Fig 1: The catalytic activities of STT3A or STT3B are not required for DENV replication. (A) Huh7.5.1 cells were transduced with a lentivirus encoding a puromycin resistance marker, Cas9, and a single guide RNA (sgRNA) targeting the indicated genes. The sgRNAs targeting GFP act as a negative control. Two independent sgRNAs (indicated by 1 or 2 below the bars in the bar graph) were used per target gene to generate knockout cell pools. Cells were infected with luc-DENV for 3 days, and luciferase activity was measured in relative light units (RLU). Western blots for the indicated proteins are shown below the bar graph. Arrows indicate nonspecific bands. (B and C) Knockout cell pools were transduced with lentiviral vectors to rescue expression of the indicated protein. “STT3A dead” is a catalytically inactive W526A D527A double mutant. “STT3B dead” is a catalytically inactive W605A D606A double mutant with a C-terminal V5 tag. Transient transfection of constructs to express pSAP and SHBG were used to assess STT3A and STT3B catalytic activity. Western blots below the bar graph show the expression of the indicated proteins. wt and WT, wild type. (D) Knockout cell pools were transiently transfected with a luc-DENV replicon, and luciferase activity was measured at 4 h, 48 h, and 72 h posttransfection (hpt). Data are plotted as a ratio of RLU to the RLU at 4 hpt to control for transfection efficiency. GDD indicates a polymerase dead replicon with replacement of the GNN active site sequence in the NS5 RNA polymerase by GDD. (E) Cell viability for knockout cell pools was measured by Cell-Titer Glo over the course of 4 days for three independent wells. Data are normalized to day 1 values. The values for the parental Huh7.5.1 cells (P) are shown. In panels A to E, data are expressed as means plus standard deviations (SD) (error bars) for three independent biological replicates. In panels A to D, statistical significance was determined by Student’s t test and indicated by asterisks as follows: *, P < 0.05; **, P < 0.005; ***, P < 0.0005. In panel A, the means were compared to the mean for GFP sgRNA 2. In panels B and C, the means were compared to the means of their respective knockouts. In panel D, the means were compared to the mean for the GFP control.