Metabolic investigation of the sterol pathway in infection.

(A) NIH/3T3 cells were infected with mCMV-GFP (MOI of 0.2) and subsequently treated with Simvastatin (SMV) (2.5 µM) and mevalonate (MEV) (300 µM) or geranylgeraniol (GGOH) (15 µM) or farnesol (FOH) (15 µM) or squalene (SQE) (15 µM) or of water soluble cholesterol (complexes of cholesterol with methyl-β-cyclodextrin, CHO/MCD) (5 µg/ml) for 72 h. The level of infection was determined by measuring GFP fluorescence at 76 hpi (Material and Methods). Graph represents the relative level of infection compared to the untreated cells, and bars represent mean values ± SD of three independent experiments with five biological replicates for each experiment. (B) NIH/3T3 cells were transfected for 48 h with either non-targeted, eGFP, Hmgcs1, Hmgcr, Sqle, Fdft1, or Dhcr7 On-target plus siRNA smart pool and then infected with mCMV-GFP (MOI of 0.2). (C) NIH/3T3 cells were transfected for 48 h with either Risc Free, M54, and M86 (knocking down mCMV viral genes), Hmgcr, Dhcr7, Fdps, Fntb, Pggt1b, or Rabggtb On-target plus siRNA smart pool, and then infected with mCMV-GFP (MOI of 0.2). The level of non-targeted siRNA (B) and Risc Free (C) treated cells was used as a baseline estimate for the cutoff point (two standard deviations and a p value t test) as significant). Bars represent means ± SD of two independent experiments with three biological replicates for each experiment. (D) NIH/3T3 cells were incubated with various doses of IFNβ for 18 h in the presence or absence of 15 and 150 µM GGOH. The graph represents the inhibition of viral replication (in percentages) as a function of drug concentration. Bars represent mean ± SD of biological triplicates for each experiment. *pppt test.