FIG 1.

Construction and in vitro replication of D10R mutants. (A) Representation of D10R and flanking ORFs of WT and mutant viruses. The D10 deletion mutant (vΔD10) was constructed by replacing the D10R ORF of WT virus with EGFP regulated by the VACV P11 promoter (40). The revertant virus (vD10rev) containing the intact D10R ORF, catalytic site mutant (vD10mu), and stop codon mutant (vD10stop) were derived from vΔD10 by homologous recombination. The crosses in vD10mu denote the point mutations in the D10 nudix catalytic domain. The signs in vD10stop indicate the locations of the stop codons. (B) Plaque formation. Monolayers of BS-C-1 cells and MEFs were infected with the indicated VACV strains for 48 h. The cells were washed, incubated with anti-VACV polyclonal rabbit antibody, washed again, and incubated with protein A conjugated with peroxidase followed by the substrate dianisidine for visualization of plaques. (C) Growth curves. BS-C-1 cells and MEFs were grown in 12-well plates and infected with 5 PFU/cell of VACV strains. BS-C-1 cells were harvested at 3, 6, 13, 26, and 38 h postinfection, and MEFs were harvested at 2, 12.5, 26, and 38 h postinfection. Harvested cells were lysed and diluted, and virus titers were determined by plaque assay on BS-C-1 cells. The MEF titers are the averages of two independent experiments.