FIG 8.

IFI16 is retained in the nucleus during late stages of RV-VM1 infection. (A) Performance of the pp65 mutant RV-VM1 HCMV in fibroblasts compared with wild-type virus (RV-HB5). The arrows indicate large globular structures (LGS) in the nuclei typically associated with RV-VM1 replication. (B) HFF cells were mock infected or infected with RV-HB5 or RV-VM1 viruses at an MOI of 1 for 48 or 144 h and then subjected to immunofluorescence analysis. pp65 (green) and IFI16 (red) were visualized using primary antibodies followed by secondary antibody staining in the presence of 10% HCMV-negative human serum. Nuclei were counterstained with DAPI (blue). (C) PLA were performed to detect protein-protein interactions using fluorescence microscopy in HFF cells mock infected or infected with RV-HB5 or RV-VM1 virus at an MOI of 1 for 48 or 144 h. A positive signal was detected as distinct fluorescent dots in the Texas red channel when IFI16 and pp65 were in close proximity (∼40 nm). (D) HFFs were infected with RV-HB5 or RV-VM1 at an MOI of 1. Nuclear and cytoplasmic fractions were prepared at the indicated time points and subjected to Western blot analysis for IFI16, pp65, α-tubulin, and TBP. HCMV-IEA was employed as a positive control for viral infection.