Rescue and autonomous replication of adeno-associated virus type 2 genomes containing Rep-binding site mutations in the viral p5 promoter

Abstract

The Rep proteins encoded by the adeno-associated virus type 2 (AAV) play a crucial role in the rescue, replication, and integration of the viral genome. In the absence of a helper virus, little expression of the AAV Rep proteins occurs, and the AAV genome fails to undergo DNA replication. Since previous studies have established that expression of the Rep78 and Rep68 proteins from the viral p5 promoter is controlled by the Rep-binding site (RBS) and the YY1 factor-binding site (YBS), we constructed a number of recombinant AAV plasmids containing mutations and/or deletions of the RBS and the YBS in the p5 promoter. These plasmids were transfected in HeLa or 293 cells and analyzed for the potential to undergo AAV DNA rescue and replication. Our studies revealed that (i) a low-level rescue and autonomous replication of the wild-type AAV genome occurred in 293 but not in HeLa cells; (ii) mutations in the RBS resulted in augmented expression from the p5 promoter, leading to more efficient rescue and/or replication of the AAV genome in 293 but not in HeLa cells; (iii) little rescue and/or replication occurred from plasmids containing mutations in the YBS alone in the absence of coinfection with adenovirus; (iv) expression of the adenovirus E1A gene products was insufficient to mediate rescue and/or replication of the AAV genome in HeLa cells; (v) autonomously replicated AAV genomes in 293 cells were successfully encapsidated in mature progeny virions that were biologically active in secondary infection of HeLa cells in the presence of adenovirus; and (vi) stable transfection of recombinant AAV plasmids containing a gene for resistance to neomycin significantly affected stable integration only in 293 cells, presumably because rescue and autonomous replication of the AAV genome from these plasmids occurred in 293 cells but not in HeLa or KB cells. These data suggest that in the absence of adenovirus, the AAV Rep protein-RBS interaction plays a dominant role in down-regulating viral gene expression from the p5 promoter and that perturbation in this interaction is sufficient to confer autonomous replication competence to AAV in 293 cells.

FIG. 1

Schematic structure of the AAV genome containing various mutations and/or deletions in the RBS and the YBS in the viral p5 promoter. Mutations and deletions are denoted by the prefixes “m” and “d.” Asterisks indicate the deleted nucleotides. The AAV p5 promoter is represented by the closed circle, and the RNA start site is indicated by the arrow. The hairpin (HP) and the D sequences that constitute the viral ITRs are represented by open and closed rectangles, respectively. Each of the recombinant plasmids was constructed as described in Materials and Methods.

FIG. 2

Southern blot analyses of rescue and replication of AAV genomes containing various mutations and/or deletions in the RBS and YBS in 293 cells in the absence of adenovirus. Ten micrograms of each indicated plasmid DNA was transfected separately in approximately 70% confluent 293 cells in a 10-cm-diameter dish, and low-M_r DNA isolated at 24 h (lanes 1, 4, 7, 10, and 13), 48 h (lanes 2, 5, 8, 11, and 14), and 72 h (lanes 3, 6, 9, 12, and 15) posttransfection was digested with DpnI and analyzed on Southern blots, using an AAV-specific DNA probe. Autoradiography was performed at −70°C for 16 h. m and d denote the monomeric and dimeric replicative forms of the AAV genome.

FIG. 3

Southern blot analyses of rescue and replication of AAV genomes containing various mutations and/or deletions in the RBS and YBS in adenovirus-infected 293 cells. Ten micrograms of each indicated plasmid DNA was transfected separately in approximately 70% confluent 293 cells, and low-M_r DNA isolated at 24 h (lanes 1, 4, 7, 10, and 13), 48 h (lanes 2, 5, 8, 11, and 14), and 72 h (lanes 3, 6, 9, 12, and 15) posttransfection was digested with DpnI and analyzed on Southern blots, using an AAV-specific DNA probe as described in the legend to Fig. 2. Autoradiography was performed at room temperature for 15 min. m and d denote the monomeric and replicative forms of the AAV genome.

FIG. 4

Northern blot analyses of expression of AAV genes containing various mutations and/or deletions in the RBS and YBS in 293 cells. Total cellular RNA was isolated from mock-transfected cells or cells transfected with 10 μg of each indicated plasmid DNA in two separate experiments, and 20 μg RNA from each transfectant was analyzed on Northern blots, using an AAV-specific DNA probe. The three viral transcripts initiating from p5, p19, and p40 promoters are indicated. The same blots were stripped of the AAV probe and rehybridized with the GAPDH gene probe to ascertain the equivalence of RNA loads and transfer. Autoradiography was performed at −70°C for 72 h.

FIG. 5

DNA slot blot analyses of efficiency of plasmid transfection in 293 cells. Twofold serial dilutions of 2 μg each low-M_r DNA isolated from mock-transfected cells or cells transfected with 10 μg of each indicated plasmid DNA from the same two separate experiments described in the legend to Fig. 4 were analyzed on quantitative DNA slot blots, using an AAV-specific DNA probe as described in Materials and Methods. Autoradiography was performed at −70°C for 2 h.

FIG. 6

Southern blot analyses of rescue and replication of AAV genomes containing various mutations and/or deletions in the RBS and YBS in HeLa cells in the absence or presence of the adenovirus E1A gene products. Ten micrograms of each indicated plasmid DNA was transfected separately in HeLa cells, either in the absence (−pAd2-E1A) or in the presence (+pAd2-E1A) of an adenovirus E1A expression plasmid. Low-M_r DNA isolated at 72 h posttransfection was digested with DpnI and analyzed as described in the legend to Fig. 2.

FIG. 7

Southern blot analyses of replication of the AAV genome in secondary infections following autonomous rescue, replication, and encapsidation in 293 cells. Ten-micrograms of each of plasmids pXS-70A and pSub201 DNA was transfected separately in 293 cells, and progeny virions were purified on CsCl equilibrium density gradients as described in Materials and Methods. The viral stocks were used to infect HeLa cells in the presence of adenovirus. Low-M_r DNA isolated at 24 h (lanes 1 and 4), 48 h (lanes 2 and 5), and 72 h (lanes 3 and 6) postinfection was analyzed on Southern blots, using an AAV-specific DNA probe. The monomeric (m) and dimeric (d) replicative forms and the single strands of the AAV genome (ss) are indicated. Autoradiography was performed at −70°C for 2 h.

FIG. 8

Schematic structures of recombinant AAV plasmids containing the neo^r selectable marker gene and deletion or substitution in the D sequence in the viral ITRs. The hairpin (HP) and D sequences are denoted by the open and closed boxes, respectively. The adenovirus ITRs are denoted by boxes with vertical lines, and the neo^r gene is represented by cross-hatched boxes. Each of the recombinant plasmids was constructed as described in Materials and Methods.