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. 2018 Aug 17;14(8):e1007261. doi: 10.1371/journal.ppat.1007261

Fig 6. Infectious particles grown in the presence of the SCD1 inhibitor are slower to infect new cells.

Fig 6

(A) Schematic of the experimental design: Step 1: Huh7 cells (set 1) were infected with DENV2, MOI = 3 (Virus A) and treated with DMSO or the SCD1 inhibitor. At 48hr the virus was titrated (Virus B and C). Step 2: This virus was used to infect naïve Huh7 cells (set 2) at a MOI of 0.1. The concentration of inhibitor remaining in viral supernatant C was mimicked by adding inhibitor to viral supernatant B during attachment. Cells were washed and overlaid with media without inhibitor and incubated for 36hr. Total RNA (RNA A and B) and virus supernatant (Virus D and E) were collected. (B) Viral RNA copies (RNA A and B) from Huh7 cells (set 2) were measured by qRT-PCR. The fold change of viral RNA copies in RNA B compared to RNA A is shown. (C) Huh7 cells with siRNA knockdown of SCD1 or an irrelevant control were infected with DENV2 (MOI = 0.1) for 48hr. Virus supernatant was collected and titrated. This virus was used to infect new Huh7 cells with an equal MOI (0.3). Virus was grown for 48hr and the supernatant was titrated. (D) Supernatants were collected from DENV infected cells with or without the inhibitor and UV-inactivated. WT DENV2 was then diluted in these UV-treated supernatants and used to infect new cells with an equal MOI. (E) Virus grown with the SCD1 inhibitor was again titrated and used to infect BHK cells with 100pfu/well. Attachment was allowed to occur at 4°C for 2hr, the temperature was shifted to 37°C and the cells were treated with acid glycine at the indicated time points after infection to inactivate un-internalized virus. Cells were overlaid with agarose and plaques were counted at 6 days. A linear regression was performed. The slope of the entry of the virus grown in the presence of the SCD1 inhibitor was 0.14PFU/min and DMSO was 0.27 PFU/min. (F) Huh7 cells were infected with DENV2 (MOI = 3) and treated with SCD1 inhibitor or DMSO. Supernatants were collected at 48hr, RNA was extracted, viral RNA copies were measured by qRT-PCR. Equal RNA copies were transfected into BHK cells to allow plaques to form. (ns = not significant, *** = p<0.001, from a two-tailed t-test).