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.