Fig 2.

Analyses of recombinant SeV-RFP strains with mutations at P2 and P3 of the F protein. (A) Detection of wt- and mutant-SeV-RFP-infected cells. Monolayers of Huh7/TMPRSS2-18 and Huh7/TMPRSS2m-4 cells were infected with wt and mutant SeV-RFPs at an MOI of 0.01, cultured in the absence of trypsin, and observed daily using a fluorescence microscope. Data at 3, 5, 7, and 9 days postinfection are shown. (B) Replication kinetics of wt and mutant SeV-RFPs in Huh7/TMPRSS2-18 and Huh7/TMPRSS2m-4 cells. Cells were infected with wt and mutant SeV-RFPs at an MOI of 0.01, cultured in the absence of trypsin, and examined for their virus titers (CIU) daily. (C) Pulse-chase labeling, immunoprecipitation, and SDS-PAGE for detection of SeV F proteins. Huh7/TMPRSS2-18 and Huh7/TMPRSS2m-4 cells infected with wt or mutant SeV-RFP were pulse-labeled with [³⁵S]methionine for 15 min, cultured (chased) in normal medium for 30 or 120 min, and subjected to immunoprecipitation and SDS-PAGE for detection of the F proteins. (D) Detection of wt- and mutant-SeV-RFP-infected cells. Monolayers of Calu-3 and Caco-2 cells were infected with wt and mutant SeV-RFPs at an MOI of 0.001, cultured in the absence of trypsin, and observed daily using a fluorescence microscope. Data at 3, 5, and 6 days postinfection are shown. (E) Virus production in Calu-3 cells. Calu-3 cells were infected with wt and mutant SeV-RFPs at an MOI of 0.001 and examined for their virus titers (CIU) at 5 days postinfection.