FIG 6.

CRISPRi-mediated knockdown of SIRT1 inhibits MERS-CoV replication. (A) Two populations of Huh7 cells stably transfected with doxycycline (DOX)-inducible dCas9 were further transfected with two different lentiviral preparations containing a sgRNA sequence targeting SIRT1 to produce two independent populations of stable Huh7-dCas9-sgRNA cells. Each of the two populations was divided into two groups and either treated with DOX or grown in normal culture media for 7 days. Cells were lysed and samples subjected to Western blotting for SIRT1. (B) The stable Huh7-dCas9-sgRNA cells were grown for 7 days with or without DOX treatment and infected with MERS-CoV at an MOI of 0.1. For all cell groups, media were changed to normal growth media at the time of infection and samples were collected 24 h later to determine titer by TCID₅₀ assay on Vero cells. Data are from three independent infections, with error bars representing standard deviations. ****, P < 0.001 (by Student’s t test comparing DOX-treated and untreated samples). (C) The two populations of stable dCas9-Huh7-sgRNA cells were cultured as described for panel B and infected with MERS-CoV at an MOI of 0.1 or 1 and simultaneously treated with 200 μM resveratrol or ethanol (EtOH) vehicle control. Virus titer in supernatant was determined as described for panel B. Data are from a single infection; error bars represent standard deviations in calculations of TCID₅₀ per milliliter from three samples.