FIG. 1.

(A) Replication of AAV 5 viral DNA in 293 cells in the presence of various combinations of Ad5 gene products. 293 cells were infected with AAV5 at an MOI of 10 and transfected with equal molar quantities of CMV E2a, CMV E4Orf6, and a VAI RNA-expressing clone in the combinations indicated. pBS-SK was used to compensate for the absence of any individual plasmid. Monomer length (mRF), dimer length (dRF), and single-strand progeny (ssDNA) DNA replication forms, either cell associated (C) or released into the media (M), are indicated. Markers (m; lane 1) are a mixture of untreated and denatured 4.6-kb Acc65I fragments from pAV5, which corresponds to the full-length AAV5 genome. (B) Quantification of cell-associated AAV5 generated in 293 cells in the presence of various combinations of Ad5 gene products: Slot blot analysis to detect cell-associated virus production, performed as described in the Materials and Methods, during the same experiment in which replication was assayed in panel A, is shown. The various combinations of Ad5 helper functions are shown on the left, and dilutions of samples are shown to the right. (C and D) Analysis of AAV5 Rep protein (C) and capsid protein (D) production in 293 cells in the presence of various combinations of Ad5 gene products: Immunoblot analysis to determine AAV5 protein expression during the same experiment in which viral replication (A) and virus production (B) was determined as described above, using either antibody against the viral Rep proteins (C) or the viral capsid proteins (D), is shown. The locations of the AAV5 proteins, as well as endogenously expressed 14-3-3 and actin proteins serving as loading controls, are shown.