FIG. 5.

Effect of different mutations on the formation of packaging complexes. 293T cells were transfected with AAV2 wt, newly mutated genomic plasmids (L336W, T329A/T330A, and R432A mutants) or previously described mutated genomic plasmids (N335A pore mutant without a packaging defect and del219/220, D219A, and S338A pore mutants with packaging defects [3]; pTAV2.1, a small Rep-deficient mutant [25]; delRep, plasmid pCMV-VP expressing only VPs [43]). (A) Cell extracts were prepared in RIPA buffer and analyzed for viral protein expression by Western blot analysis using monoclonal anti-Rep (303.9) or anti-VP (B1) antibodies. (B) Rep-capsid complexes were immunoprecipitated from RIPA extracts by using a polyclonal anti-Rep antiserum and analyzed by Western blotting to detect precipitated Rep proteins and coprecipitated capsid proteins. A control reaction was carried out using extracts of cells that had been transfected with a plasmid only expressing the three VPs (lane delRep). (C) The amounts of coprecipitated VPs were quantified by calculating the ratios of precipitated Rep78 to coprecipitated VP3 by using the program ImageQuant (Molecular Dynamics), and these were compared to the ratio of wt AAV2. Means ± standard deviations from at least three independent experiments are shown.