Figure 7. FASN is an RBP that Plays a Proviral Role in CHIKV Replication and Release.
(A) SDS-PAGE and silver stain (top), agarose gel (middle) or immunoblot (bottom) of CLASP performed on lysates from U2OS cells cultured in the presence or absence of 100 μM 4SU, and with or without IFN treatment.
(B) Phosphorimage (top) or immunoblot (bottom) of PAR-CLIP performed on lysates from U2OS cells overexpressing Flag-HA-FASN and cultured in the presence or absence of 100 μM 4SU.
(C) Top, Immunoblot of CLASP performed on lysates from HEK293T cells transiently expressing truncated FASN. Cells were cultured in the presence or absence of 100 μM 4SU. Bottom, Schematic of the protein domains of FASN and its truncated versions. Data represent two biologically independent repeats in A–C.
(D) Strand-specific RT-qPCR of viral RNA levels in U2OS cells treated with siRNA for FASN or control. CHIKV infection was performed 72 hr after siRNA transfection. MOI, multiplicity of infection. n = 4, error bars, mean ± SD.
(E) Rescue of viral RNA replication effect by re-expression of FASN. n = 3, error bars, mean ± SD. *p < 0.05.
(F) Top, CHIKV RNA in supernatants harvested from U2OS cells 24 hpi at an MOI of 10−4 after 72 hr siRNA treatment, quantified by RT-qPCR. Bottom, CHIKV titer in the supernatants as measured by plaque assay in Vero cells. n = 3, error bars, mean ± SD.
(G) Luciferase assay of lysates from cells treated with siRNA for FASN or control. Cells were infected with CHIKV-nLuc at MOI = 10. n = 4, error bars, mean ± SD.
(H) Luciferase assay of cell lysates from cerulenin treated or untreated cells. Cells were infected with CHIKV-nLuc at MOI of 10. Cells were pretreated with 10 μM cerulenin for 2 hr. n = 8, error bars, mean ± SD.
(I) Rescue of viral translation by re-expression of wild-type (FASN) or catalytically-dead mutant (FASNmut) on (G) experimental conditions. n = 4, error bars, mean ± SD.
See also Figure S6