AKT2 reduces IFNβ1 production to modulate antiviral responses and systemic lupus erythematosus

Introduction

I-IFN and IFN-induced transcription of a wide range of IFN-stimulated genes (ISGs) are both critical to elicit innate and adaptive immune responses against various infections (Sadler & Williams, 2008; Schneider et al, 2014; Wu & Chen, 2014; Boxx & Cheng, 2016). However, I-IFN is pathogenic during the development of autoimmune disorders, which might be triggered by multiple genetic and environmental factors. Especially, the persistent presence of I-IFN has been proved to accelerate the pathogenesis of SLE (Munz et al, 2009; Bengtsson & Ronnblom, 2017). It is therefore vital to understand the precise mechanism of regulating I-IFN production, which can offer new drug targets to treat infections or SLE.

The induction of I-IFN is elicited by pattern recognition receptors (PRRs) and a cascade of downstream molecules. IRF3 is a master transcription factor for I-IFN production, and IRF3 transcriptional activity and other biological functions are precisely regulated via its phosphorylation (Taniguchi et al, 2001; Saitoh et al, 2006; Chattopadhyay et al, 2016; Huai et al, 2019). Phosphorylated IRF3 undergoes conformational change to form dimers, which then enters the nuclei and associates with interferon transcriptional elements to initiate I-IFN transcription, mainly Ifnb1 (Honda et al, 2006; Sadler & Williams, 2008). Phosphorylation sites of the cluster 1 (Ser385/Ser386) and the cluster 2 (Ser396, Ser398, Ser402, Thr404, and Ser405) in the C terminus of IRF3 have been demonstrated to indicate IRF3 activation status in antiviral immunity (Lin et al, 1998, 1999; Panne et al, 2007). Further studies suggest that Ser386 is critical, while Ser396 plays a moderate role in IRF3 dimerization and activation (Dalskov et al, 2020; Jing et al, 2020), and PTEN regulates the phosphorylation of IRF3 at Ser97 to prevent IRF3 nuclear import (Li et al, 2016). Despite these findings, it is still unclear whether IRF3 phosphorylation at other sites could affect the intracellular vs. nuclear translocation of IRF3.

The serine/threonine kinase AKT (also named protein kinase B/PKB) represents a critical kinase family that can phosphorylate different target proteins and control a variety of cellular functions. AKT family contains three highly conserved isoforms in mammals, AKT1/PKBα, AKT2/PKBβ, AKT3/PKBγ (Fayard et al, 2010). There are growing researches about the distinct role of AKTs in the induction of I-IFN. After herpes simplex virus-1 (HSV-1) infection, AKT1 phosphorylates TBK1 to attenuate STING signaling (Wu et al, 2019); others also suggest that AKT1 phosphorylates cGAS to suppress its enzymatic activity and results in downregulation of IFNβ1 production (Seo et al, 2015). In contrast to the negative role of AKT1 on I-IFN production, our group has recently demonstrated that in response to viral infection, AKT3 expression is significantly enhanced in macrophages and promotes IFNβ1 induction (Xiao et al, 2020). It is not fully understood whether and how AKT2 could affect I-IFN production.

In this study, we discovered that Akt2 expression was downregulated in macrophages upon treatment with IFNβ1, the Toll-like receptor7/9 (TLR7/9) agonists, and during viral infection or during TMPD (N, N, N′, N′-tetramethyl-p-phenylenediamine)-induced SLE. Disruption of Akt2 enhanced the production of IFNβ1 and protected mice from viral infection. Conversely, Akt2 deficiency led to the much worse SLE for increased IFNβ1 production. Mechanismly, AKT2 could directly bind and phosphorylate IRF3 at Thr207, which worked together with 14-3-3ε to restrain the nuclear translocation of IRF3. In addition, AKT2 but not AKT2 kinase-dead mutant overexpression aggravated viral infection in zebrafish larvae, while overexpression of the IRF3-T207A promoted IFNβ1 production and reduced viral infection which was not reversed by AKT2 in zebrafish larvae. These data have demonstrated that AKT2 phosphorylates IRF3-T207 to reduce IRF3 nuclear localization and IFNβ1 induction, providing either protective or pathogenic role in SLE and viral infections.

Results Akt2 expression is negatively correlated with IFNβ1 production

We firstly examined the distribution of AKT members in different organs. In contrast to the dominant expression of Akt3 in brain, Akt1 was widely expressed and Akt2 was mainly expressed in heart, liver, and kidney (Fig EV1A). The expression levels of Akt1 and Akt2 were downregulated, while Akt3 levels were upregulated in livers from vesicular stomatitis virus (VSV)-infected mice (Fig EV1B). We also confirmed the decreased Akt2 expression in spleen and lung from VSV-infected mice (Fig EV1C), as well as from hepatitis B virus (HBV)-infected hepatocellular carcinoma (HCC) patients (Fig EV1D) that is consistent with the reduced AKT2 expression in livers from HBV-infected patients provided by the GEO database (Fig 1A, left panel). Akt2 mRNA levels were also substantially downregulated in brain or lung from Japanese encephalitis virus (JEV)-infected or the influenza virus (H7N9, H1N1/PR8)-infected mice, respectively (Fig 1B). Moreover, the reduced Akt2 mRNA expression was detected in bronchial epithelial cells upon H1N1 infection (Fig 1A, right panel), and in murine peritoneal elucidated macrophages (PEMs) after treated with lipo-poly(I:C) to activate the RIG-1/MAVS pathway or treated with lipo-poly(A:T), lipo-ISD and HSV-1 to activate the cGAS/STING pathway (Fig 1C). Then, we also detected the decreased expression of total AKT2 and the phosphorylated AKT2-Ser474 at protein levels in VSV-infected PEMs (Fig EV1E).

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Figure EV1. The reduced Akt2 expression and the verification of Akt2 siRNAs efficacy (related to Fig 1)

A. qRT–PCR analysis for the mRNA expression of Akt1 (n ≥ 3), Akt2 (n ≥ 3), Akt3 (n = 4) in the different tissues of at least three different WT C57BL/6 mice. B, C. The mRNA expression of Akt1, Akt2, or Akt3 in the liver, spleen, or lung from WT mice after the VSV infection for 6 h (1 × 106 PFU/g, i.v.) were detected by qRT–PCR. Akt1 in liver, n ≥ 5; Akt2 in liver, n ≥ 7; Akt3 in liver, n ≥ 5; Akt2 in spleen, n ≥ 7; Akt2 in lung, n ≥ 7. D. qRT–PCR analysis for the mRNA expression of AKT2 in the liver tissue of the normal individuals (n = 3) or HBV-infected different HCC patients (n = 8). E. Immunoblot analysis of p-AKT2 (Ser473), AKT2, p-TBK1 (Ser172), TBK1, p-IRF3 (Ser396), IRF3 and GAPDH in the VSV-stimulated PEMs at indicated time points. F. The mRNA expression of Ifnb1 and Cxcl10 were measured by qRT–PCR in the WT and Ifnar1 KO PEMs stimulated with VSV and lipo-ISD for 6 h. n = 3, respectively. G. qRT–PCR analysis for the mRNA expression of Ifnb1 (n = 3, left panel) and then, the mRNA change of Akt2 (n = 3, right panel) were further calculated by compared with “siNC mock” in the PEMs with Socs1 or Socs3 knockdown followed by stimulation of VSV for 6 h. H. The mRNA level (left panel, by qRT–PCR) and protein expression (right panel, by immunoblot) of AKT2, AKT1, AKT3 in WT PEMs transfected with siNC, siAkt2-1, or siAkt2-2 for 48 h.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; using unpaired t-test (B–D), or two-way ANOVA test (G). Data are pooled from at least three independent experiments (B, C, F, G) or representative of three independent biological replicates (A, E, H). Error bars (A-D, H, mean ± SD; F and G, mean ± SEM).

Source data are available online for this figure.

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Figure 1. Akt2 expression is negatively correlated with IFNβ1 production

A. The mRNA value of AKT2 was analyzed in the GEO Profiles from the liver explant of hepatitis B virus (HBV)-associated acute liver failure (ALF) patients (GDS4387/225471_s_at) and from bronchial epithelial cells with pandemic and seasonal H1N1 influenza virus infections in vitro (GDS4855/203808_at). normal, n = 10; HBV, n = 17; control, n = 3; influenza A, n = 9. B. The mRNA expression of Akt2 in the brain homogenates of WT mice with JEV injection (5.0 × 106 PFU/g, i.v.) or in the lung of WT mice with H7N9 (105.5 EID50, i.n.) and PR8 infection (10 LD50, i.n.) were detected by quantitative reverse transcription-PCR (qRT–PCR). Mock, n = 4; JEV, n = 12; PBS, n = 6; H7N9, n = 4; PR8, n = 8. C. The qRT–PCR analysis of Akt2 mRNA in PEMs stimulated with lipo-poly(I:C) (1 μg/ml), lipo-poly(A:T) (1 μg/ml), lipo-ISD (3 μg/ml), or HSV-1 (MOI, 1) for 6 h. n = 3, respectively. D. The mRNA of Akt2 (red line) and Ifnb1 (black line) were analyzed by qRT–PCR in the mock and VSV (MOI, 1) or LM (MOI, 1)-stimulated PEMs or BMDMs at the indicated time. Mock and VSV, n = 4; mock and LM, n ≥ 3. E. The mRNA levels of Akt2 in WT (n = 3) and Ifnar1 KO (n = 3) PEMs stimulated with VSV and lipo-ISD for 6 h were detected by qRT–PCR. F. PEMs were pretreated with the indicated inhibitors for 2 h and stimulated with recombined of IFNβ1 (1 μg/ml) for another 3 h, followed by qRT–PCR for detection of the Akt2 mRNA levels. DMSO, n = 2; inhibitors, n = 3. G, H. PEMs were pretreated with DMSO or AKT2 inhibitor CCT128930 (10 μM) (G) for 2 h, or knocked down of Akt2 with siAkt2-1 (20 nM) for 48 h (H), then the mRNA level of Ifnb1 was measured by qRT–PCR after lipo-poly(I:C), lipo-poly(A:T), lipo-ISD, VSV, or HSV-1 stimulation for another 6 h. n = 3, respectively.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using unpaired t-test (A, B left panel), or one-way ANOVA test (B right panel, C and F), or two-way ANOVA test (E, G and H). Data are from two (F) or at least three independent biological replicates (C–E, G, H). Error bars (A and B, mean ± SD; C-H, mean ± SEM).

Source data are available online for this figure.

Notably, we found that the mRNA levels of Akt2 were negatively correlated with Ifnb1 mRNA levels in VSV-infected PEMs, or in Listeria monocytogenes (LM) infected bone marrow-derived macrophages (BMDMs) (Fig 1D). Therefore, to investigate whether the IFNβ1/IFNAR signaling was responsible for Akt2 expression, we obtained PEMs from the I-IFN receptor-deficient (Ifnar1 KO) mice which hardly produced Ifnb1 and Cxcl10 in response to VSV infection or lipo-ISD treatment (Fig EV1F), ensuring PEMs from Ifnar1 KO mice were defective in response to IFNβ1 stimulation. Interestingly, Akt2 expressions were no longer downregulated in VSV-infected or lipo-ISD-treated PEMs from the Ifnar1 KO mice (Fig 1E).

The IFNβ1/IFNAR signaling activates several key downstream effectors, and which one could participate in the suppression of Akt2 expression? To answer this, WT PEMs were pretreated with various inhibitors targeting IFNβ1/IFNAR downstream proteins, including signal transducer and activator of transcriptions (STATs), p38, activator protein-1 (AP-1), nuclear transcription factor kappa-B (NF-κB), and only the inhibitors targeting STAT3 and STAT6 (not STAT5) could prevent the downregulation of Akt2 expression (Fig 1F). Furthermore, in the cytokine-activated Janus kinase (JAK)/STAT signaling cascade, the suppressors of cytokine signaling (SOCS) family members, especially SOCS1 and SOCS3, are induced by I-IFN stimulation, which then inhibit STAT activity to form a negative feedback loop (Morris et al, 2018). Knockdown of Socs1 or Socs3 expression by siRNAs further reduced Akt2 mRNA levels and enhanced I-IFN production after VSV treatment (Fig EV1G). Those results indicate that during viral infection, the IFNβ1/IFNAR signal decreases Akt2 expression via STAT3/STAT6, and knockdown of SOCS1/SOCS3 could release their inhibition on STAT3/6 and further reduce Akt2 expression.

To elucidate whether AKT2 regulated Ifnb1 production, PEMs were treated with the AKT2 selective inhibitor CCT128930 (Fig 1G) or Akt2 was knocked down by siRNA (Figs 1H and EV1H). After stimulated with lipo-poly(I:C), lipo-poly(A:T), lipo-ISD or infected with VSV and HSV-1, inhibition of AKT2 or knockdown of Akt2 both enhanced the mRNA levels of Ifnb1 in PEMs (Fig 1G and H). These data suggest that Akt2 expression is reduced upon stimuli or viral infection, and Akt2 negatively regulates IFNβ1 production.

AKT2 kinase activity is indispensable to attenuate I-IFN production in macrophages and in zebrafish larvae

To better understand the AKT2 function, we next used the Akt2 knock-out (KO) mice. PEMs, BMDMs, and embryonic fibroblasts (primary-MEFs) were prepared from WT and Akt2 KO mice. Akt2 KO macrophages showed enhanced Ifnb1 production after treated with various stimuli to activate the RIG-1/MAVS pathway or the cGAS/STING pathway (Figs 2A and EV2A). The ISGs including Ifna4, Cxcl10, and Ccl5 were also induced at higher levels in Akt2 KO PEMs upon VSV infection (Fig 2B). In addition, TLR3 and TLR4 pathway both can converge to induce Ifnb1, and Akt2 KO PEMs also produced higher levels of Ifnb1 upon treated with poly(I:C) to activate the TLR3 pathway or treated with LPS to activate the TLR4 pathway (Fig 2C). Following I-IFN production in Akt2 KO macrophages, we further asked whether this affected bystander cells. WT or Akt2 KO PEMs were stimulated with lipo-poly(I:C) for 3 h and washed several times to remove any residual lipo-poly(I:C). Fresh medium was added to WT or Akt2 KO PEMs for another 9 h to collect cell medium, which was used to stimulate MEFs or PEMs. The culture medium from Akt2 KO PEMs indeed induced higher Ifnb1, Ifna4 production in MEFs or Cxcl10, Ifit1, Mx1 production in PEMs (Fig EV2B). This suggests that Akt2 KO macrophages not only enhance I-IFN production but also enhance the ISG expression in bystander cells such as PEMs and MEFs, therefore better preventing the viral infection and propagation. Consistent with Akt2 KO PEMs (Fig 2A–C), Akt2 KO primary-MEFs also enhanced Ifnb1 mRNA level and reduced the amount of GFP-fused VSV infection shown by immunofluorescence microscopy or FACS assays (Figs 2D and EV2C). Moreover, after VSV infection, overexpression of AKT2 in MEFs and HEK293T cells restrained Ifnb1 expression (Fig 2E), and IFNβ1-reporter luciferase assay confirmed that AKT2 inhibited IFNβ1 transcription in a dose-dependent manner (Fig EV2D).

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Figure 2. AKT2 kinase activity is indispensable to attenuate I-IFN production in macrophages and in zebrafish larvae

A, B. qRT–PCR detection for the mRNA expression of Ifnb1 (A), Ifna4, Cxcl10 and Ccl5 (B) in WT (n = 3) and Akt2 KO (n = 3) PEMs stimulated with lipo-poly(I:C), VSV, lipo-poly(A:T), lipo-ISD or HSV-1 for 6 h. C. The expression of Ifnb1 mRNA level by qRT–PCR in WT (n = 3) and Akt2 KO (n = 3) PEMs treated with poly(I:C) (10 μg/ml) for 6 h (left panel) or LPS (1 μg/ml) for 2 h (right panel). D. The expression of Ifnb1 in WT (n = 3) and Akt2 KO (n = 3) primary-MEFs with VSV treatment (MOI, 0.1) for 6 h was measured by qRT–PCR (left panel). After VSV treatment for 3 h, the culture suspension was discarded and washed 3 times with PBS, then primary-MEFs were cultured with fresh medium for another 15 h for analysis of the VSV-infected (GFP) primary-MEFs by fluorescence microscope (middle panel, representative images) and by FACS assay (right panel). Bar, 100 μm. E. MEF (left panel, n = 3) and HEK293T cells (right panel, n = 3) were transfected with AKT2 for 24 h and stimulated with VSV for 6 h, then cells were harvested for the detection of mRNA expression of Ifnb1 by qRT–PCR. F. IFNβ1 luciferase assays of HEK293T cells with transfection of AKT2/AKT2-T309A/S474A and TBK1 (n = 3) or infection of VSV (n = 3). Protein expression levels are shown in Fig EV2E. G. Zebrafish larva at 48 h after fertilization were micro-injected GFP-fused VSV (1 × 103 PFU/larvae) for 18 h, then the representative images of VSV-infected zebrafish larva were collected by fluorescence microscope. The infected area (GFP) and macrophages (Red) are indicated by arrows. Bars, 200 μm. H. Zebrafish larvae were overexpressed the indicated protein for 48 h and challenged with VSV for another 6 h, then the mRNA levels of ifn1 in zebrafish larvae were measured by qRT–PCR (left panel). Every dot represents three zebrafish embryos. Horizontal square bracket shows the statistical analysis of the comparison with “PBS mock”, the rest shows the comparison with “PBS VSV”. PBS mock, n = 5; PBS VSV, n = 13; AKT2 VSV, n = 7; AKT2-T309A/S474A VSV, n = 9. H&E staining (middle panel) and survival rates (Kaplan–Meier curve) (right panel) were collected from zebrafish larvae after VSV micro-injection for 18 h or longer. The arrows indicated the VSV-infected eye and skeletal muscle in zebrafish larvae. Bars, 100 μm.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test (E, F, H left panel), or two-way ANOVA test (A–C, D left and right panel), or log-rank (Mantel–Cox) test (H right panel). Data are from three independent experiments (A–C, D left and right panel, E, F) or representative of three independent biological replicates (D middle panel, G, H). Error bars (A–F, mean ± SEM; H, mean ± SD).

Source data are available online for this figure.

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Figure EV2. Akt2 absence facilitates the cellular antiviral responses and zebrafish larva is a suitable model for protein overexpression or VSV infection (related to Fig 2)

Determination of the Ifnb1 mRNA expression by qRT–PCR in M-CSF (20 ng/ml) induced BMDMs stimulated with lipo-poly(I:C), VSV, lipo-poly(A:T), lipo-ISD or HSV-1 for 6 h. n = 3, respectively. The mRNA expression levels of indicated genes were analyzed by qRT–PCR in MEFs (Ifnb1 and Ifna4) and PEMs (Cxcl10, Ifit1 and Mx1) which were treated with conditional medium for 3 h. FACS analysis of the viral load indicated by GFP in primary WT and Akt2 KO MEFs. IFNβ1 luciferase activity was measured in HEK293T cells transfected with empty vector or AKT2 at increasing amount for 24 h and infected by VSV for another 6 h, respectively. Immunoblot analysis showed the indicated constructs in HEK293T cells. The protein levels of indicated constructs as showed in Fig 2F were measured by immunoblot analysis. The in vitro transcriptional mRNAs were verified by agarose gel electrophoresis (left panel) and its translation in zebrafish larvae for 48 h were verified by immunoblot analysis (right panel). Representative images of mpeg1-mCherry macrophages (Red) and VSV-infected area (GFP) were collected by fluorescence microscopy in the brain and abdominal vessel (arrows) of zebrafish larvae as showed in Fig 2G. Bars, 200 μm.

Data information: **P < 0.01, ***P < 0.0011, ****P < 0.0001; using a one-way ANOVA test (B) or two-way ANOVA test (A). Data are from three independent experiments (A) or representative of three independent biological replicates (B–G). Error bars (A, mean ± SEM; B and D, mean ± SD).

Source data are available online for this figure.

Because AKT2 kinase activity is pivotal for its biological function in various cell types, we next overexpressed the kinase-dead mutant AKT2-T309A/S474A in HEK293T cells to explore whether AKT2 kinase activity was functioned in suppressing IFNβ1 production. When co-expressed with TBK1 or infected with VSV, AKT2 reduced the IFNβ1 luciferase readings, while the AKT2-T309A/S474A failed to inhibit this (Figs 2F and EV2E).

To verify the in vivo antiviral function of AKT2 or its kinase-dead mutant AKT2-T309A/S474A, we employed the zebrafish model as previously described (Meng et al, 2016; Guerra-Varela et al, 2018). Zebrafish can be generated in short term to express the interested proteins and is responsive to VSV infection. We used mpeg1-mCherry transgenic zebrafish larvae in which macrophage-lineage cells express mCherry and could be monitored in vivo. AKT2 mRNA was transcripted in vitro and injected into zebrafish embryos for 48 h to ensure AKT2 expression at protein levels (Fig EV2F), followed by VSV infection. We observed the colocalization of GFP-fused VSV (green) with mpeg1-mCherry macrophages (red) in the dorsal muscle, brain, and abdominal vessels of zebrafish larvae (Figs 2G and EV2G, indicated by arrows). Zebrafish larvae overexpressing AKT2 reduced ifn1 mRNA levels, displayed severer tissue damages in eye and trunk muscle, and decreased the survival rates (Fig 2H). In contrast, zebrafish larvae overexpressing the AKT2-T309A/S474A no longer aggravate viral infection than PBS control (Fig 2H). Together, we have demonstrated that AKT2 inhibits Ifnb1 expression and this is dependent on AKT2 kinase activity.

AKT2 restrains IRF3 nuclear translocation via 14-3-3ε

To gain mechanistic insight into the function of AKT2 on suppression of IFNβ1 production, we employed the IFNβ1 reporter luciferase system in HEK293T cells. Overexpression of AKT2 reduced MAVS-, TRIF-, or TBK1-induced IFNβ1 luciferase readings, but could not affect the constitutively active form IRF3-5D-mediated IFNβ1 transcription (Figs 3A and EV3A). When IRF3 or IRF3-5D was reconstituted into HEK293T-IRF3-KO cell line (Fig EV3B), AKT2 repressed IRF3 but not IRF3-5D-induced IFNβ1 luciferase activity in response to VSV infection (Fig 3B). Furthermore, when Irf3 was knocked down by siRNAs (Fig EV3C), Akt2 KO PEMs failed to further enhance Ifnb1 levels under VSV infection (Fig 3C). These data indicate that AKT2 inhibits IFNβ1 production via IRF3.

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Figure 3. AKT2 restrains IRF3 nuclear translocation via 14-3-3ε

A, B. IFNβ1 luciferase assays (upper panels) in HEK293T (A) and HEK293T-IRF3 KO (B) cells transfected with the indicated plasmids or infected with VSV. Immunoblot analysis showed the indicated constructs in HEK293T cells (bottom panels). n = 3, respectively. C. The mRNA levels of Ifnb1 were measured by qRT–PCR in WT (n = 3) and Akt2 KO (n = 3) PEMs with Irf3 knockdown for 48 h and VSV treatment for 6 h. D. WT and Akt2 KO PEMs were infected with or without VSV for 6 h. Then, cell lysates of WT or Akt2 KO PEMs were prepared for immunoprecipitation using anti-AKT2 antibody, followed by immunoblotting using anti-IRF3 antibody (left panel). Alternatively, cell lysates were prepared for immunoprecipitation using anti-IRF3 antibody, followed by immunoblotting using anti-AKT2 antibody (right panel) to detect the endogenous interaction between AKT2 and IRF3. E. Immunoblot analysis of GST-AKT2 and His-IRF3 interaction in a GST pull-down assay. F. Immunoassay of IRF3 in dimer or monomer form by native-gel and p-IRF3 (Ser396), p-TBK1 (Ser172), AKT2 and ACTIN by SDS-gel in WT and Akt2 KO PEMs with VSV stimulation for 6 h. G. Immunofluorescent microscopic imaging (left panel) and statistics analysis (right panel) for IRF3 nuclear translocation in WT and Akt2 KO PEMs at 6 h post-VSV infection. IRF3 (red), Nuclei (Hoechst, green). The white arrows indicate the nuclei with IRF3 translocation. Mock, n = 2; VSV, n = 3. Bar, 20 μm. H. Immunoassay of IRF3, AKT2, LaminB1, and Tubulin in the nuclear and cytoplasmic fractions from the WT and Akt2 KO PEMs after VSV treatment for 6 h. Tubulin and LaminB1 were used as cytoplasmic and nucleic protein loading control, respectively. I. Immunoassay of IRF3, SP1, and Tubulin in the nuclear and cytoplasmic fractions of the HEK293T cells overexpressed with indicated constructs for 24 h and stimulated with VSV for 6 h. Tubulin and SP1 served as cytoplasmic and nucleic protein loading control, respectively. J. IFNβ1 luciferases assays were performed in HEK293T cells transfected with AKT2 and 14-3-3ε followed by VSV treatment. n = 3, respectively. K. qRT–PCR analysis for the Ifnb1 mRNA levels in VSV-stimulated WT and Akt2 KO PEMs with 14-3-3ε siRNA pretreatment. n = 3, respectively.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA (A, B, J), or two-way ANOVA test (C, G right panel, K). Data are from three independent experiments (A–C, J and K), or representative of two or three independent biological replicates (D–I). Error bars (A–C, G, J and K, mean ± SEM).

Source data are available online for this figure.

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Figure EV3. AKT2 interacts with 14-3-3ε and IRF3 to slack IFNβ1 production (related to Fig 3)

IFNβ1 luciferase activity was measured (top panel, n = 3) in HEK293T cells transfected with indicated constructs. Immunoblot analysis showed the indicated constructs in HEK293T cells (bottom panels). Immunoblot analysis of IRF3 in HEK293T and HEK293T-IRF3 KO cells. qRT–PCR analysis for knockdown efficiency of Irf3 siRNAs (siIrf3-1 and siIrf3-2). WT and Akt2 KO PEMs were treated with or without VSV or HSV-1 for 6 h, and then, the Irf3 mRNA level was measured by qRT–PCR. n = 3, respectively. Schematic diagram of constructs expressing HA-tagged human AKT2, Flag-tagged human IRF3 and their mutants (left panel). HEK293T cells were co-transfected with indicated constructs for 24 h. Anti-HA and anti-Flag antibody-conjugated agarose beads were used for immunoprecipitation, and the interaction domain was mapped by immunoblot analysis with the indicated antibodies (right panel). Immunoassay of IRF3 in dimer or monomer form by native-gel and p-IRF3 (Ser396), p-TBK1 (Ser172), AKT2 and GAPDH by SDS-gel in WT and Akt2 KO PEMs with lipo-ISD stimulation for 6 h. Immunofluorescent microscopic imaging (left panel) and statistics analysis (right panel, n = 3) for IRF3 nuclear translocation in lipo-ISD stimulated WT and Akt2 KO PEMs. IRF3 (red), Nuclei (Hoechst, green). The white arrows indicate the nuclei with IRF3 translocation. Bar, 10 μm. Immunoprecipitation with anti-Myc antibody-conjugated agarose beads and immunoblot analysis about the interactions between HA-AKT2 and Myc-14-3-3ζ or Myc-14-3-3ε in HEK293T cells after overexpression of indicated constructs for 24 h. The mRNA level of 14-3-3ζ (n = 4) was measured by qRT–PCR in WT PEMs transfected the two 14-3-3ζ siRNAs (si14-3-3ζ-1 and si14-3-3ζ-2) for 48 h (left panel). The mRNA level of Ifnb1 (n = 3) was determined by qRT–PCR in VSV-stimulated PEMs that were transfected with si14-3-3ζ-1 (middle panel). IFNβ1 luciferase activity (n = 3) was measured in HEK293T cells after being transfected with empty vector or 14-3-3ζ for 24 h and treated with VSV for 6 h, and immunoblot analysis showed the indicated constructs (right panel). The functional verifications of 14-3-3ε (left and right panels) as those of 14-3-3ζ in Fig EV3I (left and middle panels, respectively). HEK293T cells were transfected with Flag-IRF3, HA-AKT2, and Myc-14-3-3ε. 24 h post-transfection, cells were collected and subjected to immunoprecipitation (anti-Flag antibody-conjugated agarose beads) and immunoblot. Immunoassay of the p-IRF3 (Ser396) and dimerization of IRF3 in HEK293T cells with the overexpression of the indicated plasmids.

Data information: *P < 0.05, **P < 0.01 and ns, not significant (P > 0.05); using a one-way ANOVA test (A left panel, I right panel) or two-way ANOVA test (D, G right panel, I middle panel and J right panel). Data are from at least three independent experiments (A left panel, D, G right panel, I and J) or representative of three independent biological replicates (A right panel, B, C, E right panel, F, G left panel, H, K and L). Error bars (A left panel, D, G right panel, I and J, mean ± SEM; A right panel and C, mean ± SD).

Source data are available online for this figure.

AKT2 did not affect IRF3 transcription and translation (Fig EV3, 3-right panel). To explore whether AKT2 interacted with IRF3 endogenously, cell lysates of WT or Akt2 KO PEMs were prepared for immunoprecipitation using anti-AKT2 antibody, followed by immunoblotting using anti-IRF3 antibody (Fig 3D, left panel). Alternatively, cell lysates were prepared for immunoprecipitation using anti-IRF3 antibody, followed by immunoblotting using anti-AKT2 antibody (Fig 3D, right panel). Both sets of data confirmed the endogenous association between AKT2 and IRF3. To map their interaction, HA-tagged AKT2 and Flag-tagged IRF3 or their truncations were co-expressed in HEK293T cells as indicated (Fig EV3E). Immunoprecipitation with anti-HA or anti-Flag antibodies was performed, which showed that the N terminus of AKT2 interacted with the IAD-domain of IRF3 (Fig EV3E). Next, GST-tagged AKT2 and His-tagged IRF3 were purified from E.coli to verify their direct interaction via the pull-down assay (Fig 3E). As a master transcription factor, IRF3 is phosphorylated and undergoes dimerization to translocate into the nuclei and induce Ifnb1 transcription. Surprisingly, upon VSV infection or ISD treatment, Akt2 deficiency did not affect IRF3 Ser396 phosphorylation and dimerization (Figs 3F and EV3F), but Akt2 KO PEMs elevated the amount of IRF3 in the nuclei as measured by immunofluorescence (Figs 3G and EV3G) and immunoblotting (Fig 3H).

In previous reports, AKT2 can act as an anchor and cooperate with 14-3-3 to restrain the transcription-related factor p27Kip1 in cytoplasm (Fujita et al, 2002; Sekimoto et al, 2004). Based on the abundant expression of the 14-3-3 family members, that is, 14-3-3ε (14-3-3epsilon, 14-3-3Ε) and 14-3-3ζ (14-3-3zeta, 14-3-3Ζ) in macrophages (Munier et al, 2002), we performed IP and found that both 14-3-3ε and 14-3-3ζ interacted with AKT2 (Fig EV3H). We further explored whether 14-3-3ε and 14-3-3ζ might assist AKT2 to regulate the cellular location of IRF3 and expression of Ifnb1. Knockdown or overexpression of 14-3-3ζ did not affect Ifnb1 mRNA level or IFNβ1 luciferase activity (Fig EV3I). In contrast, knockdown of 14-3-3ε could enhance Ifnb1 production (Fig EV3J), implying that 14-3-3ε, but not 14-3-3ζ, might work together with AKT2 to inhibit Ifnb1 transcription. Interestingly, 14-3-3ε also interacted with IRF3 (Fig EV3K) and reduced the amount of IRF3 in the nuclei (Fig 3I), but did not affect IRF3-Ser396 phosphorylation and dimerization (Fig EV3L). In addition, co-expression of 14-3-3ε with AKT2 further suppresses IFNβ1 in the IFNβ1 luciferase assays (Fig 3J), and knockdown of 14-3-3ε enhanced the Ifnb1 mRNA levels in WT PEMs, but this promotion was disappeared in Akt2 KO PEMs (Fig 3K). These data together support that AKT2 directly binds IRF3, which cooperates with 14-3-3ε, to prevent IRF3 translocation into the nuclei and suppress IFNβ1.

AKT2 phosphorylates IRF3 at Thr207 and blocks IRF3 activation

Our above findings indicated that inhibition of IFNβ1 expression was dependent on kinase activity of AKT2; therefore, we speculated that AKT2 kinase activity was critical to inhibit IRF3 translocation into the nuclei. Next, Flag-tagged IRF3 was overexpressed in HEK293T-IRF3 KO cells together with HA-tagged AKT2 or the kinase-dead mutant AKT2-T309A/S474A. As expected, the AKT2 kinase-dead mutant failed to decrease the amount of IRF3 in the nuclei (Fig 4A). Using the Phos-tag gel, we detected the enhanced IRF3 phosphorylation levels in Akt2 KO macrophages upon VSV challenge (Fig EV4A). To confirm that AKT2 regulated IRF3 phosphorylation in the in vitro kinase assay, HA-tagged AKT2 were enriched from HEK293T cells via immunoprecipitation and incubated with the purified His-tagged IRF3 protein from E. Coli. In the presence of AKT2, IRF3 was indeed phosphorylated that was shown as the shifted bands in the Phos-tag gel (Fig 4B, red arrow). To further identify the potential phosphorylation sites in IRF3, Flag-tagged IRF3 was overexpressed into HEK293T cells with AKT2 or the GFP control, next, anti-Flag immunoprecipitation was performed for the mass spectrometry (MS) analysis. Compared to the control, AKT2 overexpression induced IRF3 phosphorylations at Ser14, Ser173, S175, Thr180, Thr207 (Fig EV4B). We next mutated these residues into alanine (S14A, S173A/S175A, S180A, and T207A) to prevent phosphorylation. Only when the IRF3-T207A mutant was co-expressed, AKT2 no longer reduced the IFNβ1 luciferase activity (Figs 4C and EV4C). Moreover, the nuclear location and phosphorylation of IRF3-T207A were not changed by AKT2 (Fig 4D and E), and overexpression of IRF3-T207A did not change IRF3 Ser396-phosphorylation and dimerization after VSV stimulation (Fig EV4D). To verify the important function of IRF3-T207, IRF3-T207D (D, aspartic acid) was generated to mimic the phosphorylation status. Overexpression of IRF3-T207D inhibited IFNβ1 production, and AKT2 co-expression showed no further inhibition (Fig EV4E).

Details are in the caption following the image

Figure 4. AKT2 phosphorylates IRF3 at Thr207 and blocks IRF3 activation

Immunoblot analysis of IRF3 in the cytoplasmic and nucleic fractions of HEK293T-IRF3 KO cells with overexpression of IRF3 and AKT2 or AKT2-S309A/T474A. Immunoblot analysis of in vitro kinase assay by the phosphorylation gel. HA-AKT2 was immunoprecipitated from HEK293T cells by anti-HA/IgG antibody and Protein-G beads, and His-IRF3 was purified from E.coli cells by anti-His antibody-conjugated agarose beads. His-IRF3 alone and anti-IgG-beads added with His-IRF3 served as controls. The red arrow indicated the phosphorylated IRF3. IFNβ1 luciferase assays and immunoblot analysis showed the HEK293T-IRF3 KO cells with overexpression of the indicated plasmids for 24 h and VSV infection for 6 h. n = 3, respectively. Immunofluorescence microscopy (top panel) of AKT2-, Flag-IRF3-, or Flag-IRF3-T207A-overexpressed MEF cells after poly(A:T) stimulation for 6 h. Flag (IRF3, red) and Hoechst (nuclei, blue). Immunoassay of nuclear–cytoplasm extractions (bottom panel) from HEK293T-IRF3 KO cells with overexpression of the indicated plasmids for 24 h followed by VSV infection for 6 h. GAPDH and SP1 were used as cytoplasmic and nucleic protein loading control, respectively. Bar, 20 μm. HEK293T-IRF3 KO cells were overexpressed with indicated plasmids, and the cell lysates were conducted to immunoblot analysis by phosphorylation gel for the assay of phosphorylated IRF3 (the upper band) by anti-Flag antibody. Survival rates (left panel) until 72 h and H&E staining (right panel) at 18 h of the zebrafish larvae with indicated protein expression and VSV challenge. The arrows indicated the VSV-infected eye and skeletal muscle in zebrafish larvae. Bars, 100 μm. qRT–PCR analysis of the VSV copies in overexpressed zebrafish embryos with the VSV challenge at 18 h later. Every dot represents three zebrafish embryos. Horizontal vertical square bracket shows the statistical analysis of comparison with “PBS mock”, the rest shows the comparison with “PBS VSV”. PBS mock, n = 15; PBS VSV, n = 9; IRF3 VSV, n = 16; IRF3-T207A VSV, n = 11; AKT2 VSV, n = 14; AKT2-T309A/S474A VSV, n = 9; IRF3 + AKT2 VSV, n = 9; IRF3 + AKT2-T309A/S474A VSV, n = 13; IRF3-T207A + AKT2 VSV, n = 10. The survival rates of over-expressed zebrafish larvae as long as 72 h after the VSV challenge.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test (G), or two-way ANOVA test (C), or log-rank (Mantel–Cox) test (F left panel and H). Data are from at least three independent experiments (C), or representative of three independent experiments (A, B, D–H). Error bars (C, mean ± SEM; G, mean ± SD).

Source data are available online for this figure.

Details are in the caption following the image

Figure EV4. The identification of possible phosphorylated sites of IRF3 by AKT2 (related to Fig 4)

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