FIG 6.

RNF4 affects establishment of viral replication centers. (A) H1299 cells were infected with wt virus (H5pg4100) at a multiplicity of 20 FFU per cell and fixed with methanol after 48 h postinfection. Cells were labeled with α-PML pAb NB 100-59787 (Novus Biologicals, Inc.) and rabbit MAb α-E2A/DBP, detected with Alexa 488 (α-PML; green)- and Cy3 (α-E2A/DBP; red)-conjugated secondary antibody. Nuclei are labeled with DAPI (4,6-diamidino-2-phenylindole). Overlays of images (merge; d and j) and corresponding enlarged overlay (merge; e and f) staining patterns are shown. E2A/DBP staining was quantified and analyzed by counting the ratio between diffuse and replication center localization in infected cells. (B) Model of cross talk between RNF4, Daxx, and E1B-55K. Schematic representation illustrating a proposed model linking E1B-55K-dependent Daxx restriction and modulation by cellular factor RNF4. Upon HAdV infection, RNF4 is recruited to the nucleus in an E1B-55K-dependent manner to promote Daxx PTM prior to proteasomal degradation to counteract the cellular chromatin complex and ensure efficient viral gene expression.