Fig 4. The mutant rep-D371Y gene is not compatible with the production of recombinant AAV2 virus stocks.
(A) Wild-type (wt AAV2, a) and recombinant (rAAV2-D371Y, b) virus stocks were visualized and analyzed with transmission electron microscopy. Red arrows: empty particles (electron dense), green arrow: fully packaged particle (electron light). Scale bars; 50nm. (B) Southern analysis to assess replication of wt and recombinant AAV2 virus stocks. Vero 2–2 cells were co-infected with either wt (wt AAV2) or recombinant (rAAV2-D371Y) (MOI 20) and HSV-1 (MOI 1) followed Hirt DNA extraction 48hrs later. Southern blotting was performed as described in Fig 2F with a DIG-labeled probe specific for Rep. The ITR ssDNA, the monomeric (ITRm) and the dimeric (ITRd) AAV2 replication intermediates are indicated on the right. (C, D) The capability of the mutant Rep68-D371Y proteins to bind ssDNA is reduced, whereas specific binding of dsDNA is not affected. Gel filtration chromatography profiles of either wt Rep68 or mutant Rep68-D371Y proteins were utilized to assess the binding capacity to (C) unspecific ssDNA or (D) specific dsDNA (RBS) templates. V0 is the void volume where aggregates are eluted with a molecular mass larger than the exclusion limit of the column (indicating complex Rep multimers). The other species at P1 represent the Rep-dsDNA or Rep-ssDNA complexes respectively. The P2 species represent unbound Rep proteins.