Fig 1.
Adaptation of ΔB1 virus and identification of mutation within the B12R gene.
(A) ΔB1 virus was serially passaged in CV1 cells in triplicate and named A1-A3 for adapted ΔB1 viruses. Virus harvested at each passage was titrated on CV1-B1myc cells. (B) Deep sequencing data for WT (Wiebe), ΔB1, ΔB1-A1, and ΔB1-A3 viruses was used to graph insertion/deletion mutations at each nucleotide site for the entire vaccinia genome when comparing ΔB1, ΔB1-A1, and ΔB1-A3 viruses to the change in indel mutations of the WT (Wiebe) compared to the WT WR (reference sequence). (C) Graphed insertion/deletion mutations for ΔB1-A3 compared to WT WR (reference sequence) for reads 170,015–175,094bp. The dotted line indicates indel mutations that occur in 5% of the total reads at a single nucleotide. Indel mutations above 5% were considered significant mutations in the mixed ΔB1 adapted virus population. Locations of encoded genes are labeled below, corresponding to the base pairs on the x-axis of the graph.
Fig 2.
Rescued DNA replication block and viral yield for ΔB1mutB12 virus in CV1 cells.
(A) CV1 cells infected with WT (black), ΔB1 (red), ΔB1mutB12-A1 (light green), ΔB1mutB12-A3 (dark green) at a MOI of 3 were harvested 24h post infection for qPCR of relative DNA accumulation or (B) for titration on CV1-B1myc cells for viral yield. (C) WT (black), ΔB1 (red), or ΔB1mutB12-A3 (green) infections of CV1 cells were performed at a MOI of 3 and harvested at 3, 7, 16, or 24h post infection for relative DNA accumulation or (D) viral yield quantification on CV1-B1myc cells. (E) A multi-step viral yield assay was completed by infecting CV1 cells at a MOI of 0.01 with WT (black), ΔB1 (red) or ΔB1mutB12-A3 (green) and harvested at 48h post infection for titration on CV1-B1myc cells.
Fig 3.
Depletion of B12 rescues ΔB1 virus growth in CV1 cells.
(A) CV1 cells were transfected with pJS4-HA-B12wt or pJS4-HA-B12ΔA690 plasmid and infected 6h post transfection with WT virus at a MOI of 3. 24h post infection cells were harvested for immunoblot analysis. The HA-B12Δ690 represents the indel mutation within the ΔB1mutB12 virus B12R gene. (B) B12 proteins were expressed from the pJS4 vector during WT infection (representative immunoblot in Fig 1A). Relative protein levels for HA-B12wt and HA-B12ΔA690 were averaged from five independent experiments, and normalized to control protein levels set to 1. The denoted * p-value equals 0.02. (C) 200PFU/well WT, ΔB1, or ΔB1mutB12-A3 infections were carried out on CV1 cells 24h following transfection with siCtrl or siB12. Cells were fixed 72h post infection. (D) Multi-step viral yield assay was conducted in siCtrl or siB12 CV1 cells for WT (black), ΔB1 (red), and ΔB1mutB12-A3 (green) infections at a MOI of 0.01. Cells were harvested at 7h or 48h post infection and titration on CV1-B1myc cells. (E) Growth assays on siCtrl or siB12 transfected CV1 cells were completed for WT (black), ΔB1 (red), and ΔB1mutB12 (green) viruses at a MOI of 3 for relative DNA accumulation and (F) viral yield titration on CV1-B1myc cells.
Fig 4.
Rescue of DNA replication block using siB12 is specific for viruses lacking a functional B1.
(A) Diagram of hypothesis that B12 is either a general inhibitor of DNA replication or specific to B1 kinase mutant viruses. (B) CV1 cells treated with siCtrl of siB12 were infected with WT (black), ΔB1 (red), ts2 B1 mutant (pink), ts24 D5 mutant (blue), or ts42 E9 mutant (purple) at a MOI of 3 and harvested 24h post infection for quantification of relative DNA accumulation. Infections were carried out at 31.5°C, 37°C or 39.7°C to provide permissive, semi-nonpermissive and nonpermissive temperatures respectively for the temperature sensitive mutant viruses.
Fig 5.
B12 reconstitution during infection with B1 and B12 naïve viruses repressed vaccinia replication.
(A) Immunoblot analysis of control or HA-B12 (GeneArt) lentivirus transduced CV1 cells was completed to detect tubulin (loading control) and HA (HA-tagged B12). (B) CV1 control cells or cells stably expressing B12 or HA-tagged B12 were infected with WT or ΔB1mutB12-A3 at 300PFU/well and fixed 72h post infection. (C) 24h relative DNA accumulation quantification was completed for CV1 control or HA-B12 expressing cells transfected with siCtrl or siB12 and infected with WT (black), ΔB1 (red), or ΔB1mutB12-A3 (green) at a MOI of 3.
Fig 6.
B12 exhibits a nuclear localization in uninfected and infected cells.
(A) CV1 cells with or without HA-B12 (GenScript) mRNA transfection were used for immunofluorescence detection of HA-tagged B12 (red, top row). CV1 control and CV1-B1myc expressing cells were incubated with αmyc for B1myc detection (red, bottom row). All cells were stained with DAPI nuclear stain (blue). (B) B1myc expressing CV1 cells were also transfected with HA-B12 (GenScript) mRNA and separately incubated with a primary antibody to detect HA-tagged B12 (αHA, top red image) or myc-tagged B1 (αmyc, bottom red image) and DAPI (blue) nuclear stain. (C) CV1 cells were infected with WT or WT/HA-B12 virus at a MOI of 5 and fixed at 4hpi or (D) 7hpi for immunofluorescence analysis of HA-B12 detection (red), I3 ssDNA binding protein (green) and DAPI nuclear stain (blue). The scale bars represent 100μm.
Fig 7.
B12 nuclear localization is distinct from chromatin bound proteins.
(A) CV1 cells were transfected with no mRNA, HA-GFP mRNA, or HA-B12 (GenScript) mRNA. Cells were either fixed then permeabilized to detect HA-tagged proteins or (B) prepermeabilized, fixed and then permeabilized again for detection of HA-tagged proteins remaining in cells following washes to remove unbound protein. The DAPI nuclear stain (blue) and αHA antibody (green) for detection of HA-GFP and HA-B12 were used. The scale bars in 7A and 7B represent 200μm. (C) Subcellular fractionation of CV1 control or HA-B12 (GeneArt) stably expressing cells was completed to separate cells into cytoplasmic extract (Cyto.), membrane extract (Memb.), soluble nuclear extract (Nuc.), chromatin-bound extract (Chrom.), and cytoskeleton extract (Cytoskel.). Lamin A/C, GAPDH and BAF protein detection were used as fractionation controls and HA was used to detect HA-tagged B12 protein.
Fig 8.
The ΔB1mutB12 virus restriction of BAF antiviral activity is greater than the ΔB1 virus.
(A) Immunoblot analysis of total BAF protein (top panel) or phosphorylated BAF (bottom panel) in CV1 cells uninfected or infected with WT, ΔB1, ΔB1mutB12-A1, or ΔB1mutB12-A3 at a MOI of 10. Cells were collected at 6h post infection. (B) Control or CV1 cells expressing 3XFlag-tagged BAF in excess were infected with WT (black), ΔB1 (red), or ΔB1mutB12 (green) at a MOI of 3 and harvested at 24h post infection for analysis of relative DNA accumulation or (C) viral yield titration on CV1-B1myc cells.
Fig 9.
Subcellular localization of B12 and working model of B1/B12/BAF signaling during vaccinia infection.
(A) Vaccinia B1 kinase overlaps with cytoplasmic localized host BAF protein whereas vaccinia B12 pseudokinase shares a nuclear subcellular localization with the nuclear fraction of BAF. (B) The B1 kinase participates in restriction of BAF’s antiviral function against vaccinia DNA replication in the cytoplasm, while also repressing B12 negative regulation of vaccinia DNA replication through an unknown mechanism that is partly mediated via BAF regulation. Direct interactions and/or signaling through additional factors may be required for B1-B12 signaling and B12-BAF signaling, and are depicted using gold lines. B1-BAF interaction and BAF binding to dsDNA are direct interactions and denoted in black lines.