Zinc-finger antiviral protein (ZAP) is a restriction factor for replication of modified vaccinia virus Ankara (MVA) in human cells
Fig 5
Effect of ZAP on MVA genome replication and gene expression.
(A) A549 and A549 ZAP-KO cells were infected in triplicate with 5 PFU/cell of MVA or MVA 51.2. After 6 h, DNA was isolated and genome copies determined by ddPCR. DNA copies from each sample are shown as dots, and the bar represents the mean value. n.s., not significant, p>0.05. (B, C) A549 and A549 ZAP-KO cells were infected in triplicate with MVA or MVA 51.2 for 8 h. RNA was extracted, poly(A) selected, and cDNA prepared. Copy numbers of I1 and A3 transcripts per μg of RNA were determined with specific primers using ddPCR and shown as dots; the bar represents the mean value. (D, E) A549+C12 and A549 ZAP-KO+C12 cells were infected in triplicate as in above panels and RNA read counts at 8 and 19 h determined by RNAseq. Abundances of individual viral RNAs were plotted after normalization to total read counts in each sample. (F) Ratios of individual MVA mRNAs from A549 ZAP-KO+C12 cells/A549+C12 cells at 8 h after infection and CpG densities of individual mRNAs were plotted. (G) Expression and processing of viral proteins. A549, A549 ZAP-KO cells stably transfected with C12 or an empty vector (vec) were mock infected or infected with MVA at 5 PFU/cell. Total proteins were collected after 8 or 24 h and resolved by SDS-PAGE. Viral early I3, intermediate/late A3 virion core and A17 virion membrane proteins and GAPDH loading control were resolved by SDS-PAGE and detected by probing Western blots with specific antibodies. (H) Abundances of individual viral proteins from A549 and A549 ZAP-KO cells infected with MVA or MVA 51.2 for 18 h determined by TMT mass spectrometry.