FIG 7.

NS2 fully replaces the function of NS4 for AAV2 DNA replication following transfection of the AAV2 duplex genome as well as following AAV2 infection. HEK293 cells (A and B) or HeLa cells (C and D) were cotransfected with SSV9 and Ad pHelper or different combinations of HBoV1 helper genes or were first infected with AAV2 and then transfected with pHelper or HBoV1 helper genes. Forty-eight hours after transfection with SSV9 or 72 h after infection with AAV2, 90% of the collected cells were lysed for Hirt DNA extraction, and 10% of the collected cells were lysed for protein analysis. (A and C) Hirt DNA samples were subjected to Southern blotting with a ³²P-labeled AAV2 probe. dRF, mRF, DpnI-digested DNA, and a DNA size marker are indicated. (B and D) Cell lysates for protein analysis were subjected to Western blotting. AAV2 proteins (Rep78, Rep52, VP1, VP2, and VP3) were probed with anti-Rep and anti-VP antibodies. HBoV1 proteins were probed with anti-NS1C and anti-NP1 antibodies. β-Actin was probed as a loading control.