EFTUD2 Is a Novel Innate Immune Regulator Restricting Hepatitis C Virus Infection through the RIG-I/MDA5 Pathway

Abstract

The elongation factor Tu GTP binding domain-containing protein 2 (EFTUD2) was identified as an anti-hepatitis C virus (HCV) host factor in our recent genome-wide small interfering RNA (siRNA) screen. In this study, we sought to further determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2 and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and JAK-STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver tissue from in HCV-infected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. EFTUD2 inhibited HCV infection by inducing expression of the interferon (IFN)-stimulated genes (ISGs) in Huh7 cells. However, its impact on HCV infection was absent in both RIG-I knockdown Huh7 cells and RIG-I-defective Huh7.5.1 cells, indicating that the antiviral effect of EFTUD2 is dependent on RIG-I. Furthermore, EFTUD2 upregulated the expression of the RIG-I-like receptors (RLRs) RIG-I and MDA5 to enhance the innate immune response by gene splicing. Functional experiments revealed that EFTUD2-induced expression of ISGs was mediated through interaction of the EFTUD2 downstream regulators RIG-I, MDA5, TBK1, and IRF3. Interestingly, the EFTUD2-induced antiviral effect was independent of the classical IFN-induced JAK-STAT pathway. Our data demonstrate that EFTUD2 restricts HCV infection mainly through an RIG-I/MDA5-mediated, JAK-STAT-independent pathway, thereby revealing the participation of EFTUD2 as a novel innate immune regulator and suggesting a potentially targetable antiviral pathway.

Importance: Innate immunity is the first line defense against HCV and determines the outcome of HCV infection. Based on a recent high-throughput whole-genome siRNA library screen revealing a network of host factors mediating antiviral effects against HCV, we identified EFTUD2 as a novel innate immune regulator against HCV in the infectious HCV cell culture model and confirmed that its expression in HCV-infected liver tissue is inversely related to HCV infection. Furthermore, we determined that EFTUD2 exerts its antiviral activity mainly through governing its downstream regulators RIG-I and MDA5 by gene splicing to activate IRF3 and induce classical ISG expression independent of the JAT-STAT signaling pathway. This study broadens our understanding of the HCV innate immune response and provides a possible new antiviral strategy targeting this novel regulator of the innate response.

FIG 1

EFTUD2 restricts HCV infection in Huh7 and Huh7.5.1 cells. (A) Huh7 and Huh7.5.1 cells were transfected with pEFTUD2 or empty vector for 48 h before infection with HCV JFH1(MOI of 0.2) for another 24 h. HCV infection was determined by qPCR and Western blotting of HCV core protein. EFTUD2 expression was assessed by Western blotting. (B) Huh7 and Huh7.5.1 cells were transfected with siRNA specifically targeting EFTUD2 (siEFTUD2) or a negative-control siRNA (siNeg) for 48 h. EFTUD2 expression was assessed by qPCR and Western blotting. (C) Huh7 and Huh7.5.1 cells were transfected with siEFTUD2 or siNeg for 48 h before infection with HCV JFH1 (MOI of 0.2) for another 24 h. HCV infection was determined by qPCR and Western blotting of HCV core protein. (D and E) Huh7 and Huh7.5.1 cell lines stably expressing EFTUD2 (D) or shEFTUD2 (E) were generated as described in Materials and Methods and infected with HCV JFH1. HCV infection was assessed by qPCR at 24 h postinfection. (F to H) Huh7 or Huh7.5.1 cells were infected with HCV Jc1/Gluc (MOI of 1) 2 days after transfection with empty plasmid (pEmpty)/EFTUD2 (pEFTUD2) or with a negative-control siRNA (siNeg)/siRNA targeting EFTUD2 (siEFTUD2). HCV infection was assessed by luciferase activity at 6, 12, 24, 48, and 72 h postinfection.*, P < 0.05; **, P < 0.01; ***, P < 0.001. RLU, relative light units.

FIG 2

Decreased expression of EFTUD2 in liver specimens from patients with chronic HCV infection and in hepatic cells from an HCV cell culture model. The expression of EFTUD2 (A), RIG-I (B), and MDA5 (C) was assessed by qPCR and normalized to the level of the endogenous control (GAPDH) using liver biopsy specimens from eight chronically HCV-infected patients (HCV) and eight HCV-uninfected controls. Details of patient characteristics are described in Table 2. (D) The expression of EFTUD2 in Huh7 and Huh7.5.1 cells with or without HCV JFH1 infection was assessed by qPCR. *, P < 0.05; **, P < 0.01.

FIG 3

EFTUD2 upregulates expression of RIG-I and MDA5 with or without HCV JFH1 infection. Huh7 and Huh7.5.1 cells were transfected with pEFTUD2 or empty vector for 48 h (A, B, and E) or with an siRNA specifically targeting EFTUD2 (siEFTUD2) or siNeg for 48 h (C, D, and F) before infection with HCV JFH1 (MOI of 0.2) or a mock control for another 24 h. (A to D) mRNA expression of RIG-I and MDA5 was measured by qPCR. (E and F) Protein expression of RIG-I, MDA5, and EFTUD2 was assessed by Western blotting with β-actin as a loading control. **, P < 0.01; ***, P < 0.001.

FIG 4

EFTUD2 siRNA significantly reduces RIG-I and MDA5 mature mRNA levels in Huh7 and Huh7.5.1 cells. To assess RIG-I or MDA5 pre-mRNAs, we designed primer F1 or R1 (Table 1), which located within intron 1. To monitor RIG-I or MDA5 mature mRNA, we designed primer F2 or R2 (Table 1) that located across exons 1 and 2. We found that EFTUD2 siRNA significantly reduced RIG-I and MDA5 mature mRNAs, but not pre-mRNAs, in both Huh7 and Huh7.5.1 cells.

FIG 5

EFTUD2 induces ISG expression through RIG-I/MDA5. Huh7 and Huh7.5.1 cells were cotransfected with EFTUD2 siRNA (siEFTUD2) or a negative siRNA (siNeg) and pΔRIG-I, pMDA5, pRIG-I, or empty vector for 48 h before infection with HCV Jc1/Gluc at an MOI of 1 for 24 h. (A) HCV infection was assessed by luciferase activity. Results are expressed in relative light units (RLU). (B and C) mRNA expression of MxA and OAS1 was measured by qPCR. **, P < 0.01; ***, P < 0.001.

FIG 6

The RLR inhibitor BX795 abrogates ISG production and reverses EFTUD2-mediated inhibition of HCV in an RIG-I dependent manner. Huh7 and Huh7.5.1 cells were transfected with pEFTUD2 vector and treated with the RLR signaling inhibitor BX795 or a mock control for 48 h and then infected with JFH1 (MOI of 0.2) for 24 h. HCV infection (A) and mRNA expression of MxA (B) and OAS1 (C) were determined by qPCR. (D) Protein expression of EFTUD2, MxA, OAS1, IRF3, phosphorylated-IRF3 (P-IRF3), and HCV core protein was assessed by Western blotting. *, P < 0.05;**, P < 0.01; ***, P < 0.001.

FIG 7

EFTUD2 activates TBK1 and IRF3 through RIG-I/MDA5 to promote ISG production and inhibit HCV infection. Huh7 and Huh7.5.1 cells were cotransfected with pEFTUD2 or empty vector together with one of the following siRNAs: IRF3 (siIRF3), TBK1 (siTBK1), MDA5 (siMDA5), or RIG-I (siRIG-I) siRNA or with a negative siRNA (siNeg) for 48 h before infection with HCV JFH1 (MOI of 0.2) for 24 h. HCV infection (A) and mRNA expression of MxA (B) and OAS1 (C) were determined by qPCR. (D) Protein expression of RIG-I, MDA5, TBK1, IRF3, P-IRF3, MxA, OAS1, and HCV core protein was analyzed by Western blotting. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 8

EFTUD2 overexpression does not induce JAK-STAT component expression and phosphorylation of STAT1/STAT2 in JFH1-infected Huh7 and Huh7.5.1 cells. (A) Huh7 and Huh7.5.1 cells were incubated with IFN-α or mock treated for 24 h. mRNA expression of EFTUD2 was determined by qPCR. (B to D) Huh7 and Huh7.5.1 cells were transfected with pEFTUD2 or empty vector for 48 h and infected with HCV JFH1 (MOI of 0.2) for 12 h and then incubated with IFN-α or mock treated for another 24 h. mRNA expression of IRF9, IFNR1, and JAK1 was determined by qPCR. (E) HCV JFH1-infected Huh7 and Huh7.5.1 cells were transfected with pEFTUD2 or empty vector for 24 h before incubation with 30 IU/ml IFN-α or mock treated for 15 min. STAT1, STAT1 phosphorylation (P-STAT1), STAT2, and STAT2 phosphorylation (P-STAT2) were assessed by Western blotting.

FIG 9

A proposed model for EFTUD2 in inhibition of HCV infection. EFTUD2 upregulates RIG-I/MDA5 through mRNA editing to activate TBK1 and IRF3, thus inducing ISG production to inhibit HCV infection. dsRNA, double-stranded RNA.