Fig 4.

E4orf6-mediated induction of B19V DNA synthesis requires the BC box motifs involved in formation of a cullin 5-based ubiquitin E3 ligase complex. 293 cells were cotransfected with 1 μg of SalI-linearized pB19-M20 DNA and increasing concentrations (25, 100, and 250 ng) of wild-type E4orf6 or constructs with various point mutations in the E4orf6 BC box motifs, which are required for the formation of a functional E3 ligase complex. The upper panels show the Southern blot detection of DpnI-resistant B19V replicative intermediates after isolation of Hirt DNA's as described for Fig. 2A, whereas the immunoblot analysis for B19V VP expression with MAB8293 as described for Fig. 1B is displayed in the lower panels. For the lanes marked “no B19V-DNA,” 4 μg of pYFP-C2 was transfected as a negative control.