The Chicken cGAS–STING Pathway Exerts Interferon-Independent Antiviral Function via Cell Apoptosis

Simple Summary The Interferon-independent antiviral function of stimulator of Interferon genes has recently received attention. However, the nature of this Interferon-independent antiviral function is not yet clear, although autophagy induced by stimulator of Interferon genes has been shown to mediate the antiviral activity of stimulator of Interferon genes. In birds, research is still nonexistent. Here, we first validated the antiviral activity of chicken stimulator of Interferon genes and determined the Interferon-independent antiviral activity of chicken stimulator of Interferon genes. Furthermore, it was found that autophagy induced by chicken stimulator of Interferon genes did not affect its antiviral activity. Finally, apoptosis induced by chicken stimulator of Interferon genes was involved in its antiviral process. Our results indicate that chicken stimulator of Interferon genes induces multiple signaling activities, which may be correlated during the antiviral process. This study demonstrates the complexity of the antiviral activity of chicken stimulator of Interferon genes and provides a unique approach for the study of antiviral activity other than that of Interferons. Abstract It has been recently recognized that the DNA sensing innate immune cGAS-STING pathway exerts an IFN-independent antiviral function; however, whether and how chicken STING (chSTING) exerts such an IFN-independent antiviral activity is still unknown. Here, we showed that chSTING exerts an antiviral activity in HEK293 cells and chicken cells, independent of IFN production. chSTING was able to trigger cell apoptosis and autophagy independently of IFN, and the apoptosis inhibitors, rather than autophagy inhibitors, could antagonize the antiviral function of chSTING, suggesting the involvement of apoptosis in IFN-independent antiviral function. In addition, chSTING lost its antiviral function in IRF7-knockout chicken macrophages, indicating that IRF7 is not only essential for the production of IFN, but also participates in the other activities of chSTING, such as the apoptosis. Collectively, our results showed that chSTING exerts an antiviral function independent of IFN, likely via apoptosis.

In addition to IRF3-and NF-κB-mediated gene transcriptions, STING also triggers cell autophagy and apoptosis [12,13].Recent studies have shown that STING can cause canonical autophagy and non-canonical autophagy [12].STING was shown to induce endoplasmic reticulum (ER) stress and unfolded protein response (UPR) through a unique UPR motif in the cyclic dinucleotide binding (CBD) domain, which negatively regulates the AKT/TSC/mTOR pathway to enhance canonical autophagy [14].In addition, STING is also able to induce non-canonical autophagy by directly interacting with microtubule-associated protein light chain 3 (LC3) through its LC3 interacting regions (LIRs) [15], and the ER-Golgi intermediate compartment (ERGIC) is used as the membrane source of LC3 lipidation and autophagy formation [15][16][17].Apoptosis, also known as the non-inflammatory type of programmed cell death, is one of the most widely studied cell death pathways [13,18].The mechanism of STING inducing apoptosis is not clear, but most studies point to ER stress [19][20][21].Additionally, STING may induce apoptosis through downstream IRF3 or NF-κB, where the phosphorylation of IRF3 and formation of the IRF3-Bax complex occur [19,20,22].
The IFN-independent activity of STING has been widely appreciated in the last years [23].Recently, we demonstrated that porcine STING (pSTING) can play its antiviral function independently of both IFN and autophagy, while relating to apoptosis [24].Our previous study also found that chicken STING (chSTING) can induce broad-spectrum antiviral activity, but the IFN-independent antiviral activity of chSTING has not been elucidated [25].In this study, we investigated the resistance of chSTING against a variety of viruses and showed that chSTING can induce a robust IFN-independent antiviral activity.We further found that apoptosis, rather than autophagy, participates in the IFN-independent antiviral activity of chSTING.

Molecular Cloning and Gene Mutations
The HA-tagged pcDNA DEST plasmids of chicken cGAS (chcGAS) and chicken STING (chSTING) were made as in our previous studies [25,26].The mutation PCR primers of chSTING were designed using the QuickChange Primer Design online tool (https://www.agilent.com,accessed on 1 October 2020), shown in Supplementary Table S1.The mutation PCR was performed with KOD plus neo polymerase (Toyobo, Shanghai, China) and pcDNA-chSTING-2HA as the template.The PCR products were transformed into competent DMT Escherichia coli after Dpn I digestion, and the resultant mutants were sequence, confirmed by DNA sequencing.

Promoter-Driven Luciferase Reporter Gene Assay
In 96-well plates (2-3 × 10 4 cells/well), 293T cells were co-transfected using Lipofectamine 2000 with ISRE or ELAM (NF-κB) firefly luciferase (Fluc) reporter (10 ng/well) and β-actin Renilla luciferase (Rluc) reporter (0.2 ng/well), together with the indicated plasmids or vector control (5-40 ng/well).DF-1 cells were similarly transfected with chicken IFNβ Fluc using TransIT-LT1 transfection reagent (MIR 2300).The total DNA per well was normalized to 50 ng by adding corresponding empty vectors.Twenty-four hours after transfection, the cells were lysed, and the Fluc and Rluc activities were measured successively by the double-luciferase reporter assay kit.The results were expressed as the fold induction of IFNβ, ISRE or ELAM (NF-κB)-Fluc compared with those of vector controls after Fluc normalization by the corresponding Rluc.Total RNA was extracted using the TRIpure reagent, and total DNA was extracted using a DNA extraction kit following the instructions.The extracted RNA was reversetranscribed into cDNA using a HiScript 1st strand cDNA synthesis kit, and then the target gene expressions were measured using quantitative PCR with a SYBR qPCR master Mix using StepOne Plus equipment (Applied Biosystems, Waltham, MA, USA).The qPCR program included a denaturation step at 94 • C for 30 s, followed by 40 cycles at 94 • C for 5 s and 60 • C for 30 s.The relative mRNA levels were normalized to RPL32 or GAPDH mRNA levels, and calculated using the 2 −∆∆CT method.The efficiencies of the qPCR assays were always at 90 to 110%, requiring use of the 2 −∆∆CT method.The sequences of qPCR primers used are shown in Supplementary Table S2.

Western Blotting Analysis
Whole-cell proteins were extracted from cells lysed using the radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris pH 7.2, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100).The protein lysate samples were 3:1 mixed with 4× loading buffer and boiled for 5-10 min.After centrifugation, the lysate supernatants were run by SDS-PAGE, and then the proteins in gel were transferred to the polyvinylidene fluoride (PVDF) membrane.The membrane was incubated with 5% skim milk TBST at room temperature (RT) for 1 h, probed with the indicated primary antibodies at 4 • C overnight, washed, and then incubated with secondary antibodies for 1 h at RT.The protein signals were detected via the electrochemiluminescence (ECL) detection substrate and imaging system (Tanon, Shanghai, China).

Plaque Assay
Vero cells were seeded into 24-well plates (3 × 10 5 cells/well).Once the cells were grown into a monolayer, cells were infected with the tenfold serially diluted cell supernatants from HSV1-, VSV-or SeV-infected cells for 2 h.Then, the infected cells were washed with PBS and overlaid by immobilizing a medium of a 1:1 mixture of warmed 2× DMEM with 4% FBS and a stock solution of heated 1.6% low-melting agarose (Sigma-Aldrich, St. Louis, MO, USA).Plague formations took 4 days for HSV1, and 2 days for VSV and SeV, respectively.Upon completion, the immobilizing medium was discarded by carefully tipping and the cells were fixed and stained with a crystal violet cell colony staining solution (0.05% w/v crystal violet, 1% formaldehyde, 1× PBS and 1% methanol) for 1 h at RT. Finally, cells were gently washed with tap water until the clear plaques appeared.The plaques were counted, and photos were taken.

Viral Fluorescence Imaging
The viral fluorescence was imaged using the OLYMPUS IX53 fluorescence microscope, with the objective lens parameter of 10 × 0.30 ph1.For red fluorescent protein (RFP) imaging, the excitation wavelength and emission wavelength were 532 nm (green light) and 588 nm, respectively.For green fluorescent protein (GFP) imaging, the excitation wavelength and emission wavelength were 488 nm (blue light) and 507 nm, respectively.The microscopic image processing software used was OLYMPUS cellsens Entry 2.2 (Build 17989) (https://www.olympus-sis.com).The same set of samples were exposed for the same time, and three typical images were collected for each sample.

Flow Cytometry for Apoptosis Detection
The level of cell apoptosis was examined using the Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit.Briefly, the treated cells were harvested via trypsin digestion and washed with the binding buffer, and then resuspended in the binding buffer.The staining solution of Annexin V-FITC and PI were added successively.The cells were incubated with fluorescein dyes at RT for 15 min in the dark, and the stained cells were immediately detected using flow cytometry.About 10,000 cell events were collected for analysis of FITC and PI signals detected by channels FL1 and FL3, respectively.The FITC and PI signals were processed using the FlowJo software v10.9 (https://www.flowjo.com),and presented as dot plots.The flow cytometry analyzer used was CytoFLEX S (Beckman Coulter, Brea, CA, USA), equipped with four laser beams (488 nm laser, 638 nm laser, 405 nm laser, 561 nm laser).

Statistical Analysis
All the results were the representatives of two or three similar experiments.The data in bar graphs were presented as the mean ± standard deviation (SD) and analyzed using GraphPad Prism 8.0.The normality of data distribution was tested using the Shapiro-Wilk test.Student's t-test and ANOVA were used for statistical analysis, and p < 0.05 was considered statistically significant.Statistical signs are indicated as follows: *, p < 0.05; **, p < 0.01 and ns, not significant.

chSTING Exerts an IFN-Independent Antiviral Activity in Mammalian 293T Cells
Our previous study showed that chSTING could not induce IFN in mammalian cells; however, substituting its pLxVS motif with a porcine pLxIS motif enables the induction of IFN in mammalian 293T cells by chSTING [26].We examined the antiviral activity of chSTING WT and its pLxVS sub-mutant in 293T cells (Figure 1).Despite the difference in IFN induction, both chSTING and its pLxVS sub-mutant exhibited antiviral activity against a DNA virus (HSV1) (Figure 1A) and two RNA viruses (VSV and SeV) (Figure 1B,C), as evidenced by GFP fluorescence, viral GFP blotting and viral plaque assay, when cotransfected with chcGAS.These results indicated the IFN-independent antiviral activity of chSTING in mammalian 293T cells.

chSTING Exhibits a Robust IFN-Independent Antiviral Function in Chicken Cells
Considering the physiological relevance, we selected chicken cells (chicken fibroblast cell line DF-1) for further research.Unlike 293T, chSTING alone has significant signaling activity when transfected into DF-1 alone, and its activity does not increase further after co-transfection with cGAS [26].Accordingly, ectopic chSTING in DF-1 cells exhibited significant antiviral responses to NDV, vaccinia viruses SMV and VACV, whereas coexpression with chcGAS did not enhance such antiviral responses (Figure 3A).As expected, chcGAS alone had no observable antiviral effect as evidenced by RFP fluorescence and RFP immune blotting for NDV, and by qPCR for VACV and SMV (Figure 3B).
As for antiviral function, compared with chSTING WT, the chSTING pLxVS sub and chSTING S366A exhibited robust anti-NDV activity, whereas chSITNG ∆CTT also showed anti-NDV activity despite it being to a lower degree (Figure 3E).On the other hand, chSTING WT and all the chSTING mutants (pLxVS sub, S366A and ∆CTT) exhibited significant antiviral activity against VACV and SMV (Figure 3F).Notably, the above results could be reproduced in the chicken macrophage HD11 cell line (Figure 3G).This result suggested that chSTING exhibits an IFN-independent antiviral activity in chicken cells as well, and indicated that chSTING possesses other antiviral mechanisms in addition to IFN. or both (0.25 µg each) for 24 h, with the pcDNA3.1 vector transfection and mock treatment used as controls.The transfected cells were infected with NDV-RFP (0.01 MOI) for 12 h.The red fluorescence of RFP was observed, and the RFP protein was detected via Western blotting.(B) DF-1 cells were transfected as in A, and the transfected cells were infected with SMV and VACV (0.01 MOI), respectively, for 48 h, followed by qPCR detection of the viral replication.(C) DF-1 cells in a 96-well plate (6 × 10 4 cells/well) were transfected with chSTING WT/pLxVS sub/S366A/∆CTT (40 ng each), or empty vector (40 ng), together with IFNβ Fluc (10 ng) plus Rluc (0.2 ng) for 24 h, followed by the measure of luciferase activities.(D) DF-1 cells grown in a 12-well plate (6 × 10 5 cells/well) were transfected with chSTING WT/pLxVS sub/S366A/∆CTT (1 µg each) or empty vector (1 µg) for 48 h.The RNA expressions of chIFN-β, chOASL and chIL-8 were analyzed using RT-qPCR.(E) DF-1 cells in a 24-well plate (3 × 10 5 cells/well) were transfected with chSTING WT/pLxVS sub/S366A/∆CTT (0.5 µg each) for 24 h.The transfected cells were infected with NDV-RFP (0.01 MOI) for 12 h, followed by visualization of RFP and immunoblotting analysis of RFP and STING protein expressions.(F) DF-1 cells were transfected as in E, and transfected cells were infected with SMV and VACV (0.01 MOI) for 48 h, followed by qPCR detection.(G) STING −/− HD11 cells in a 24-well plate (3 × 10 5 cells/well) were transfected as indicated for 24 h, and infected with NDV for 12 h, followed by visualization of red fluorescence of RFP and immunoblotting analysis of RFP and STING protein expressions.The gray values of RFP/actin were shown below the RFP bands in Western blotting.* p < 0.05, ** p < 0.01 versus Mock controls.ns, not significant.

Apoptosis Rather Than Autophagy Is Involved in the Antiviral Function of chSTING
Since it was determined that chSTING can play its antiviral role without IFN in different types of cells, the inherent autophagy and apoptosis activity of chSTING and its three mutants were tested in 293T cells (Figure 4).The Western blotting showed that chSTING and all three mutants (pLxVS sub, pLxVS sub S366A and ∆CTT) upon chcGAS stimulation induced LC3 lipidation (LC3-II) and p62 phosphorylation (p-p62), indicative of the autophagy occurrence (Figure 4A).Flow cytometry showed that chSTING and all chSTING mutants were able to induce significant apoptosis (Figure 4B).Then, the statistical analysis was performed, and it was found that all chSTING mutants, including IFN-deficient mutants, could induce early and late apoptosis (Figure 4C).These results suggested that chSTING-induced apoptosis and autophagy were both independent of IFN.
In order to determine whether the apoptosis and/or autophagy of chSTING participates in the antiviral activity of chSTING, two autophagy inhibitors (3-MA and NH 4 Cl) (Figure 4D) and two apoptosis inhibitors (DEVD and VAD) (Figure 4E) were applied to antiviral experiments in chicken HD11 cells.STING agonist 2 3 -cGAMP triggered an obvious anti-NDV response in HD11 cells (Figure 4D,E).The two autophagy inhibitors did not significantly affect the antiviral effect of 2 3 -cGAMP stimulation (Figure 4D), whereas the two apoptosis inhibitors could reverse the antiviral response activated by 2 3 -cGAMP to some extent, compared with DMSO control group (Figure 4E).The above results suggested that apoptosis, rather than autophagy, induced by chSTING is likely involved in the IFN-independent antiviral function of chSTING, which was consistent with the results of pSTING we previously studied [24].

IRF7 Is Required for STING Antiviral Function in Chicken HD11 Cells
In order to further investigate the antiviral function of chSTING, STING −/− HD11 cells were used for NDV infection experiment.Compared with the WT HD11, STING −/− HD11 had a much higher NDV replication level (Figure 5A).The STING agonist, 2 3 -cGAMP, suppressed the NDV replication potently in WT HD11 cells, but lost the antiviral activity in STING −/− HD11 cells (Figure 5A).The results confirmed and reinforced the critical role of chSTING in antiviral innate immunity.Chicken IRF7 (chIRF7) is a transcription factor that mediates IFN induction by chST-ING; therefore, the IRF7 +/− HD11 cells were also examined for NDV infection.Surprisingly, IRF7 +/− HD11 cells had a much higher NDV replication level compared with the WT HD11 cells and the STING agonist, 2 3 -cGAMP, lost its antiviral activity in IRF7 +/− HD11 cells (Figure 5B).These results indicated that chIRF7 plays a significant role in the process of anti-NDV replication likely via both IFN-dependent and IFN-independent ways.To explore whether chIRF7 is involved in chSTING-induced apoptosis, we analyzed the cell apoptosis in IRF7-knockout cells.The results showed that the cleaved caspase 3 in IRF7 +/− HD11 cells was substantially decreased with or without cGAMP stimulation compared with those in WT HD11 cells (Figure 5C).Furthermore, when the IRF7 +/− HD11 cells were treated with two apoptosis inhibitors, no further increase in NDV replication was observed (Figure 5D).These results implicated that chIRF7 is directly involved in cell apoptosis.

Discussion
STING, as the signaling adaptor downstream of cGAS, plays a key role in antiviral innate immunity [4].In recent years, chSTING has also been reported to resist Marek's disease virus (MDV, member of the Herpesviridae family) and fowl adenovirus [27-29], both avian DNA viruses.Later on, we showed that chSTING also resists the replications of a broad spectrum of viruses including DNA virus, RNA virus and retrovirus [25].The broad-spectrum antiviral activity of chSTING has also been further confirmed in this study: chSTING could reduce the replications of HSV1, VSV and SeV in mammalian 293T cells (Figures 1 and 2), and suppress the replications of NDV, SMV and VACV in chicken cells (Figures 3-5).
Despite that, the IFN-independent antiviral activity of STING has been appreciated [23,30,31].In this study, we first demonstrated the IFN-independent antiviral activity of chSTING in both mammalian and chicken cells (Figures 1-3).It is known that STING can not only induce IFN, but also activate NF-κB, autophagy and apoptosis [12,13,32].First, chSTING pLxVS sub S366A and chSTING ∆CTT have differential NF-κB signaling (Figure 2C), but the antiviral activity of these two chSTING mutants are similar (Figure 2F,G).Thus, it indicates that NF-κB does not affect apparently the antiviral activity of chSTING.Second, although some studies pointed out that STING-induced autophagy was involved in the antiviral process [16,31,33], we did not find any involvement of autophagy in the antiviral activity of chSTING (Figure 4).Third, the two kinds of apoptosis inhibitors could reverse STING agonist 2 3 -cGAMP triggered antiviral activity, suggesting that apoptosis is likely involved in the IFN-independent antiviral process of chSTING (Figure 4).The results are consistent with those obtained in our previous study on porcine STING (pSTING), reflecting the conservation of STING during evolution.
Cell apoptosis is the key to control and eliminate viral infection [34].When IFN cannot control the influenza virus, the host can induce cell apoptosis to inhibit viral replication [35].Many viruses have evolved a variety of apoptosis-inhibiting proteins to evade cell apoptosis at an early stage of infections and thus contribute to their own replications, including porcine respiratory and reproductive syndrome virus (PRRSV) [36], human cytomegalovirus (HCMV) [37], α-herpesvirus [38] and ectromelia virus [39].Thus, apoptosis induced by chSITNG serving as the IFN-independent antiviral machinery might be counteracted by chicken viruses, as well for immune evasion.
Here, we found that chIRF7 is required for STING antiviral response to NDV in chicken HD11 cells (Figure 5).Several DNA viruses including HSV1 and Vaccinia Viruses SMV and VACV did not replicate well in HD11 cells, and other relevant viruses need to be tested for this interesting phenomenon.In fact, in our previous pSTING work, we found that IRF3 is also required for STING antiviral activity [24].The similar phenotypes seem contradictory to the IFN-independent antiviral activity of STING.However, it will be reconciled if the IRF3/IRF7, required for IFN induction by STING, also participated in the IFN-independent activity of STING, such as apoptosis.Although we showed here that chIRF7 is likely involved in chSTING triggered cell apoptosis directly, the hypothesis needs to be confirmed in the future.In addition, the mechanism of apoptosis by STING is not clear currently.In particular, the responsible sites and/or molecular switch of STING for apoptosis need to be identified and clarified.

Figure 1 .
Figure 1.chSTING does not rely on IFN to play its anti-viral functions in 293T.(A) The 293T cells in a 24-well plate (1.5 × 10 5 cells/well) were co-transfected with chcGAS (0.25 µg) combined, with chSTING WT and pLxVS sub (0.25 µg each) for 24 h, with pcDNA3.1 empty vector transfection and mock as controls.The transfected cells were infected with HSV1-GFP (0.1 MOI) for another 24 h.The viral GFP fluorescence was observed by microscopy.The expressions of viral GFP protein were detected by Western blotting.HSV1 plaques and the numbers of plaques were presented.(B,C) The 293T cells were transfected as in HSV1 infection.The transfected cells were infected with VSV-GFP (0.01 MOI) (B) and SeV-GFP (0.01 MOI) (C) for another 12 h.The viral GFP fluorescence was observed via microscopy.The expressions of viral GFP protein were detected through Western blotting.The gray values of GFP protein bands in Western blotting were shown below the corresponding bands after normalization of actin expressions.VSV and SeV plaques and the number of plaques were presented.* p < 0.05 versus Mock controls.

Figure 4 .
Figure 4. Apoptosis, rather than autophagy, is involved in the antiviral process of chSTING.(A) The 293T cells grown in a 24-well plate (1.5 × 10 5 cells/well) were transfected with the indicated combinations of chcGAS (0.25 µg) and chSTING WT/pLxVS sub/pLxVS sub S366A/∆CTT (0.25 µg each) for 24 h, and the protein expressions were analyzed via Western blotting with the indicated antibodies.The protein bands separated by vertical lines were from non-contiguous portions of one gel.(B,C) The 293T cells in a 24-well plate (1.5 × 10 5 cells/well) were transfected with chcGAS (0.25 µg) combined with chSTING WT/pLxVS sub/pLxVS sub S366A/∆CTT, as indicated (0.25 µg each), for 48 h.The apoptosis of the transfected cells was analyzed using Annexin V-FITC/PI staining followed by flow cytometry.The dot plots of cell apoptosis in various samples are presented in B. As indicated bt Q3 (Annexin + /PI − ), the early apoptosis and late as indicated by Q2 (Annexin + /PI + ) were plotted as bar graphs in C. (D) HD11 cells grown in a 24-well plate (3 × 10 5 cells/well) were treated with two autophagy inhibitors, 3-MA and NH 4 CL, with the indicated concentrations for 30 min.Cells were washed and stimulated by the transfection of 2 3 -cGAMP (2 µg/mL) for 12 h.The stimulated cells were infected with NDV-RFP (0.01 MOI) for another 12 h, followed by detections of viral RFP fluorescence and RFP protein expression.(E) HD11 cells were treated with two apoptosis inhibitors, Ac-DEVD-CHO and Z-VAD-FMK, with the indicated concentrations for 30 min.Cells were washed and stimulated by transfection of 2 3 -cGAMP (2 µg/mL) for 12 h.The stimulated cells were infected with NDV-RFP (0.01 MOI) for another 12 h, followed by the detection of viral replications.The gray values of p-p62/actin, LC3II/LC3I and RFP/GAPDH are shown below the corresponding bands in Western blotting.* p < 0.05 versus Mock controls.