Inducible MicroRNA-3570 Feedback Inhibits the RIG-I-Dependent Innate Immune Response to Rhabdovirus in Teleost Fish by Targeting MAVS/IPS-1

Abstract

Effectively recognizing invading viruses and subsequently inducing innate antiviral immunity are essential for host antiviral defense. Although these processes are closely regulated by the host to maintain immune balance, viruses have evolved the ability to downregulate or upregulate these processes for their survival. MicroRNAs (miRNAs) are a family of small noncoding RNAs that play vital roles in modulating host immune response. Accumulating evidence demonstrates that host miRNAs as mediators are involved in regulating viral replication and host antiviral immunity in mammals. However, the underlying regulatory mechanisms in fish species are still poorly understood. Here, we found that rhabdovirus infection significantly upregulated host miR-3570 expression in miiuy croaker macrophages. Induced miR-3570 negatively modulated RNA virus-triggered type I interferon (IFN) and antiviral gene production, thus facilitating viral replication. Furthermore, miR-3570 was found to target and posttranscriptionally downregulate mitochondrial antiviral signaling protein (MAVS), which functions as a platform for innate antiviral signal transduction. Moreover, we demonstrated that miR-3570 suppressed the expression of MAVS, thereby inhibiting MAVS-mediated NF-κB and IRF3 signaling. The collective results demonstrated a novel regulation mechanism of MAVS-mediated immunity during RNA viral infection by miRNA.IMPORTANCE RNA viral infection could upregulate host miR-3570 expression in miiuy croaker macrophages. Induced miR-3570 negatively modulates RNA virus-triggered type I IFN and antiviral gene production, thus facilitating viral replication. Remarkably, miR-3570 could target and inhibit MAVS expression, which thus modulates MAVS-mediated NF-κB and IRF3 signaling. The collective results of this study suggest a novel regulation mechanism of MAVS-mediated immunity during RNA viral infection by miR-3570. Thus, a novel mechanism for virus evasion in fish is proposed.

Keywords: MAVS; miR-3570; microRNA; teleost fish; virus evasion.

FIG 1

Viral infection upregulates miR-3570 expression in macrophage. Miiuy croaker macrophages were transfected with various MOIs of SCRV for 36 h (A) or different times (MOI, 5) (B), and the miR-3570 expression level was determined with qRT-PCR. (C) Miiuy croaker macrophages were stimulated with poly(I·C) for different times, and the miR-3570 expression level was measured by using qRT-PCR. The results are standardized to 1 in control cells. All data are representative of at least three independent experiments.

FIG 2

miR-3570 is involved in modulating SCRV-triggered antiviral gene production. (A) Miiuy croaker macrophages were transfected with control mimics (Ctrl), miR-3570 mimics (miR-3570), control inhibitors (Ctrl-i), or miR-3570 inhibitors (miR-3570-i) for 48 h, and then miR-3570 expression was determined by qRT-PCR. (B) Macrophages were infected with SCRV at an MOI of 5 for 36 h, and the mRNA levels of TNF-α, IFN-β, MX1, ISG15, and Viperin were detected by qRT-PCR. (C and D) Macrophages were transfected with Ctrl or miR-3570 (C) and Ctrl-i or miR-3570-i (D) for 48 h and then were infected with SCRV at an MOI of 5 for 36 h. The expression level of miR-3570 was analyzed by qRT-PCR. (E) Ctrl or miR-3570 and Ctrl-i or miR-3570-i were transfected into macrophages for 48 h and then stimulated with poly(I·C) for 12 h. The expression levels of the above-described genes were determined by qRT-PCR. The results are standardized to 1 in control cells. All data are representative of three independent experiments. (**, P < 0.01).

FIG 3

miR-3570 targets miiuy croaker MAVS. (A) HEK293 cells were cotransfected with MAVS-3′UTR (WT), together with Ctrl, miR-3570, Ctrl-i, or miR-3570-I, for 24 h, and the luciferase activity was determined. For each transfection, the total amount of oligonucleotides was controlled and normalized (final concentration, 100 nM). (B) HEK293 cells were cotransfected with pIZ/EGFP-MAVS-3′UTR WT or pIZ/EGFP-MAVS-3′UTR mutant type (MT), together with miR-3570 or Ctrl. At 48 h posttransfection, the fluorescence intensity was evaluated. (C) Schematic diagram of the predicted target sites of miR-3570 in 3′UTR of miiuy croaker MAVS. HEK293 cells or HeLa cells were transfected with miR-3570 or Ctrl, along with wild-type MAVS-3′UTR or the mutant type of MAVS-3′UTR for 24 h, and the luciferase activity was determined. (D) The miR-3570 mimics (0, 30, 60, and 90 nM) together with the control mimics (90, 60, 30, and 0 nM) were cotransfected with WT reporter plasmid into HEK293 cells. After 24 h or 48 h, the luciferase activity was determined. Luciferase activity was normalized to renilla luciferase activity. All data are representative of at least three independent experiments. (**, P < 0.01).

FIG 4

pre-miR-3570 regulates miiuy croaker MAVS. (A) Sequence alignment of pre-miR-3570 and the construction of the pre-miR-3570 plasmid. HEK293 cells were transfected with the pre-miR-3570 plasmid, along with wild-type MAVS-3′UTR (WT) or the mutant type of MAVS-3′UTR (MT) for 24 h, and the luciferase activity was determined. (B) HEK293 cells were transfected with MAVS-3′UTR, together with the pre-miR-3570 plasmid and miR-3570 inhibitors or its controls, for 24 h, and the luciferase activity was determined. For each transfection, the total amounts of the transfections were controlled and normalized. (C) HEK293 cells were transfected with the WT, together with the concentration gradient of pre-miR-3570, for 24 h. pcDNA6.2 was used to ensure that the same amounts of plasmid DNA are transfected for each transfection. The luciferase activity value was measured by using the Dual-Luciferase reporter assay system. (D) The time gradient was conducted for transfection with the pre-miR-3570 plasmid. Luciferase activity was normalized to renilla luciferase activity. All data are representative of at least three independent experiments (**, P < 0.01).

FIG 5

miR-3570 suppresses the expression of MAVS at posttranscriptional level. (A and B) The miiuy croaker macrophages were transfected with miR-3570 or Ctrl (A) and Ctrl-i or miR-3570-i (B). After 48 h of transfection, the protein and mRNA levels of MAVS were determined by Western blotting and qRT-PCR, respectively. (C and D) HEK293 cells were cotransfected with MAVS expression plasmid, along with miR-3570 mimics (C) and the pre-miR-3570 plasmid (D), in a concentration gradient manner, and control mimics (C) and pcDNA6.2 plasmid (D) were used to control the same amount of molecules for transfections. After 48 h, MAVS protein and mRNA levels were determined by Western blotting and qRT-PCR, respectively. (E and F) After transfection of Ctrl, miR-3570, Ctrl-i, and miR-3570-I for 48 h, the macrophages were treated with SCRV for 36 h (E) or poly(I·C) for 12 h (F). The mRNA expression of MAVS was analyzed by qRT-PCR and normalized to that of β-actin. The results are standardized to 1 in control cells. All data are representative of at least three independent experiments (**, P < 0.01; *, P < 0.05).

FIG 6

Overexpression of miR-3570 inhibits MAVS-mediated NF-κB and IRF3 signaling. (A) HEK293 cells were cotransfected with the MAVS expression plasmid, pRL-TK renilla luciferase plasmid, together with luciferase reporter gene NF-κB, ISRE, or IRF3 for 48 h, and the luciferase activity was measured. (B) HEK293 cells were cotransfected with Ctrl, miR-3570, Ctrl-i, or miR-3570-i, together with pRL-TK renilla luciferase plasmid, MAVS expression plasmid, and luciferase reporter NF-κB, IRF3, or ISRE for 48 h, and the luciferase activity was measured. For each transfection, the total amounts of oligonucleotides were controlled and normalized (final concentration, 100 nM). (C) HEK293 cells were transfected with pre-miR-3570 in a concentration gradient manner, together with NF-κB, ISRE, or IRF3, for 48 h, and pcDNA6.2 plasmid was use to ensure the same amounts of molecules were used for transfections. The luciferase activity was measured by using the Dual-Luciferase reporter assay system. (D) Time gradient experiment of the pre-miR-3570 plasmid. Luciferase activity was normalized to renilla luciferase activity. All data are representative of at least three independent experiments (**, P < 0.01; *, P < 0.05).

FIG 7

Expression levels of MAVS and antiviral genes after MAVS interference. (A) Miiuy croaker macrophages were transfected with control siRNA (si-Ctrl) or siRNA against MAVS (si-MAVS). After 48 h, MAVS mRNA and protein levels were determined by qRT-PCR and Western blotting, respectively. (B to F) After 48 h of transfection with si-Ctrl or si-MAVS, miiuy croaker macrophages were infected with SCRV for 36 h or poly(I·C) for 12 h. The expression levels of TNF-α (B), IFN-β (C), Mx1 (D), ISG15 (E), and Viperin (F) were determined. The results are standardized to 1 in control cells. All data are representative of at least three independent experiments (**, P < 0.01; *, P < 0.05).

FIG 8

miR-3570 regulates expression of components of MAVS-dependent signaling cascade. (A) Schematic outline of MAVS-mediated pathway in mammals. (B) Miiuy croaker macrophages were transfected with si-Ctrl and si-MAVS for 48 h, and then the mRNA levels of TRAF3, TRAF6, TBK1, IKKi, and IRF3 were detected by qRT-PCR. (C and D) Miiuy croaker macrophages were transfected with Ctrl or miR-3570 (C) or Ctrl-i or miR-3570-i (D) for 48 h, and the mRNA levels of TRAF3, TRAF6, TBK1, IKKi, and IRF3 were detected by qRT-PCR. The results are standardized to 1 in control cells. All data are representative of at least three independent experiments (**, P < 0.01; *, P < 0.05).

FIG 9

miR-3570 promotes SCRV replication. (A) Miiuy croaker macrophages were transfected with Ctrl, miR-3570, Ctrl-i, or miR-3570-i, infected by SCRV at an MOI of 5 for 1 h, washed, and then added to fresh medium. After 72 h, SCRV TCID₅₀ in cultural supernatants was measured with MKC cells. (B and C) qRT-PCR analysis was conducted for intracellular SCRV RNA (B) or supernatant SCRV RNA (C). All data are representative of at least three independent experiments (**, P < 0.01; *, P < 0.05; N.D., not detected).

FIG 10

Proposed model for the miR-3570 regulatory mechanism in fish species. (A) Schematic diagram of the predicted target sites of miR-3570 in the 3′UTR of Danio rerio MAVS. HEK293 cells were transfected with miR-3570 or Ctrl, along with the wild-type Danio rerio MAVS-3′UTR (DrMAVS 3′UTR WT) or the mutant type of Danio rerio MAVS-3′UTR (DrMAVS 3′UTR MT) for 24 h, and the luciferase activity was determined. (B) Schematic diagram of the predicted target sites of miR-3570 in 3′UTR of Larimichthys crocea MAVS. HEK293 cells were transfected with miR-3570 or Ctrl, along with the wild-type Larimichthys crocea MAVS-3′UTR (LcMAVS 3′UTR WT) or the mutant type of Larimichthys crocea MAVS-3′UTR (LcMAVS 3′UTR MT) for 24 h, and the luciferase activity was determined. (C) Proposed model for the mechanism by which induced miR-3570 negatively regulates inflammatory cytokines and antiviral gene production in feedback manner by targeting MAVS and inhibiting NF-κB and IRF3 signaling, thereby promoting virus replication.