 sequences. (A) A schematic representation of viral-cellular junctions in the plasmid pHA16-3 is shown. The plasmid pHA16-3 was isolated from a genomic library of latently infected HeLa cells (HA16C5) by selection for hybridization to an AAV probe. Subsequently, the viral–cellular junctions were sequenced. pHA16-3 contains a 14-kb insert with one junction between AAV2 (light gray) and AAVS1 (dark gray) and a second one between AAV (light gray) and TNNT1 (intermediate gray). At each junction, one nucleotide (white box) is common to both sequences. Underlined nucleotide numbers correspond to sequences at the recombination junctions (AAV2, accession no. AF043303; TNNT1, accession no. AJ011713; AAVS1, accession no. S51329). Nucleotide 1046 in TNNT1 corresponds to the cloning site. The DNA sequences between the junctions have not been determined (thin line). (B) DNA sequences at the AAV-TNNT1 junction are shown. The AAV RBS and TRS sites are indicated.

Figure 4

The organization of the troponin genes is shown relative to AAVS1. Boxes within TNNI3 and TNNT1 indicate the positions of the exons (30). Dark boxes indicate coding regions within exons, and open boxes indicate noncoding regions. The positions of TRS and RBS are indicated as lines within AAVS1. The sizes are shown in kb.