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. 2020 Mar 24:11:368.
doi: 10.3389/fmicb.2020.00368. eCollection 2020.

RIPK3 Promotes JEV Replication in Neurons via Downregulation of IFI44L

Peiyu Bian  1 , Chuantao Ye  1 , Xuyang Zheng  1 , Chuanyu Luo  2 , Jiali Yang  3 , Mengyuan Li  1 , Yuan Wang  4 , Jing Yang  4 , Yun Zhou  1 , Fanglin Zhang  4 , Jianqi Lian  1 , Ying Zhang  1 , Zhansheng Jia  1 , Yingfeng Lei  4
Affiliations

RIPK3 Promotes JEV Replication in Neurons via Downregulation of IFI44L

Peiyu Bian et al. Front Microbiol. .

Abstract

Japanese encephalitis virus (JEV), the leading cause of viral encephalitis in Asia, is neurovirulent and neuroinvasive. Neurons are the main target of JEV infection and propagation. Receptor interacting serine/threonine-protein kinase 3 (RIPK3) has been reported to contribute to neuroinflammation and neuronal death in many central nervous system diseases. In this study, we found that the progression of JE was alleviated in RIPK3-knockout (RIPK3-/-) mice in both peripheral and intracerebral infection. RIPK3-knockdown (RIPK3-RNAi) neuro2a cells showed higher cell viability during JEV infection. Moreover, the JEV load was significantly decreased in RIPK3-/- mouse-derived primary neurons and RIPK3-RNAi neuro2a cells compared with wild-type neurons, but this was not observed in microglia. Furthermore, RNA sequencing of brain tissues showed that the level of the interferon (IFN)-induced protein 44-like gene (IFI44L) was significantly increased in JEV-infected RIPK3-/- mouse brains, RIPK3-/- neurons, and RIPK3-RNAi-neuro2a cells. Then, it was demonstrated that the propagation of JEV was inhibited in IFI44L-overexpressing neuro2a cells and enhanced in IFI44L and RIPK3 double knockdown neuro2a cells. Taken together, our results showed that the increased expression of RIPK3 following JEV infection played complicated roles. On the one hand, RIPK3 participated in neuroinflammation and neuronal death during JEV infection. On the other hand, RIPK3 inhibited the expression of IFI44L to some extent, leading to the propagation of JEV in neurons, which might be a strategy for JEV to evade the cellular innate immune response.

Keywords: Japanese encephalitis virus (JEV); cellular innate immune response; interferon-induced protein 44-like gene (IFI44L); neurons; receptor interacting serine/threonine-protein kinase 3 (RIPK3).

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Figures

FIGURE 1
FIGURE 1
Receptor interacting serine/threonine-protein kinase 3 (RIPK3)-knockout mice showed decreased morbidity and mortality after Japanese encephalitis virus (JEV) infection via peripheral and intracerebral infection. (A) RIPK3–/– (n = 17) and wild-type (WT; n = 14) C57BL/6 mice (8–10 weeks) were infected with JEV-P3 at 5 × 106 plaque-forming units (PFUs) in 20 μl phosphate-buffered saline (PBS) via footpad injection. The data were analyzed and shown as Kaplan–Meier survival curves. (B) The mean behavior score of each mouse measured at 8:00–9:00 and 16:00–17:00 was calculated and analyzed. Data are shown as the mean ± SEM of all mice in each group. (C) The mean weight of each mouse at 8:00–9:00 and 16:00–17:00 was calculated and analyzed. Data are shown as the mean ± SEM of all mice in each group. (D) RIPK3–/– (n = 9) and WT (n = 13) C57BL/6 mice (8–10 weeks) were infected with JEV-P3 at 100 PFU in 2 μl PBS via intracerebral injection. The death cases of each group were recorded every day, and then the data were analyzed and shown as Kaplan–Meier survival curves.
FIGURE 2
FIGURE 2
Japanese encephalitis virus (JEV) infection induced receptor interacting serine/threonine-protein kinase 3 (RIPK3) expression which contributed to neuronal death. (A) C57BL/6 mice were infected with JEV (n = 5) or PBS (n = 3) via footpad injection, and the brains were harvested. The expression of RIPK3 was evaluated by qPCR. The data represent the change relative to RIPK3 expression in phosphate-buffered saline (PBS)-treated mice. Data are shown as the mean ± SD. (B) The expression of RIPK3 in primary neurons at 24, 48, and 72 h post infection (hpi) after JEV infection was evaluated by qPCR. The data represent the change relative to the level in neurons at 0 hpi. Data are shown as the mean ± SD. Three independent experiments were performed. (C) The expression of RIPK3 in Neuro2a cells at 24, 48, and 72 hpi was tested by qPCR. The data represent the change relative to the level in neuro2a cells at 0 hpi. Data are shown as the mean ± SD. Three independent experiments were performed. (D) RIPK3 knockdown neuro2a cells were constructed by RIPK3-specific RNA interference mediated by a lentiviral vector, and positive cells were purified by puromycin selection. The expression of RIPK3 in RIPK3-RNAi-neuro2a cells and vehicle-neuro2a cells was detected by Western blotting (WB). The level of RIPK3 in RIPK3-RNAi-neuro2a cells decreased significantly. (E,F) The effect of RIPK3 on the viability of neuro2a cells during JEV infection. The survival rates of RIPK3-RNAi-neuro2a cells and vehicle-neuro2a cells after JEV infection at multiplicities of infection (MOIs) of 0.1, 0.5, 1.0, and 5 with six replicates were tested by cell viability assay kits at 24 and 48 hpi. The survival rate of RIPK3-RNAi-neuro2a cells was increased relative to that of vehicle-neuro2a cells after JEV infection, especially at 48 hpi. Data are presented as the mean ± SEM.
FIGURE 3
FIGURE 3
The Japanese encephalitis virus (JEV) load was lower in receptor interacting serine/threonine-protein kinase 3 (RIPK3)-knockout mice after JEV infection via intracerebral (IC) injection. RIPK3–/– and wild-type (WT) C57BL/6 mice (8–10 weeks) were infected with 100 plaque-forming units (PFUs) JEV-P3 in 2 μl phosphate-buffered saline (PBS) via intracerebral injection. Mice were sacrificed, and the brains of each group were harvested at 3, 4, and 5 days post infection (dpi). The viral load in the brains was tested by qPCR. Data are presented as the mean ± SD. (A) The viral load in the brains of RIPK3–/– (n = 4) mice and WT (n = 5) C57BL/6 mice at 3 dpi. (B) The viral load in the brains of RIPK3–/– (n = 8) and WT (n = 7) C57BL/6 mice at 4 dpi. (C) The viral load in the brains of RIPK3–/– (n = 6) and WT (n = 5) C57BL/6 mice at 5 dpi.
FIGURE 4
FIGURE 4
Receptor interacting serine/threonine-protein kinase 3 (RIPK3) promoted the propagation of Japanese encephalitis virus in neurons. (A) Vehicle-neuro2a cells and RIPK3-RNAi-neuro2a cells were infected with JEV-p3 at a multiplicity of infection (MOI) of 0.1 and collected at 24, 48, 72 h post infection (hpi) for RNA extraction. The expression of JEV was evaluated by qPCR. The data represent the change relative to the level in vehicle-neuro2a cells at 24 hpi. Data are presented as the mean ± SD. The experiments were repeated three times. (B) Protein from vehicle-neuro2a cells and RIPK3-RNAi-neuro2a cells was extracted at 24, 48, and 72 h after JEV infection, and the E protein of JEV was detected by Western blotting (WB). Representative images from three independent experiments are shown. (C) The supernatant from vehicle-neuro2a cells and RIPK3-RNAi-neuro2a cells was collected at 24, 48, and 72 h post JEV infection. The infectious JEV particles in the supernatant were detected by plaque assay with double wells at a dilution of 1:100. Representative images from three independent experiments are shown. (D) Neuro2a cells were transfected with pCMV-GFPSpark or pCMV-RIPK3-OFPSpark, and then GFP-neuro2a cells and RIPK3-neuro2a cells were infected with JEV-p3 at an MOI of 0.1 and collected at 12 and 24 hpi for RNA extraction. The expression of JEV was evaluated by qPCR. The data represent the change relative to the level in GFP-neuro2a 12 hpi. Data are shown as the mean ± SEM of three independent experiments. (E) The supernatant from GFP-neuro2a cells and RIPK3-neuro2a cells was collected at 12 and 24 h after JEV infection. The infectious JEV particles in the supernatant were detected by plaque assay at a dilution of 1:1,000. Representative images from three independent experiments are shown. (F) RIPK3-RNAi-Neuro2a cells were transfected with pCMV-GFPSpark or pCMV-RIPK3-OFPSpark, and then GFP-RIPK3-i-neuro2a cells and RIPK3-RIPK3-i-neuro2a cells were infected with JEV-p3 at an MOI of 0.1 and collected at 12 and 24 hpi for RNA extraction. The expression of JEV was evaluated by qPCR. The data represent the change relative to that in GFP-RIPK3-i-neuro2a cells at 12 hpi. Data are shown as the mean ± SEM of three independent experiments. (G) The supernatant from GFP-RIPK3-i-neuro2a cells and RIPK3-RIPK3-i-neuro2a cells was collected at 12 and 24 h post JEV infection. The infectious JEV particles in the supernatant were detected by plaque assay at a dilution of 1:100. Representative images from three independent experiments are shown.
FIGURE 5
FIGURE 5
Receptor interacting serine/threonine-protein kinase 3 (RIPK3) knockdown had a limited effect on the level of Japanese encephalitis virus (JEV) in microglia. To explore whether RIPK3 knockout affected the level of JEV in microglia, RIPK3-RNAi-N9 cells were constructed and were infected with JEV at a multiplicity of infection (MOI) of 1. (A) The expression of RIPK3 in vehicle-N9 and RIPK3-RNAi-N9 cells was evaluated by qPCR. The expression of RIPK3 was decreased significantly in RIPK3-RNAi-N9 cells. (B). RNA from vehicle-N9 and RIPK3-RNAi-N9 cells was extracted at 24 and 48 h after JEV infection. The JEV level was evaluated by qPCR. Data are presented as the mean ± SD. The experiments were repeated three times. (C) The supernatant from vehicle-N9 and RIPK3-RNAi-N9 cells was collected at 24 and 48 h post JEV infection. The infectious JEV particles in the supernatant were detected by plaque assay with double wells at a dilution of 1:100. Representative images from three independent experiments are shown. (D) Protein from vehicle-N9 and RIPK3-RNAi-N9 cells was extracted at 24 and 48 h after JEV infection. The JEV E protein, proCaspase1, and pCaspase1 were detected by Western blotting (WB). Representative images from three independent experiments are shown. (E). The level of interleukin (IL)-1β in the supernatant from RIPK3-RNAi-N9 cells and vehicle-N9 cells was detected by ELISA. Data are presented as the mean ± SEM of three independent experiments.
FIGURE 6
FIGURE 6
Interferon (IFN)-stimulated genes (ISGs), especially IFN-induced protein 44-like gene (IFI44L), were upregulated in receptor interacting serine/threonine-protein kinase 3-knockout (RIPK3–/–) mouse brains and neurons after Japanese encephalitis virus (JEV) infection. Wild-type (WT) and RIPK3–/– mice (8–10 weeks) were injected intracerebrally with phosphate-buffered saline (PBS) or JEV 100 plaque-forming units (PFUs) in 2 μl PBS. Brains were harvested at 3 days post infection (dpi), and total RNA was extracted for RNA-sequencing. (A) The analysis of volcano plots of differentially expressed genes in the brains of WT and RIPK3–/– mice after JEV infection. (B) The change in a series of ISGs between WT and RIPK3–/– brains after JEV infection was analyzed according to RNA sequencing. In addition to IFI44L, the expression of oas1h, ifi1, zbp1, etc. was increased to some extent in RIPK3–/– mouse brains after JEV infection. (C) WT (n = 3) and RIPK3–/– (n = 3) mice were infected with JEV by intracerebral (IC) injection again, brains were collected at 3 dpi, and the viral load was evaluated by qPCR. Data are presented as the mean ± SD. (D). The level of IFI44L in the brains of WT and RIPK3–/– mice was detected by qPCR. Data are presented as the mean ± SD. (E) WT and RIPK3–/– mouse-derived primary neurons were infected with JEV at a multiplicity of infection (MOI) of 0.1, and the expression of IFI44L was detected at 24, 48, and 72 hpi by qPCR. Data are presented as the mean ± SD. The experiments were repeated three times. (F) RIPK3-RNAi-neuro2a cells and vehicle-neuro2a cells were infected with JEV at an MOI of 0.1, and the expression of IFI44L was tested at 24, 48, and 72 hpi by qPCR. Data are presented as the mean ± SD. The experiments were repeated three times.
FIGURE 7
FIGURE 7
The increase in interferon-induced protein 44-like gene (IFI44L) was independent of the phosphorylation of receptor interacting serine/threonine-protein kinase 3 (RIPK3) or mixed lineage kinase domain-like pseudokinase (MLKL). The phosphorylation of RIPK3 and subsequently MLKL formed the classical signal of necroptosis. To explore whether the inhibition of ifi44l was dependent on the phosphorylation of RIPK3 or MLKL, Neuro2a cells were treated with 1.5 nM RIPK3 kinase inhibitor GSK872 (RD, United States) or 1 μM MLKL inhibitor necrosulfonamide (RD, United States) 2 h before Japanese encephalitis virus (JEV) infection, and the inhibitors remained until 48 h post infection (hpi). The experiments were repeated three times. (A) RNA from vehicle-neuro2a cells, RIPK3-RNAi-neuro2a cells, and inhibitor-treated neuro2a cells was extracted at 24 and 48 hpi, and the expression of IFI44L was evaluated by qPCR. Data are presented as the mean ± SD. (B) The viral load in vehicle-neuro2a cells, RIPK3-RNAi-neuro2a cells, and inhibitor-treated neuro2a cells was detected by qPCR. Data are presented as the mean ± SD. (C) Protein from RIPK3-RNAi-neuro2a cells, vehicle-neuro2a cells, and inhibitor-treated neuro2a cells was extracted, and the JEV E protein was tested by Western blotting (WB). (D) Supernatants from RIPK3-RNAi-neuro2a cells, vehicle-neuro2a cells, and inhibitor-treated neuro2a cells were collected after JEV infection for 24 and 48 h. The infectious JEV particles in the supernatant were detected by plaque assay at a dilution of 1:100.
FIGURE 8
FIGURE 8
The propagation of Japanese encephalitis virus (JEV) was inhibited in interferon-induced protein 44-like gene (IFI44L)-overexpressing neuro2a cells and enhanced in IFI44L- and receptor interacting serine/threonine-protein kinase 3 (RIPK3) double knockdown neuro2a cells. (A) To identify the effect of IFI44L on JEV propagation, IFI44L-overexpressing neuro2a cells (IFI44L-neuro2a) were constructed, and GFP-Zeocin-overexpressing neuro2a cells (GZ-neuro2a) were constructed as a control. The experiments were repeated three times. The expression of IFI44L was tested by qPCR. Data are presented as the mean ± SD. (B) IFI44L-neuro2a cells and GZ-neuro2a cells were infected with JEV at a multiplicity of infection (MOI) of 0.1, and the virus load was detected by qPCR at 24 and 48 h post infection (hpi). (C) The expression of IFI44L in neuro2a cells after JEV infection for 24 and 48 h was tested by qPCR. (D,E) Neuro2a cells were treated with three different IFI44L-interfering lentiviruses targeting different segments of IFI44L. The expression of IFI44L and viral mRNA was tested by qPCR at 48 hpi. Data are presented as the mean ± SD. The experiments were repeated three times. (D) The expression of IFI44L was decreased in IFI44L-RNAi-neuro2a cells compared to vehicle-neuro2a cells. (E) The virus load in IFI44L-RNAi-neuro2a cells was higher than that in vehicle-neuro2a cells. (F,G). To identify the role of IFI44L in JEV propagation in RIPK3-RNAi-neuro2a cells, IFI44L knockdown was performed in RIPK3-RNAi-neuro2a cells. The expression of IFI44L (F) and viral mRNA (G) in RIPK3-RNAi-neuro2a cells and IFI44L/RIPK3-RNAi-neuro2a cells was tested by qPCR at 24 and 48 hpi. Data are presented as the mean ± SD. The experiments were repeated three times.
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