We evaluated this hypothesis using Huh7.5.1 cells, which are RIG-I pathway signaling defective and more permissive for HCV infection compared to their parental Huh7 cells. Methods: We performed siRNA knockdown of EFTUD2, RIG-I, or MDA5 in uninfected or JFH1-infected Huh7.5.1 or Huh7 cells. Selected cells were incubated with learn more the RIG-I-like receptor (RLR) signaling inhibitor BX795.Effects on IFN signaling were
monitored by a luciferase reporter system driven by ISRE. Selected gene mRNA levels and HCV replication were monitored by qPCR. Results: JFH1 HCV replicated more efficiently in Huh7.5.1 than in Huh7 cells (281808±13506 vs 10402±574) at 24hr JFH1 infection. Treatment with BX795 increased JFH1 HCV replication from 9918±494 to 31208±1612 (P<0.001) in Huh7 cells, but had no significant effect on HCV replication [295893±22768 (BX795) vs 249740±19938 (1%DMSO)] in Huh7.5.1 cells. EFTUD2 siRNA increased HCV replication by 1.8- and 2.8-fold in JFH1-infected Huh7.5.1 and Huh7 cells respectively. EFTUD2 siRNA significantly decreased RIG-1 and MDA5 mRNA transcription in Huh7.5.1 and Huh7 cells. However, EFTUD2 siRNA did not affect IFNAR1 or IRF9 mRNA expression, or IFN stimulated ISRE-signaling in either Huh7.5.1 or Huh7 cells, suggesting that EFTUD2 does not regulate classical ISGs through Jak-STAT or ISRE signaling. Overexpression of EFTUD2 reduced HCV replication
from 12731 ±785 to 4243±265 (P<0.001) in JFH1-infected Huh7 cells. Silmitasertib Interestingly, BX795 abrogated EFTUD2-mediated inhibition of HCV replication [11, 406±1486 (pEFTUD2+BX795) vs 4160±532 (pEFTUD2), P=0.0013]. Overexpression of EFTUD2 more modestly inhibited JFH1 HCV replication from 302649±21437 to 226986±14577 (P=0.007) in Huh7.5.1 cells. In contrast, BX795 did not rescue the observed EFTUD2-mediated
inhibition of HCV replication [260059±30564 (pEFTUD2+BX795) vs 208694±17938 (pEFTuD2), BCKDHA P=0.07] in these cells. Conclusions: EFTUD2 exerts its anti-HCV action primarily through regulation of the RIGI/MDA5 pathways, since overexpression of EFTUD2 suppresses HCV replication in a RIG-I competent cell line, and this suppression rescued by RLR inhibition. Conversely, EFTUD2 has no effect on Jak-STAT and ISRE signaling. These findings suggest a novel role for EFTUD2 in its interaction with the viral RNA sensor pathway. Disclosures: Raymond T . Chung – Advisory Committees or Review Panels: Idenix; Consulting: Enanta; Grant/Research Support: Gilead, Merck, Mass Biologic, Gilead The following people have nothing to disclose: Chuanlong Zhu, Jian Hong, Lei Zhao, Pattranuch Chusri, Nikolaus Jilg, Dahlene N. Fusco, Esperance A. Schaefer, Cynthia Brisac, Stephane Chevaliez, Daniel Wambua, Lee F. Peng, Wenyu Lin Objective: The response of chronic hepatitis C (CHC) to IFN treatment is hampered in patients with advanced liver fibrosis, and IFN might also affect the efficacy of triple therapy (PegIFN+RBV+DAA).