Characterization of the mouse adeno-associated virus AAVS1 ortholog

Abstract

The nonpathogenic human adeno-associated virus (AAV) has developed a mechanism to integrate its genome into human chromosome 19 at 19q13.4 (termed AAVS1), thereby establishing latency. Here, we provide evidence that the chromosomal signals required for site-specific integration are conserved in the mouse genome proximal to the recently identified Mbs85 gene. These sequence motifs can be specifically nicked by the viral Rep protein required for the initiation of site-specific AAV DNA integration. Furthermore, these signals can serve as a minimal origin for Rep-dependent DNA replication. In addition, we isolated the mouse Mbs85 proximal promoter and show transcriptional activity in three mouse cell lines.

FIG. 1.

(A) Nucleotide sequence alignment of the human, mouse, and monkey AAVS1 sequences (MacVector; Oxford Molecular Limited). The TRS motif is italicized and underlined; the RBS motif is in bold. M and S indicate the translation initiation site and serine residue, respectively. Dashes indicate gaps between sequences. (B) Schematic representation of the genetic organization of the region surrounding the TRS and RBS motifs within the mouse (top) and human (bottom) genomes. The lengths and distances between the TNNI3, TNNT1, MBS85, and DRC3 genes are shown in kilobases. The positions of the TRS-RBS motifs are indicated by stars. The arrows indicate the direction of transcription. The center section shows the genomic organization of the mouse and human MBS85 transcripts. Black boxes indicate coding regions, white boxes indicate noncoding regions. (C) Tissue distribution of Mbs85 mRNAs in the mouse. A multiple tissue Northern blot derived from mouse tissues was hybridized with a cDNA probe consisting of exons 5 to 22 (ex5-22) of the mouse Mbs85 cDNA. The ex5-22 probe was generated by digestion of clone BF540586 with EcoRI/HindIII. The bottom panel shows hybridization with a β-actin cDNA probe. The blot was stripped between hybridizations. Sizes are indicated in kilobases.

FIG. 2.

Cell-free endonuclease assays. (A) Fully double-stranded and (B) partially single-stranded origin substrates (as a specificity control) (5 fmol) containing the AAV, human (hS1), mouse (mS1), and mouse origin mutant (mS1mut) sequences were incubated for 45 min at 37°C in either the absence (−) or presence of 1 pmol of AAV Rep68 protein (68) or 1 pmol of AAV Rep68 endonuclease mutant (Y156F). M, 5′ end-labeled marker oligonucleotides corresponding in sequence and length to the expected reaction product (14 nt). (C) Synthetic oligonucleotide substrates used in the nicking and replication assays. Locations of the TRS (^) and RBS (boxed) are indicated. The TRS-containing strand was first kinase labeled (positions of the 5′ radiolabel are indicated by stars) and then annealed to its complementary strand.

FIG. 3.

Cell-free DNA replication assay. Schematic representation of the pBSori substrates used in this study (bottom). The AAV, human (hS1), and mouse (mS1) origins (consisting of the TRS and RBS sequences) were cloned into pBluescript via XbaI and SalI sites. Prior to the assay, plasmids were linearized with XmnI. Each linear, origin-containing substrate was incubated in the presence (+) or absence (−) of AAV Rep68 protein (top). An arrow indicates the position of the full-length replication product. Sizes are in base pairs.

FIG. 4.

(A) Nucleotide sequence alignment of the human and mouse AAVS1 sequences upstream of the ATG (MacVector). The recognition sites for the TRS, RBS, translation initiation codon (M), and transcription factor binding elements for Sp1, CRE, and ATF-2 (white boxes) are indicated. Dashes indicate gaps between the human and mouse sequences. The SmaI sites present in the human AAVS1 sequence are denoted by the line above, and the NaeI sites present in the mouse AAVS1 sequence are underlined. (B) Expression of Mbs85 by Northern blot analyses. The Northern blot of C2C12, N2A, and NIH 3T3 cells was hybridized with a cDNA probe consisting of exons 5 to 22 (top panel). The blot was stripped and rehybridized with a β-actin cDNA probe (bottom panel). Positions of the 28S and 18S rRNAs are indicated. (C) Transcriptional activity of the mouse Mbs85 proximal promoter. Plasmid pDsRed2-N1 (red fluorescent protein under the cytomegalovirus promoter; Clontech) and the sense and antisense plasmids were transfected into C2C12, NIH 3T3, and N2A cells. Forty-five hours posttransfection, the cells were visualized for red fluorescent protein expression and DAPI staining by using an epifluorescent microscope (Leica DMRA2) and a Hamamatsu digital camera. M.T., mock transfection.