Adeno-associated virus type 2 DNA replication in vivo: mutation analyses of the D sequence in viral inverted terminal repeats

Abstract

The adeno-associated virus type 2 (AAV) genome contains inverted terminal repeats (ITRs) of 145 nucleotides. The terminal 125 nucleotides of each ITR form palindromic hairpin (HP) structures that serve as primers for AAV DNA replication. These HP structures also play an important role in integration as well as rescue of the proviral genome from latently infected cells or from recombinant AAV plasmids. Each ITR also contains a stretch of 20 nucleotides, designated the D sequence, that is not involved in HP structure formation. We have recently shown that the D sequence plays a crucial role in high-efficiency rescue, selective replication, and encapsidation of the AAV genome and that a host cell protein, designated the D sequence-binding protein (D-BP), specifically interacts with this sequence (X.-S. Wang, S. Ponnazhagan, and A. Srivastava, J. Virol. 70:1668-1677, 1996). We have now performed mutational analyses of the D sequences to evaluate their precise role in viral DNA rescue, replication, and packaging. We report here that 10 nucleotides proximal to the HP structure in each of the D sequences are necessary and sufficient to mediate high-efficiency rescue, replication, and encapsidation of the viral genome in vivo. In in vitro studies, the same 10 nucleotides were found to be required for specific interaction with D-BP, but viral Rep protein-mediated cleavage at the functional terminal resolution site is independent of these sequences. These data suggest that AAV replication and terminal resolution functions can be uncoupled and that the lack of efficient replication of AAV DNA may not be a consequence of impaired resolution of the viral ITRs. These studies further illustrate that the D sequence-D-BP interaction plays an important role in the AAV life cycle and indicate that it may be possible to develop the next generation of AAV vectors capable of encapsidating larger pieces of DNA.

Enhanced Transgene Expression from Recombinant Single-Stranded D-Sequence-Substituted Adeno-Associated Virus Vectors in Human Cell Lines In Vitro and in Murine Hepatocytes In Vivo

“…Given these built-in features, it would appear that the WT single-stranded AAV is less than ideal to be used as a recombinant vector for gene therapy to achieve high-level transgene expression. Our initial attempts to develop rAAV vectors devoid of both of the D sequences in one rAAV genome did not succeed, as these genomes failed to undergo encapsidation into rAAV capsids (18)(19)(20). In the present studies, however, in which we deleted only one of the two D sequences in two respective plasmids, abundant rescue followed by replication and encapsidation of the viral genomes ensued, albeit at ϳ50% efficiency because only single-polarity S-sequence-substituted progeny AAV genomes are generated and packaged, in contrast to their unmodified counterparts, which are known to generate and encapsidate progeny strands of both polarities that are packaged in mature particles with equal frequency.…”

“…The variation in transduction efficiencies was not affected when cells were pretreated with MG132, a proteasome inhibitor, or Try23, a specific inhibitor of cellular epidermal growth factor receptor (EGFR) protein kinase (Fig. 4B), both of which are known to significantly increase the transduction efficiency of ssAAV2 vectors (13,20), which indicates that neither intracellular trafficking nor proteasome degradation is involved. Not surprisingly, coinfection with adenovirus type 2 (Ad2) led to similar levels of increase in transgene expression mediated by the WT and the two mutant vectors in HEK293 cells (Fig.…”

“…1A, bottom). The S sequence was chosen due to the fact that this specific oligonucleotide does not interact with the cellular proteins that bind with the D sequences (6), and that the S sequence does not compete with the D sequence (20). In addition, in each of the rAAV genomes, the terminal resolution sites (trs) in both ITRs remain functional (18)(19)(20), and the entire vector genome size is larger than 2.8 kb, which exceeds the packaging capacity of self-complementary AAV (scAAV) vectors (9,10), to ensure that only virions containing ssAAV genomes were packaged (28)(29)(30).…”

“…Following purification and mass spectrometry, the cellular protein binding with the ssD[ϩ] sequence was found to have partial amino acid homology to NF-B-repressing factor (NRF), a negative regulator of transcription (NRF) (17). Since both the cellular proteins binding with either the ssD[Ϫ] or ssD[ϩ] sequence have the potential to inhibit transgene expression mediated by ssAAV vectors and since we have previously reported that the removal of the D sequences from the viral genome impairs rescue, replication, and encapsidation of AAV DNA (18)(19)(20), we wished to examine whether restoration of one D sequence in the ssAAV genome might not only restore rescue, replication, and encapsidation but also enhance transgene expression from ssAAV vectors. We report here that the ssD[ϩ]-sequence-substituted ssAAV genomes could be successfully packaged into rAAV vectors and that the transduction efficiency of these vectors was significantly higher than that of conventional ssAAV vectors.…”

Rescue and Autonomous Replication of Adeno-Associated Virus Type 2 Genomes Containing Rep-Binding Site Mutations in the Viral p5 Promoter

“…Encapsidation and biological activity of AAV progeny virions. Equivalent amounts of plasmids pXS-70A and pSub201 were transfected in 293 cells as previously described (59)(60)(61)(62). Approximately 72 h posttransfection, cells were harvested and progeny virions were purified by one round of CsCl equilibrium density gradient followed by exhaustive digestion with DNase I as previously described (21).…”