RIOK3 and Its Alternatively Spliced Isoform Have Disparate Roles in the Innate Immune Response to Rift Valley Fever Virus (MP12) Infection

Abstract

Rift Valley fever virus (RVFV) is a pathogenic human and livestock RNA virus that poses a significant threat to public health and biosecurity. During RVFV infection, the atypical kinase RIOK3 plays important roles in the innate immune response. Although its exact functions in innate immunity are not completely understood, RIOK3 has been shown to be necessary for mounting an antiviral interferon (IFN) response to RVFV in epithelial cells. Furthermore, after immune stimulation, the splicing pattern for RIOK3 mRNA changes markedly, and RIOK3's dominant alternatively spliced isoform, RIOK3 X2, exhibits an opposite effect on the IFN response by dampening it. Here, we further investigate the roles of RIOK3 and its spliced isoform in other innate immune responses to RVFV, namely the NFκB-mediated inflammatory response. We find that while RIOK3 is important for negatively regulating this inflammatory pathway, its alternatively spliced isoform, RIOK3 X2, stimulates it. Overall, these data demonstrate that both RIOK3 and its X2 isoform have unique roles in separate innate immune pathways that respond to RVFV infection.

Keywords: NFκB; RIOK3; Rift Valley fever virus; alternative splicing; innate immunity.

Figure 1

RIG-I-mediated innate immune response to RNA virus infection. (a) Innate immune pathways activated in response to RNA viral genome. (b) IFN response. (c) Canonical NFκB pathway.

Figure 2

Paracrine communication from cells deficient in RIOK3 elicits diminished IFN response to infection with RVFV. (a) WT or RIOK3 KO cells were immune stimulated with poly(I:C) (1μg/mL), and media from these cells was transferred to identical plates of WT cells infected with RVFV (see diagram). IFNβ mRNA expression in the infected cells was measured by RT-qPCR. Cells treated with media from the WT cells elicited a stronger IFN response to infection than cells treated with media from RIOK3 KO cells. (b) Similarly, WT or RIOK3 KO cells were treated with TNFα at a concentration of 20 ng/mL, and media from these cells was transferred to identical plates of WT cells infected with RVFV (see diagram). Then, IL-8 and TNFα mRNA expression was measured by RT-qPCR. Cells treated with media from the WT or RIOK3 KO cells did not elicit statistically different inflammatory responses to infection, as measured by expression of IL-8 and TNFα. Fold induction in plots is relative to values of cells mock-infected and incubated with reduced serum media (negative controls). In (a,b), cells were grown in 6-well plates, seeded at a density ~0.3 × 10⁶ cells/well, and infected at a multiplicity of infection (MOI) ~2. Plots present the data as the mean value of 3 biological replicates +/− SEM. Student’s t-test: ** p < 0.01.

Figure 3

Effect of RIOK3 KO on inflammatory cytokine transcription during TNFα treatment or RVFV infection. (a) IL-8 mRNA expression in WT vs. RIOK3 KO cells treated with 20 ng/mL TNFα for 4 h. (b) TNFα mRNA expression in WT vs. RIOK3 KO cells treated with 20 ng/mL TNFα for 4 h. (c) IL-8 mRNA expression in WT vs. RIOK3 KO cells infected with RVFV MP12 at an MOI ~1. (d) TNFα mRNA expression in WT vs. RIOK3 KO cells infected with RVFV MP12 at an MOI ~1. (e) Luciferase assay measuring NFκB promoter activity in WT vs. RIOK3 KO HEK 293 cells treated with 20 ng/mL TNFα for 4 h. Fold induction is relative to negative control values from respective cell types (WT or RIOK3 KO) that were not treated with TNFα. (f) Luciferase assay measuring NFκB promoter activity in WT vs. RIOK3 KO HEK 293 cells infected with RVFV at an MOI ~2. Fold induction is relative to negative control values from respective cell types (WT or RIOK3 KO) that were mock-infected. In (a,b), cells were grown in 12-well plates and seeded at a density ~0.1 × 10⁶ cells/well, and fold induction is relative to expression values of respective cell types that were not treated with TNFα. In (c,d), fold induction is relative to expression values of respective cell types that were mock-infected. Plots present the data as the mean value of 3 biological replicates +/− SEM. Student’s t-test: ** p < 0.01.

Figure 4

Accumulation of alternatively spliced RIOK3 mRNA isoforms during RVFV MP12 infection correlates with a stalled IFN response but rising inflammatory response. (a) Alternative splicing of RIOK3 in RVFV MP12-infected HEK 293cells from 0 to 30 h.p.i. as detected by RT-PCR (b) IFNβ mRNA expression in RVFV MP12-infected cells from 0 to 30 h.p.i. (c) IL-8 mRNA expression in RVFV MP12-infected cells from 0 to 30 h.p.i. (d) TNFα mRNA expression in RVFV MP12-infected cells from 0 to 30 h.p.i. In plots (b–d), fold induction is relative to values of cells at 0 h.p.i. Cells were grown in 12-well plates, seeded at a density ~0.1 × 10⁶ cells/well, and infected at an MOI ~2. Plots in (b–d) present the data as the mean value of 3 biological replicates +/− SEM. Student’s t-test: * p < 0.05, ** p < 0.01.

Figure 5

RIOK3 X2 expression triggers inflammatory pathway activation. (a) IL-8 mRNA expression in X2-inducing MO- vs. standard control MO-treated WT HEK 293 cells (b) TNFα mRNA expression in X2-inducing MO- vs. standard control MO-treated WT HEK 293 cells (c) IL-8 mRNA expression in GFP- vs. RIOK3 X2-transfected WT HEK 293 cells (d) TNFα mRNA expression in GFP- vs. RIOK3 X2-transfected WT HEK 293 cells (e) Luciferase assay measuring NFκB promoter activity in GFP- vs. RIOK3 X2-transfected WT HEK 293 cells (f) Luciferase assay measuring NFκB promoter activity in GFP- vs. RIOK3 X2-transfected RIOK3 KO HEK 293 cells. In plots (a,b), fold induction is relative to values of cells transfected with standard control MO and that were not treated with TNFα. Cells were grown in 12-well plates, seeded at a density ~0.2 × 10⁶ cells/well, and treated with 80 ng/mL TNFα for 4 h. In (c,d), fold induction is relative to values of cells transfected with GFP and that were not treated with TNFα. In (c–f), cells were grown in 12-well plates, seeded at a density ~0.1 × 10⁶ cells/well, and treated with 20 ng/mL TNFα. In (c–e), cells were treated with TNFα for 4 h, and in (f), cells were treated with TNFα for 24 h. Plots in (a–f) present the data as the mean value of 3 biological replicates +/− SEM. Student’s t-test: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Current knowledge of RIOK3′s modulation of innate immunity to RNA virus infection.