Rescue and replication of adeno-associated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats: selective encapsidation of viral genomes in progeny virions

Abstract

The adeno-associated virus type 2 (AAV) genome can be successfully rescued from recombinant plasmids following transfection in adenovirus-infected human cells. However, following rescue, the AAV genome undergoes preferential replication and encapsidation, whereas little replication and packaging of the vector DNA sequences occur. In view of the crucial role in the rescue, replication, and packaging of the proviral genome played by the AAV inverted terminal repeats (ITRs), which consist of a palindromic hairpin (HP) structure and a 20-nucleotide stretch, designated the D-sequence, that is not involved in the HP formation, we evaluated the involvement of the individual ITRs as well as their components in the selective viral DNA replication and encapsidation. A number of recombinant AAV plasmids that contained deletions-substitutions in different regions of the individual ITRs were constructed and examined for their potential to allow rescue, replication, and/or packaging in adenovirus-infected human cells in vivo. The results reported here document that (i) two HP structures and one D-sequence are sufficient for efficient rescue and preferential replication of the AAV DNA, (ii) two HP structures alone allow a low-level rescue and replication of the AAV DNA, but rescue and replication of the vector DNA sequences also occur in the absence of the D-sequences, (iii) one HP structure and two D-sequences, but not one HP structure and one D-sequence, also allow rescue and replication of the AAV as well as the vector DNA sequences, (iv) one HP structure alone or two D-sequences, but not one D-sequence alone, allow replication of the full-length plasmid DNA, but no rescue of the AAV genome occurs, (v) no rescue-replication occurs in the absence of the HP structures and the D-sequences, (vi) in the absence of the D-sequences, the HP structures are insufficient for successful encapsidation of the AAV genomes, and (vii) the AAV genomes containing only one ITR structure can be packaged into biologically active virions. Thus, the D-sequence plays a crucial role in the efficient rescue and selective replication and encapsidation of the AAV genome. Furthermore, the D-sequence specifically interacts with a hitherto unknown host-cell protein that we have designated the D-sequence-binding protein (D-BP). These studies illustrate that the D-sequence-D-BP interaction constitutes an important step in the AAV life cycle.

A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle

“…The effective replication and encapsidation of the DD construct indicates the crucial role that the D sequence plays in these processes. This is consistent with observations that deletion of D sequences abolishes AAV replication and packaging (39,40,43). In addition, in vitro Rep68 binding and nicking assays are affected by mutant substrates that altered the trs sequence or spacing between A and D elements (23,34).…”

“…Rescue involves resolution of the ITR. The ITRs are the AAV rescue substrates and replication origins (39)(40)(41). Since the DD plasmids contain only a single modified ITR and replicated via an AAV mechanism in the presence of Rep proteins, we tested this substrate for the ability to rescue in vivo.…”

“…However, when one of the two ITRs is completely deleted or partially deleted in such a manner that it cannot be repaired by gene conversion, the substrate is not viable for virus production (29,32). Recently, a report has demonstrated that a cryptic trs in the AAV p5 promoter can partially substitute for replication if the left end ITR is completely deleted (39,40).…”

“…Efforts to further delineate the cis-acting DNA sequences necessary for AAV viability have determined that the 20-bp D sequence of the ITR plays a critical role in viral DNA rescue and replication. Deletion of the D sequence from both ITRs severely impairs AAV DNA replication (39,40,43). Furthermore, when infectious wild-type AAV plasmids are transfected into Ad-infected cells, only the AAV portion which contains the ITR and D sequence of the plasmid is capable of replication after rescue.…”

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

“…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.…”