FIG 1.

135 is an early gene and is nonessential for replication of GTPV in vitro. (A) Alignment of amino acid sequences of the 135 protein of GTPV (accession number AGZ95457.1) and the N1L proteins of VACV (accession number 2I39_A), myxoma virus (accession number NP_051859.1), and ectromelia virus (accession number NP_671537.1). Amino acids identical across all four viruses and three of the four viruses are highlighted in dark blue and light blue, respectively. (B) Schematic showing the genomes of wild-type and recombinant GTPV AV41 strains. Western blotting confirmed expression of the GTPV 135 protein (∼18 kDa) (C), and immunofluorescence assay indicated the subcellular localization of the 135 protein in LT cells 48 h after infection with AV41 and its r135-Flag recombinant (D). (E) The GTPV 135 gene is an early gene. LT cells were infected with r135Flag-GFP/tk at an MOI of 0.05 in the presence or absence of AraC, and expression of the 135 protein, GFP, and β-actin was detected by Western blotting. (F) The 135 gene is nonessential for GTPV replication. LT cells were infected with AV41, r135-Flag, or the Δ135 virus at an MOI of 0.05. Cells and medium were harvested at different times by freeze-thawing three times. After low-speed centrifugation, the supernatants were collected for viral titration in LT cells. Data are representative of independent experiments performed in triplicate.