FIG. 1.
Construction of recombinant vaccinia viruses expressing a GFP-EH21 fusion protein. (A) Diagram of relevant portions of recombinant vaccinia virus vEH21i. Abbreviations: TK, thymidine kinase gene; T7 pol, bacteriophage T7 RNA polymerase gene; lacO, E. coli lac operator; PL, vaccinia virus P11 late promoter; PE/L, vaccinia virus P7.5 early/late promoter; lacI, E. coli lac repressor gene; HA, vaccinia virus hemagglutinin gene; gpt, E. coli guanine phosphotransferase gene; SLO, stem-loop lacO; EMC, encephalomyocarditis virus untranslated leader sequence; GFP-EH21, ORF encoding a fusion of GFP and EH21; TT, transcription termination sequences. (B) Diagram of relevant portions of recombinant vaccinia virus vWEH21, vIEH21, vWGFP, and vIGFP. Additional abbreviations: F13 ORF, ORF encoding vaccinia virus F13 protein; F13 left flank and right flank, DNA sequences on the left and right sides of F13 ORF. Immunoblots showing inducible expression of GFP-EH21 (C), constitutive expression of GFP-EH21 fusion protein (D) or GFP alone (E). HeLa cells were infected for 20 h with the indicated wild-type or recombinant viruses. Proteins from the cell lysates were resolved by polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and detected with mouse anti-GFP MAb.