Fig 1.
Identification of host restriction factors for MVA by human genome-wide RNAi screening.
(A) A549 cells are non-permissive for MVA 47.1-GFP. BS-C-1 and A549 cells were infected with MVA 47.1-GFP or MVA44/47.1-GFP at 0.01 PFU/cell. Cells were imaged with a fluorescent microscope at 48 h post infection. (B) Outline of procedure for the genome-wide RNAi screen. MOI, multiplicity of infection. (C) Top 20 candidate genes identified from the screen and their corrected Z-scores. ZC3HAV1 is highlighted in red. (D) String analysis (https://string-db.org/) with candidate genes listed in (C). Red fill corresponds to GO term 398 (mRNA splicing via spliceosome); blue corresponds to GO term 6396 (RNA processing); white fill is ZC3HAV1.
Fig 2.
ZAP is a restriction factor for MVA in human cells.
(A-D) Human A549, HeLa, 293T and MRC-5 cells were transfected in triplicate with siRNA for 48 h and then infected with MVA 47.1-GFP at 0.01 PFU/cell. Cells were collected after 48 h and virus titers determined by plaque assay on CEF. Virus titers from each infection are shown as dots, and the bar represents the mean value. siNC: non-targeting negative control siRNA. (E) A549 and A549 ZAP-KO cells were left untreated or treated with interferon-β for 24 h. Cell lysates were analyzed by SDS-PAGE and Western blotting with anti-ZAP antibody. GAPDH was used as a loading control. Numbers on the right represent the electrophoretic positions and masses of marker proteins in kDa. (F) A549 and A549 ZAP-KO cells were infected in triplicate with the indicated viruses at 0.01 PFU/cell. Cells were collected after 48 h and virus quantitated by plaque assay on CEF. Virus titers from each infection are shown as dots and the bar represents the mean value. (G) A549 cells and A549 ZAP-KO cells stably expressing ZAP-L, ZAP-S or vector control (vec) were lysed and proteins resolved by SDS-PAGE and Western blotting with anti-ZAP antibody. (H) Cell lines described in G were infected with MVA 47.1-GFP at 0.01 PFU/cell and cells were harvested at 48 h and virus titers determined by plaque assay on CEF. Virus titers from each infection are shown as dots and the bar represents the mean value. Statistics: * p<0.05; ** p<0.01 by two-sided Student’s t-test.
Fig 3.
C16 antagonizes and interacts with ZAP.
(A) A549 cells were infected in triplicate with MVA, MVA+C12, MVA+C16 or MVA+C12/C16 at 0.01 PFU/cell. After 48 h, the cells were harvested and virus titers determined by plaque assay on CEF. Virus titers from each infection are shown as dots and the bar represents the mean value. (B) A549 and A549 ZAP-KO cells were infected with 0.01 PFU/cell of MVA 51.2 or MVA 51.2-ΔC16 and virus titers shown as above. (C) Lysates from A549, A549 ZAP-KO and A549 ZAP-KO cells stably expressing C12 (ZAP-KO+C12) were analyzed by SDS-PAGE and Western blotting with antibodies to myc tag on C12, ZAP and GAPDH, which was a loading control. Numbers on the right represent the electrophoretic positions and masses in kDa of marker proteins. (D) The cells described in C were infected with MVA or MVA 51.2 at 0.01 PFU/cell for 48 h and virus titers were determined by plaque assay on CEF. Virus titers from each infection are shown as dots and the bar represents the mean value. (E) A549 and A549 ZAP-KO cells were infected with MVA-2xMyc-C16 at 10 PFU/cell for 18 h. Cell lysates were incubated with control magnetic beads or myc-trap magnetic beads (myc), extensively washed and proteins eluted with lithium dodecyl sulfate (LDS) buffer and analyzed by SDS-PAGE and Western blotting with antibodies to ZAP and myc epitope tag. Statistics: * p<0.05; ** p<0.01 by two-sided Student’s t-test.
Fig 4.
Co-localization of C16 and ZAP.
(A) A549 cells were mock-infected, infected with MVA or MVA-2xMyc-C16 (MVA+C16) at 5 PFU/ cell for 5 h. Cells were then fixed, permeabilized, blocked and stained with primary antibodies to myc (C16) and ZAP followed by fluorescent conjugated secondary antibodies. DAPI was used to stain DNA. (B) A549 cells were infected with MVA-2xMyc-C16 and stained with antibodies to I3 and ZAP and with DAPI. Arrowheads point to virus factories. (C) A549 cells were infected with MVA-2xMyc-C16 and stained with antibodies to Trim25 and ZAP and with DAPI. (D) Uninfected A549 cells were treated with sodium arsenite or infected with MVA-2xMyc-C16 for 5 h and stained with antibodies to PABP and ZAP and with DAPI. Scale bar at bottom.
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.
Fig 6.
Transmission electron microscopy of cells infected with MVA.
A549 cells (upper and lower left panels) and A549 ZAP-KO cells (upper and lower right panels) were infected with 10 PFU/cell of MVA. After 20 h, cells were fixed, sectioned and analyzed by transmission electron microscopy. Symbols: D, dense particle; MV, mature virion; WV, wrapped virion; CEV, cell-associated enveloped virion. Magnification factors shown below each panel.