Japanese encephalitis virus activates autophagy as a viral immune evasion strategy

Abstract

In addition to manipulating cellular homeostasis and survivability, autophagy also plays a crucial role in numerous viral infections. In this study, we discover that Japanese encephalitis virus (JEV) infection results in the accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II) protein and GFP-LC3 puncta in vitro and an increase in autophagosomes/autolysosomes in vivo. The fusion between autophagosomes and lysosomes is essential for virus replication. Knockdown of autophagy-related genes reduced JEV replication in vitro, as indicated by viral RNA and protein levels. We also note that JEV infection in autophagy-impaired cells displayed active caspases cleavage and cell death. Moreover, we find that JEV induces higher type I interferon (IFN) activation in cells deficient in autophagy-related genes as the cells exhibited increased phosphorylation and dimerization of interferon regulatory factor 3 (IRF3) and mitochondrial antiviral signaling protein (MAVS) aggregation. Finally, we find that autophagy is indispensable for efficient JEV replication even in an IFN-defective background. Overall, our studies provide the first description of the mechanism of the autophagic innate immune signaling pathway during JEV infection.

Figure 1. Induction of autophagy by JEV.

(A) N2a cells were treated with Tg (1 ug/ml, 24 hours) or 3-MA (10 mM, 24 hours) or infected or mock-infected with P3 virus (MOI = 1) for 48 hours. Cell lysates from different time points were harvested for immunoblotting. (B) BHK21 cells were infected or mock-infected with JEV P3 or SA14-14-2 virus for 48 hours, and the cell lysates were harvested for immunoblotting. (C) Twelve hours after transfection with GFP-LC3 or the GFP vector plasmid, N2A cells were mock-infected or infected with JEV P3 (MOI = 1) virus for 48 hours or treated with Tg for 24 hours. The nuclei were stained with DAPI, and the cells were observed under a fluorescence microscope. The white scale bar is 20 µm. (D) Cells were treated as in (C); cells containing five or more GFP-LC3 dots were defined as autophagy-positive cells. The percentages of cells with characteristic punctate GFP-LC3 fluorescence relative to all GFP-positive cells were calculated. The results represent the mean data from three independent experiments. The statistical significance of changes in viral RNA and virus yield compared with the control were calculated by t-test. **: P<0.01. (E) The ratio of autophagy (autophagic cells/total cells) was determined by counting the number of cells containing autophagic vacuoles among a total of 30 randomly selected cells. The larger boxed images on the right represent enlargements of the smaller boxed insets of (b). The arrows indicate representative autophagosome-like structures. The cell nucleus and mitochondria are abbreviated as N and M. a) mock-infected and b) JEV-infected mouse brains. The magnification is 2500 X.

Figure 2. Autophagosome maturation induced by JEV infection.

(A) N2a cells were co-transfected with the DsRed-LC3 and LAMP1-GFP plasmids. Twelve hours after transfection, the cells were then challenged with JEV. Thirty-six hours post-infection, the cells were fixed, and the nuclei were stained with DAPI. The cells were observed under a confocal fluorescence microscope. The white scale bar is 20 µm. (B) N2a cells were mock- or JEV-infected at an MOI of 5 for 24 hours and then treated with BAF-A1 (100 nM), CQ (50 µM) or vehicle control and then lysed for analysis. N2a cells were transfected with siRNA oligonucleotides targeting the indicated gene. Twenty-four hours later, the cells were infected at an MOI of 0.1 for 72 hours and then harvested and lysed for RNA (C) and protein (D) analysis. Data for JEV RNA levels represent means with SEM of three independent experiments, compared with the control. The statistical significance of changes in viral RNA compared with the control was calculated by t-test. **: P<0.01.

Figure 3. JEV Replication is dependent upon autophagy.

N2a cells were transfected with siRNA oligonucleotides against Atg5 and Beclin1. Twenty-four hours later, the cells were infected at an MOI of 0.1. The cells were harvested at the indicated time for RNA (B) and immunoblotting analysis (A, C). The fold-induction ratio of JEV E/actin was quantified by densitometric analysis using FluorChemHD software. (D) The 72 h p.i. culture supernatants were collected for plaque assays on BHK-21 cells. Three independent replicates were performed, and the results are presented as the means with SEM. The statistical significance of changes in viral RNA and virus yield compared with the respective controls was calculated by t-test. **: P<0.01.

Figure 4. JEV infection in autophagy knock-down cells induces greater cell death.

(A) Cells were treated the same as in Fig. 3B. After 72 hours post-infection, WST-8 dye (Beyotime, C0038) was add to each well, cells were incubated at 37°C for 2 h and the absorbance was determined at 450 nm using a microplate reader. N2a cells were transfected with the indicated siRNA, and 48 hours later, the cells were infected at an MOI of 1 for 48 hours. Total cellular DNA was extracted, and the mitochondrial DNA copy number was measured by quantitative PCR and normalized to nuclear DNA levels according to the ratio of mtDNA COI over 18S rDNA (B); cell proteins were also harvested, and caspase-9, caspase-3 and actin were analyzed by immunoblotting (C). Three independent replicates were performed, and the results are presented as the means with SEM. The statistical significance of changes compared with the respective controls was calculated by t-test. **: P<0.01.

Figure 5. JEV infection up-regulates the IFN signaling pathway in autophagy-deficient A549 cells.

(A) The cells were first transfected with the indicated siRNA, and 48 hours later the cells were then transfected with IFN β-Luc Firefly luciferase and an internal control, pRL-TK (Renilla luciferase). After 24 hours, the cells were infected with JEV at an MOI of 10. Finally, the cells were harvested at the indicated time, and dual luciferase activity was determined. (B) The cells were first transfected with the indicated siRNA, and 72 hours later the cells were infected at an MOI of 10. At different time points, total RNA was extracted for analysis. (C) The experiment was processed in parallel with (B), except that proteins used for immunoblotting were harvested at 24 hours post-infection. Three replicates were performed, and the results are presented as the means with SEM. The statistical significance of variations compared with the respective controls was calculated by t-test. *: P<0.05, **: P<0.01.

Figure 6. Autophagy-related type I IFN anti-viral pathway.

(A) A549 cells were treated or mock-treated with IFN-αA/D (1,000 U/ml for 6 hours) and then infected or mock-infected with JEV (MOI = 0.1) for 48 hours. The cells were then harvested for JEV RNA and E protein analysis. (B) A549 cells were transfected with the indicated siRNA, and 48 hours later, the cells were infected or mock-infected with JEV (MOI = 0.1) for another 48 hours before being harvested for JEV RNA and E protein analysis. Three replicates were performed, and the results are presented as the means with SEM. The statistical significance of variations compared with the respective controls was calculated by t-test. **: P<0.01.

Figure 7. Autophagy is indispensable for JEV replication in the IFN-defective background.

A549 cells were transfected with the indicated siRNA, and 48 hours later, the cells were infected or mock-infected with JEV (MOI = 0.1) for 48 hours before being harvested for JEV RNA (A) and E protein analysis (B). Three replicates were performed, and the results are presented as the means with SEM. The statistical significance compared with the control was calculated by t-test. **: P<0.01.