FIG. 2.
Construction and characteristics of recombinant vaccinia virus vH2i. (A) Diagram of relevant portions of the vH2i genome. The locations of the H1L, H2R, H3L, and EGFP ORFs are shown. Additional abbreviations: P11, a vaccinia virus late promoter; P7.5, a vaccinia virus early-late promoter; PE/L, a vaccinia virus early-late synthetic promoter; PT7, bacteriophage T7 promoter; lacO, E. coli lac operator; lacI, E. coli lac repressor ORF; T7 pol, bacteriophage T7 RNA polymerase ORF. (B) Plaque formation and virus spread. BS-C-1 cell monolayers were infected with vH2i in the absence (upper panels) or presence (lower panels) of 100 μM IPTG. Cells were incubated for 48 h and then stained with crystal violet (left) or examined by fluorescence microscopy, with infected cells appearing green (right). (C) Effect of IPTG on the replication of vH2i under one-step growth conditions. Replicate samples of BS-C-1 cell monolayers were infected with 5 PFU of vH2i per cell, incubated for 1 h at 37°C, washed three times, and incubated further at the same temperature in the presence or absence of 100 μM IPTG. Cells were harvested at the indicated times postinfection (p.i.), and the virus titers were determined by plaque assay in the presence of 100 μM IPTG.