Viral RNA-independent recognition of EMCV by the NLRP3 inflammasome.
(A) LPS-primed BMDCs were inoculated with live or UV-inactivated EMCV. (B) BMDCs were primed with LPS for 3 h, and then treated with 100 µM of cycloheximide (CHX) for 1 h prior to the treatment with ATP or infection with EMCV. (C, D) BMMs were infected with EMCV or transfected with 10 µg/ml poly(dA∶dT) or various amounts (12.5, 25, 50 µg) of total RNA from uninfected cells (uRNA), total RNA from EMCV-infected cells (iRNA), or EMCV genomic RNA (vRNA) for 18 h. Total RNA was extracted from virus-infected or RNA-transfected BMMs. IFN-β mRNA levels were assessed by RT quantitative PCR. GAPDH was used as an internal control. Data are pooled from three independent experiments (C). Supernatants were analyzed for the presence of mature IL-1β by ELISA (D). (E) Viral genomic RNAs were extracted from indicated amounts of EMCV, and their concentrations were determined. (F) Virus titers in the supernatants of the cells examined in (D) are shown. Data are representative of at least three independent experiments, and indicate the mean ± S.D. N.S., not significant. **, P<0.01.