Adeno-associated virus (AAV) type 5 Rep protein cleaves a unique terminal resolution site compared with other AAV serotypes

Abstract

Adeno-associated virus (AAV) replication depends on two viral components for replication: the AAV nonstructural proteins (Rep) in trans, and inverted terminal repeat (ITR) sequences in cis. AAV type 5 (AAV5) is a distinct virus compared to the other cloned AAV serotypes. Whereas the Rep proteins and ITRs of other serotypes are interchangeable and can be used to produce recombinant viral particles of a different serotype, AAV5 Rep proteins cannot cross-complement in the packaging of a genome with an AAV2 ITR. In vitro replication assays indicated that the block occurs at the level of replication instead of at viral assembly. AAV2 and AAV5 Rep binding activities demonstrate similar affinities for either an AAV2 or AAV5 ITR; however, comparison of terminal resolution site (TRS) endonuclease activities showed a difference in specificity for the two DNA sequences. AAV2 Rep78 cleaved only a type 2 ITR DNA sequence, and AAV5 Rep78 cleaved only a type 5 probe efficiently. Mapping of the AAV5 ITR TRS identified a distinct cleavage site (AGTG TGGC) which is absent from the ITRs of other AAV serotypes. Comparison of the TRSs in the AAV2 ITR, the AAV5 ITR, and the AAV chromosome 19 integration locus identified some conserved nucleotides downstream of the cleavage site but little homology upstream.

FIG. 1

In vitro replication. (A) The effect of the MBP-Rep fusion protein from AAV-2 or AAV-5 on DNA replication was measured by TLC (see Materials and Methods). One of two templates was tested in the in vitro replication assay: AAV2RnLacZ or AAV5RnLacZ (bars 1 to 3 and 4 to 6, respectively). Background incorporation was defined as the amount of incorporation measured in the absence of Rep protein (bars 1 and 4). (B) Effects of AAV2 and AAV5 MBP-Rep78 with pMAT50 on replication. Replication reactions were performed as described in Materials and Methods, and the replication products were resolved by agarose gel electrophoresis. AAV2 and AAV5 MBP-Rep78 proteins were included in lanes 2 and 3, respectively; lane 1, extract only, no Rep. The open-circular (O) and linear (L) forms of pMAT50 are indicated.

FIG. 2

Sequence and alignment of ITR and probes. (Top) Sequence of the P1 element contained within the SmaI subfragment of AAVS1 (nt 354 to 468) (37). (Middle) Sequence of the AAV2 ITR as previously described (34). (Bottom) Sequence of the AAV5 ITR as previously described (5). Linear probes used in the EMSA and TRS assays are indicated by brackets flanking the AAV2 and AAV5 ITR stem regions. Rep binding motifs are boxed and labeled. TRSs are boxed and labeled, and the site of cleavage in each is indicated by a vertical arrow.

FIG. 3

EMSAs. Gel-purified radiolabeled probes (50,000 cpm each) were incubated with 100 ng of purified AAV2 MBP-Rep78 or AAV5 MBP-Rep78 as indicated. In the absence of protein, no shift complex was detected (lanes 1, 6, 11, and 16). The addition of purified AAV2 MBP-Rep78 (lanes 2 and 12) or AAV5 MBP-Rep78 (lanes 7 and 17) resulted in the formation of a shift complex. Binding competition studies were done in the presence of a 5-fold (lanes 3, 8, 13, and 18), 10-fold (lanes 4, 9, 14, and 19), or 20-fold (lanes 5, 10, 15, and 20) molar excess of unlabeled probe. Quantitation of the shift complex was done with a scanning densitometer (Molecular Dynamics). Bound, bound probe; free, free probe.

FIG. 4

TRS specificity. In vitro-translated AAV2 Rep78 or AAV5 Rep78 was incubated with radiolabeled probe (50,000 cpm), and the products were resolved on denaturing gels. The positions of the full-length substrate and cleavage products are indicated by arrows. Lanes: 1 and 2, probe alone; 3 and 4, AAV5 Rep78 with the AAV5 and AAV2 ITRs, respectively; 5 and 6, AAV2 Rep78 with the AAV5 and AAV2 ITRs, respectively. The upper arrow identifies the AAV5 cleavage product (lane 3), and the lower arrow indicates the AAV2 cleavage product (lane 6).

FIG. 5

TRS mapping of the AAV5 ITR. ³²P-5′-end-labeled oligonucleotide probes (A′D or D′A) were annealed with unlabeled complementary oligonucleotides and incubated with either recombinant MBP-AAV2 Rep78 (lanes 2 and 5) or MBP-AAV5 Rep78 (lanes 3 and 6). The position of the cleavage product is indicated by an arrow. The sizes of the cleavage products were determined by comparison to the products of purine-specific sequencing reactions of the labeled probe (lanes 1 and 4). The sequence of the labeled oligonucleotide probe is on the left of each panel, with the Rep binding motif and TRS homolog indicated by brackets.