A novel terminal resolution-like site in the adeno-associated virus type 2 genome

Abstract

The adeno-associated virus 2 (AAV) contains a single-stranded DNA genome of which the terminal 145 nucleotides are palindromic and form T-shaped hairpin structures. These inverted terminal repeats (ITRs) play an important role in AAV DNA replication and resolution, since each of the ITRs contains a terminal resolution site (trs) that is the target site for the AAV rep gene products (Rep). However, the Rep proteins also interact with the AAV DNA sequences that lie outside the ITRs, and the ITRs also play a crucial role in excision of the proviral genome from latently infected cells or from recombinant AAV plasmids. To distinguish between Rep-mediated excision of the viral genome during rescue from recombinant AAV plasmids and the Repmediated resolution of the ITRs during AAV DNA replication, we constructed recombinant AAV genomes that lacked either the left or the right ITR sequence and one of the Rep-binding sites (RBSs). No rescue and replication of the AAV genome occurred from these plasmids following transfection into adenovirus type 2-infected human KB cells, as expected. However, excision and abundant replication of the vector sequences was clearly detected from the plasmid that lacked the AAV left ITR, suggesting the existence of an additional putative excision site in the left end of the AAV genome. This site was precisely mapped to one of the AAV promoters at map unit 5 (AAV p5) that also contains an RBS. Furthermore, deletion of this RBS abolished the rescue and replication of the vector sequences. These studies suggest that the Rep-mediated cleavage at the RBS during viral DNA replication may, in part, account for the generation of the AAV defective interfering particles.

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

“…We surmise that excision and packaging of the subunit-length AAV genomes also occur, but the progeny virions are not infectious presumably because excision within the viral sequences would effectively abolish viral transcription and/or replication. We have indeed identified novel TRS-like sites that map outside the AAV ITRs that are involved in the excision and replication of the vector sequences (43). That AAV genomes containing only one ITR are packaged into progeny virions provides at least one clue as to how the naturally occurring defective interfering AAV particles might be generated and why only one of 100 to 200 AAV particles is infectious (2,26).…”

“…4a, panel B). A plausible mechanism that involves the Rep-mediated cleavage at a functional trs-like site in close proximity of the AAVp5 promoter sequences leading to vector rescue from plasmids that lack only the left ITR has been suggested by our recent studies (43). 12) were analyzed with the AAV (A) and the Ap r (B) DNA probes as described in the legend to Fig.…”

“…The S-sequence was inserted between the XbaI and BalI sites of plasmid pXS-27 described previously (44) to generate plasmid pXS-31. To construct plasmid pXS-32, together with the S-sequence, the XbaI-HindIII insert from a plasmid, pXS-30, which contained the PvuII-XbaI fragment from plasmid pSub201 cloned between the XbaI-EcoRV sites of pBluescriptSK(ϩ) but lacked the AAV left ITR, was ligated between the BalI-HindIII sites of a plasmid, pXS-29 (43), which contained the PvuII-XbaI fragment of plasmid pSub201 cloned into the XbaI-HincII sites of pBluescriptSK(ϩ) but lacked the viral right ITR. Standard cloning techniques were used for constructing all recombinant plasmids (31).…”

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

“…The resulting plasmids, designated pXS-69A, pXS-69B, and pXS-69C, respectively, were digested with XbaI and HindIII, and these fragments were used to replace the XbaI-HindIII fragment in plasmid pXS-26B. Plasmid pXS-48B was constructed by replacing the XbaI-HindIII fragment of pXS-26B, using the XbaI-HindIII fragment from pXS-47, also described previously (62). The wt AAV coding sequences flanked by the Ad5 ITRs were cloned between the BalI sites in plasmid pSub201 to generate plasmid pXS-37.…”

“…Both the secondary structure element of the ITR and a specific sequence at trs are required for the recognition and efficient cleavage by the Rep proteins (52,53), although a low-level cleavage can occur in the absence of the secondary structure (27). However, recent in vitro studies have documented that the purified Rep68 protein binds not only to the ITR but also to linear DNA sequences, such as the A sequence, and p5 and p19 promoter sequences (24,27,37,62). The Rep-binding site (RBS) in the p5 promoter between the TATA box and the transcription start site is responsible for the repression of expression of Rep68 (24,37).…”