INTRODUCTION
The cytosolic enzyme known as cyclic GMP-AMP synthase (cGAS) is activated by microbial double-stranded DNA (dsDNA) and serves as a pattern recognition receptor (PRR). It catalyzes the production of a unique intracellular second messenger called 2′3′-cGAMP (cGAMP), which belongs to the class of cyclic dinucleotides (CDNs). This cGAMP molecule, in turn, activates the stimulator of interferon genes protein (STING) (also named TMEM173, ERIS, MITA, or MPYS) (
1). STING is an evolutionarily conserved, endoplasmic reticulum (ER) resident, ~40 kDa dimeric transmembrane adaptor for CDNs that perform multiple functions during infections, autoimmune diseases, and cancers (
2). cGAMP-bound STING translocates from the ER to Golgi via the ER-Golgi intermediate compartment (ERGIC), in which STING recruits TANK-binding kinase 1 (TBK1); the latter then phosphorylates interferon regulatory factor 3 (IRF3). Phosphorylated IRF3 dimerizes and translocates to the nucleus to trigger expression of type I interferon (IFN-I) (
3). STING translocation to the ERGIC triggers lipidation of microtubule-associated protein 1 A/1B-light chain-3, thereby inducing autophagy to clear pathogens from the cytosol; this process is a primordial function of the cGAS-STING axis that is independent of IFN-I. The result is activation of nuclear factor κB (NF-κB) via TBK1 and inhibitor-κB kinase ε (IKKϵ), which, in turn, orchestrates cellular resistance to invading pathogens (
4,
5). Therefore, STING is a master molecule that determines the fate of microbial pathogens by sensing “foreign” DNA during infections.
African swine fever (ASF) is a notifiable, transboundary animal disease that is lethal to domestic pigs and wild boars. It is a highly contagious hemorrhagic fever for which there is no commercially available vaccine, and its unprecedented spread across Europe, Africa, and Asia is threatening the global pig industry. The ASF virus (ASFV) is a large arbovirus belonging to the family
Asfarviridae (
6). It is an icosahedral DNA virus measuring 200 nM in diameter and comprises an envelope, a capsid, an inner capsule membrane, a core shell, and an inner core. The viral genome is a linear dsDNA (170–190 kb long) molecule with covalently closed ends. The genome encodes 150–200 viral proteins, including 68 structural proteins and more than 100 non-structural proteins (
7). ASFV targets the swine monocyte/macrophage lineage for replication (
8). ASFV infection triggers a battle between the virus and the host’s innate antiviral immune responses. Viral proteins involved in immunoregulation play a significant role in virus replication (
9). In recent studies, it has been demonstrated that ASFV utilizes multiple mechanisms to evade host defense systems. For instance, M1249L inhibits interferons (
10), S273R affects inflammatory responses (
11), A224L influences apoptosis (
12), and EP402R impacts adaptive immunity (
13).
Here, we show for the first time that ASFV B175L acts as a specific negative regulator of IFN-I signaling by targeting STING and cGAMP through its conserved MYM-type Zinc finger with FCS sequence motif (zf-FCS motif). The findings reveal a novel mechanism of immune evasion that could facilitate the development of new and effective live-attenuated vaccines against ASF.
DISCUSSION
The innate immune system uses multiple germ-line encoded PRRs to recognize various pathogen-associated molecular patterns such as viral DNA to orchestrate antiviral immune responses. There are many different PRRs that can detect viral DNA in the cytosol (
19,
20). One example is the cGAS-STING-IRF3 axis, in which binding of cytosolic viral DNA to cGAS triggers its catalytic activity to generate cGAMP, which then interacts with the ER resident protein STING. The cGAMP and STING complex undergoes structural changes and traffics to the Golgi via ERGIC, where it recruits TBK1 and IRF3 prior to activating IFN-I (
21). The STING protein comprises three specific domains: a TMD, a CBD, and a CTT. The CBD and CTT domains form a butterfly-shaped domain-swapped homodimer under steady-state conditions. After binding to cGAMP, the STING homodimer undergoes extensive conformational changes, referred to as STING activation. During activation, STING releases its hidden CTT and undergoes polymerization and ubiquitination (
5). Activated STING then transverses to the Golgi apparatus, where it is palmitoylated to induce STING-dependent downstream signaling (
22). Translocated STING recruits TBK1 to its CTT through a PXPLRXD (where X refers to any residue) motif, and the TBK1 and STING complex phosphorylates IRF3, which moves into the nucleus as a dimer to induce secretion of IFN-I (
23).
Viruses have evolved specific strategies to escape host immune responses. Numerous viral proteins that interfere with STING activation and trafficking. For example, protein kinases (US3, VP24), protein-protein interaction inhibitors, deubiquitinase UL36USP, and viral ubiquitin ligase ICP0 (encoded by HSV-1) antagonize STING signaling (
24). Human papillomavirus E7 and ADV E1A obstruct STING via viral LXCXE motifs (
25). Hepatitis C virus NS4B and dengue virus NS2B3 proteins cleave STING (
26), whereas the HSV-1 VP24 and Kaposi’s sarcoma-associated herpesvirus vIRF1 block the STING-TBK1 interaction. HSV-1 VP1-2 and human T-lymphotropic virus type 1 Tax deubiquitinate STING, HSV-1 γ134.5 abrogates STING trafficking to the Golgi, and the UL82 of human cytomegalovirus iRhom2 mediates STING-TRAPβ complex assembly to interrupt TBK1 and IRF3 recruitment (
27). ASFV is a large, double-stranded DNA virus that infects domestic or feral swine of all ages causing 100% mortality. A more in-depth understanding of ASFV-host interactions requires high-quality, full-length genomic sequences of a variety of ASFV genotypes. However, most ASFV open reading frames are only predictions, and their functions are still experimentally unknown (
28).
ASFV modulates the IFN and NF-κB pathways for efficient replication. Recent studies show that ASFV virulent strains Armenia/07, 22653/14, L60, and low virulent ASFV/NHV suppress IFN and IFN-stimulated genes in infected cells (
29 – 31). Among the many ASFV-encoded proteins, MGF360-15R, I329L, E120R, I215L, A137R, MGF360-11L, and M1249L suppress IFN-I (
32 – 37). MGF360-12L and DP96R inhibit the IFN-I and NF-κB pathways (
38,
39). Yang et al. (
40) reported the negative regulation of the cGAS-STING pathway by ASFV MGF505-11R. They found that MGF505-11R interacted with STING and increased its degradation through the lysosomal, ubiquitin-proteasome, and autophagy pathways (
40). Another study revealed the role of MGF505-7R against STING-dependent antiviral responses. MGF505-7R bound to STING and inhibited the cGAS-STING pathway, while upregulating expression of ULK1 to degrade STING via autophagy (
41). Zheng et al. reported the negative effects of ASFV p17 protein on STING signaling. They found that binding of p17 to STING interfered with recruitment of TBK1 and IKKε (
42). We recently discovered a novel immune evasion mechanism mediated by ASFV EP364R and C129R, which share nuclease homology, and block cGAMP via phosphodiesterase activity (
43).
In this study, we identified a novel molecular anti-immune mechanism mediated by B175L, an uncategorized protein of ASFV. First, we found that B175L impairs immunity in transfected cells. Expression of B175L in PAM, PK-15, PIB, and MA-104 cells increased replication of GFP-tagged viruses. In addition, cytokine levels, antiviral signaling, and transcription of IFN-related genes fell. Second, pull-down assays followed by a mass spectrometry analysis identified STING (UniProt: Q86WV6) as a binding partner for B175L. HEK293T cells, which are used primarily in cellular biology experiments, are derived from HEK293 cells with stable expression of SV40 polyomavirus large T antigen (LT), which enhances replication of plasmid DNA to achieve a high copy number (
44). HEK293T cells are deficient in both cGAS and STING (
45). Previously, it was thought that lack of STING expression by HEK293T cells was caused by SV40 LT. However, a recent finding revealed that the SV40 LT is not responsible for loss of STING from HEK293T cells (
46). The authors of that study suggest that this cell-specific difference in terms of STING expression could be caused by differentially expressed genes (DEGs) or epigenetic reprogramming. Thus, the most concise explanation for STING detection upon silver staining of B175L-transfected HEK293T cells is alteration of DEGs by B175L through an unknown mechanism.
Our transient and endogenous immunoprecipitation assays confirmed the
in vitro B175L and STING interaction which was validated by confocal microscopy analysis. Third, we identified the regions of B175L and STING that are required for this interaction. Analysis of the B175L domain revealed that the zf-FCS motif of B175L is crucial. ZF proteins are one of the most abundant groups of proteins and fulfill a wide range of molecular functions. zf-FCS was first identified as an MYM family protein related to myeloproliferative syndrome and mental retardation. These proteins are present in viruses, eubacteria, archaea, metazoa, and plants. Previous reports show that zf-FCS accommodates nucleic-protein and protein-protein interactions (
47). Analysis of the STING domain revealed that the STING CBD (aa 185–270) harbors four cGAMP binding residues (R238, Y240, N242, and E260) (
17), suggesting that B175L disrupts cGAMP and STING binding. Furthermore, our experiments using cGAMP conjugates showed that B175L competes with cGAMP for binding to STING.
Finally, to get a deeper insight into the B175L and STING interaction, we transfected cGAMP dose dependently into stable PAM cells and examined phosphorylation of TBK1 and IRF3. As expected, we observed almost zero phosphorylation of TBK1 and IRF3 in B175L stable PAM cells, despite the increasing amounts of cGAMP (
Fig. 6A). Mutation analysis revealed that STING R238 and Y240 are essential for the B175L and STING interaction, while N242 and Y240 are not. This indicates that STING R238 and Y240 are key locations for cGAMP-favored activation of STING signaling. Therefore, the R238A and Y240A mutants (in which the strong affinity of cGAMP for STING is abolished) can be useful as a control when studying cGAMP and STING interactions (
17). Our sequence analysis revealed that R238 and Y240 are conserved among species. Furthermore, we found that the zf-FCS motif of B175L not only binds to STING but also interacts directly with cGAMP. Overall, binding of B175L to STING and cGAMP abrogates the STING polymerization, which could disrupt ERGIC/Golgi trafficking.
In conclusion, we show here that the previously uncategorized ASFV protein B175L is an antagonist of type-I IFN. B175L interacts with cGAMP and STING to impair STING-mediated transduction of antiviral signals. Taken together, these data increase our understanding of the diverse mechanisms underlying ASFV pathogenesis and open up new avenues of research aimed at virus attenuation and future ASFV vaccine development.
MATERIALS AND METHODS
Chemicals and antibodies
2′3′-cGAMP-Biotin conjugate (AAT Bioquest; 20316), 2′3′-cGAMP-Cy5 (AAT Bioquest; 20318), GlutaMAX Supplement (Gibco), Trypsin-EDTA (Gibco) Normocin-Antimicrobial Reagent (Invivogen), Blasticidin (Invivogen), Hygromycin B Gold (Invivogen), Zeocin (Invivogen), Puromycin (Invivogen), Poly(dA:dT) (Invitrogen), Lipofectamine 2000 (Invitrogen), Polyethyleneimine/PEI (Polysciences; 9002-98-6/26913-06-4), Protein A/G PLUS-Agarose (Santa Cruz Biotechnology; sc-2003), Halt Protease Inhibitor Cocktail (Thermo Scientific; 78429), Sepharose 6B (GE Healthcare; 17011001), Glutathione-conjugated Sepharose 4B (GST) beads (Cytiva), Strep-Tactin Sepharose resin (IBA Lifesciences; 2–1201-002), Quanti-Luc (Invivogen) were obtained commercially. The list of antibodies purchased from Cell Signaling Technology for this study included STING (D2P2F; 13647), TBK1/NAK (D1B4; 3504), STAT1 (42H3; 9175), NF-κB p65(D14E12; 8242), IRF3 (D83B9; 4302), IκBα (9242), pTBK1/NAK (D52C2; 5483), pSTAT1 (58D6; 9167), Phospho-NF-κB p65 (93H1; 3033), pIRF3 (4D4G; 4947), pIκBα (14D4; 2859), and Flag (M2) (8146). Other antibodies are as follows: Alexa Flour 488 (Abcam; 150077), Alexa Flour 647 (Abcam; 150079), StrepMAB-Classic HRP conjugate (IBA Lifesciences; 2-1509-001), Cy5 (Abcam: CY5-15), β-actin (Santa Cruz Biotechnology; sc-47778), and GST (Santa Cruz Biotechnology; sc-138).
Cell culture and transfection
PK-15 cells (ATCC CCL-33), HeLa cells (ATCC CCL-2), HEK293T cells (ATCC CRL-11268), Vero cells (ATCC CCL-81), 293-Dual hSTING-A162 cells harboring the stable transfections of the A162 isoform of human STING (S162A) and are resistant to antibiotics such as blasticidin, hygromycin, and Zeocin (Invivogen), A549 cells (ATCC CCL-185), and MA-104 cells (CRL-2378.1) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Cytiva), and PAM cells (ATCC CRL2843) were cultured in RPMI Medium (Cytiva). Both mediums were supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% Anti-Anti (Gibco). PIB isolation was performed as described previously (
48). PIB cells were cultured in RPMI Medium (Cytiva) supplemented with 10% FBS (Gibco), 1% Anti-Anti (Gibco), Normocin (100 µg/mL), Blasticidin (100 µg/mL), Hygromycin B Gold (50 µg/mL), and Zeocin (50 µg/mL). Primary PAM (Optipharm) were cultured in RPMI medium (Cytiva) supplemented with 10% FBS (Gibco) and 1% Penicillin-Streptomycin (Gibco). All the cell lines used in this study were maintained in a humidified 5% CO
2 incubator at 37°C.
For plasmid transfection, PEI was used for HEK293T cells and Lipofectamine 2000 for all other cells, following the manufacturer’s instructions. Lipofectamine RNAiMAX was chosen for cGAMP transfection. PAM, MA-104, and PIB cells stably expressing the pIRES-Flag (control) or pIRES-B175L-Flag (B175L-Flag) were generated by supplementing the cell culture media with 2.0, 4.0, and 0.3 µg/mL of puromycin, respectively.
Plasmids
Wild-type STING was amplified from template DNA using PCR and cloned into pIRES-Flag (STING-Flag), pEXPR-Strep (STING-Strep), and pEBG (STING-GST) vectors. The complete sequence of the ASFV B175L gene (FR682468.1: from 107540 to 108067) was cloned into pIRES-Flag (B175L-Flag), pEXPR-Strep (B175L-Strep), and pEBG vectors (B175L-GST); STING (STING 1–140, 1–185, 185–235, 185–270, 185–330, 185–340, and 1–340) and B175L (B175L 1–60 and 1–110) domain constructs were cloned to the pEBG vector. The cGAMP binding defective single-site (R238A, Y240A, N242A, and E260A) and the double-site (R238A + Y240A) STING mutants were generated using Mutation Generation System Kit (Thermo Scientific; F701) complying with the manufacturer’s instructions and cloned to pIRES-Flag vector. The PCR primers used for site-directed mutagenesis are listed in Table S1.
Quantitative real-time PCR
B175L-Flag-transfected PK-15 or stably expressed PAM and control cells grown in 12-well tissue culture plates (3 × 106 cells/well) were incubated in the humidified 5% CO2 at 37°C. After 12 h, cells were infected with ADV-GFP (MOI = 1.0), harvested at two time points (12 and 24 hpi), and stored at −80°C. Next, the total RNA was isolated using the RNeasy Micro Kit (Qiagen; 74004), and the complementary DNA (cDNA) was synthesized by reverse transcriptase (Toyobo; FSK-101). Finally, the qPCR was performed to quantify the different levels of cDNA using SYBR Green Q-PCR Master mix (SJ Bioscience; SG-SYBR-500) following the manufacturer’s protocol on a Rotor‐Gene Q (Qiagen). The gene-specific primer sets are listed in Table S2.
Virus infection and replication assay
GFP-tagged viruses used in this study were ADV-GFP, HSV-GFP, and VACV-GFP. These viruses were amplified in PK-15 cells and titrated by plaque assay. The virus infection into cells was done in reduced serum (1% FBS)-containing medium for 2 h. The cell supernatants retaining uninfected viruses were replaced with a fresh culture medium afterward. The GFP images were captured by fluorescence microscope (200× magnification) at 24 hpi. The fluorescence intensity was measured from cell extracts harvested at 12 hpi and 24 hpi using the fluorescence modulator (GloMax-Multi Detection System, Promega). The cell extracts and supernatants received through the freeze-thawed process were used for the standard plaque assay in A459 (ADV-GFP) and Vero cells (HSV-GFP and VACV-GFP). For the ASFV infection experiment, primary PAM cells were infected with 1.0 MOI of ASFV (Korea/wildboar/Hwacheon/2020-2287). The cell pellets were harvested at 0, 3, 6, 9, 12, 15, and 18 hpi. Subsequently, RNA extraction was conducted, and qRT-PCR analysis was performed using B175L primers (Table S2). ASFV I73R (an early gene) and B646L (a late gene) were used as controls.
Immunoblot analysis and immunoprecipitation
For immunoblot analysis, cells were harvested after 36 h post-transfection of relevant plasmids and lysed with radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris–HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 1% octylphenoxy poly(ethyleneoxy)ethanol, branched (IGEPAL)] containing phosphatase inhibitor (1 mM Na3PO4) and protease inhibitor cocktail. Then, the lysates were sonicated (30% amplitude, 10 s, three cycles for 500 µL of lysate) and centrifuged at 12,000 rpm at 4°C for 10 min. The received whole-cell lysates (WCLs) were 1:1 mixed with the sample buffer (Sigma; S3401), heated at 100°C for 10 min, and subjected to typical SDS-PAGE, followed by immunoblotting with the indicated antibodies. For immunoprecipitation, Sepharose 6B was added to WCLs (preclearance) and incubated in a rotter at 4°C for 3 h, followed by centrifugation at 12,000 rpm at 4°C for 3 min. Next, the WCLs were incubated with Strep-Tactin Sepharose resin, Glutathione-conjugated Sepharose 4B, or primary antibodies overnight at 4°C. Lastly, the immunoprecipitated beads were collected by centrifugation and washed with 300 mM NP40 lysis buffer before samples were prepared for SDS-PAGE. Concurrently, WCLs containing primary antibodies were incubated with Protein A/G PLUS‐Agarose for 4 h at 4°C. Afterward, the beads were prepared as above-mentioned. The next day, the protein-transferred polyvinylidene difluoride (PVDF) membranes were washed with Tris-buffered saline with Tween 20 (TBST) and incubated, adding the horseradish peroxidase-conjugated (HRP) mouse or rabbit secondary antibodies for 2 h at room temperature and washed again with TBST. All the PVDF membranes were visualized using an enhanced chemiluminescence detection system (ECL-GE Healthcare, Little Chalfont, United Kingdom) using a Las-3000 mini Lumino Image Analyzer.
Semi-denaturating detergent agarose gel electrophoresis (SDD-AGE) assay
The SDD-AGE assay was done as previously described (
49) with some modifications. In brief, HEK293T cells cultured in six-well plates were transfected with STING-Strep and B175L-Flag plasmids as indicated. On the following day, cells were stimulated with 4 µg/well of the cGAMP ligand for 4 h. Subsequently, the cells were rinsed with PBS, and the harvested cell extracts were lysed using RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 1% IGEPAL), which contained 1 mM Na
3PO
4 and PI, for 4 h at 4°C on a rocker. A portion of the WCLs was used for SDS-PAGE, while the proteins eluted with glycine (immunoprecipitated proteins) were subjected to 1.5% SDD-AGE. For the SDD-AGE procedure, samples were loaded onto a 1.5% vertical agarose gel (with 1× Tris-acetate-EDTA (TAE) and 0.1% SDS) and underwent electrophoresis in the running buffer (with 1× TAE and 0.1% SDS) for 50 min at a voltage of 100 V and at 4°C. Finally, the proteins were transferred onto an immunoblot membrane for subsequent immunoblotting (
50).
Confocal imaging
HeLa or PK-15 cells were seeded into an eight‐well chamber slide (ibidi; 80826) and fixed with 4% paraformaldehyde at room temperature for 20 min. After washing with PBS, cells were permeabilized with 100% methanol at −20°C for 20 min, then blocked with 2% BSA in PBS for 1 h at room temperature, followed by incubation with relevant primary antibodies at 4°C overnight. The following day, cells were washed with PBST three times and incubated with an appropriate secondary antibody. Then, cells were washed with PBST three times and stained with DAPI (Invitrogen) at room temperature for 10 min. Images were acquired under Nikon laser scanning confocal microscope (C2plus) and analyzed using NIS‐Elements software.
Luciferase assays
HEK293T cells cultured in 12-well tissue culture plates were transfected with 400 ng of IFN-β driving luciferase (firefly) plasmid, the internal control TK‐Renilla luciferase (Renilla) reporter plasmid, and relevant individual plasmids using PEI. At 24 h post-transfection, the cell layers were washed with PBS and lysed with 1× Passive Lysis buffer (Promega; E194A) for 15 min. Finally, the luciferase activity was estimated using the Dual-Luciferase Reporter Assay System (Promega; E1980) according to the manufacturer’s protocol. The values indicate the firefly luciferase activity normalized to the Renilla luciferase activity. On the other hand, the 293-Dual hSTING-A162 cells and QUANTI-Luci were used to estimate the 3× Flag cGAS or cGAMP-induced luciferase activity on hSTING with or without B175L-Flag.
Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed to detect the secreted pro-inflammatory cytokines in culture supernatants. Commercial kits for porcine IFN-β (CUSABIO, CSB-E09890p) and IL-6 (CUSABIO, CSB-E06786p) were used for the analysis according to the manufacturer’s instructions.
In vitro 2′3′-cGAMP binding assay
The protein/2′3′-cGAMP-Biotin conjugate or protein/2′3′-cGAMP-Cy5 conjugate reactions were set up in 1.5-mL microcentrifuge tubes. Per 50-µL reaction, we added 2 µL of 2′3′-cGAMP-Biotin or 2′3′-cGAMP-Cy5 (1 mM), 5 µL of 10× reaction buffer (100 mM Tris, 500 mM KCl, and 10 mM DTT), and 0.5 µL of 200 mM EDTA (pH = 8), increasing concentrations of glycine-purified proteins, and 10 µg of STING-Strep and 1–10 μg of B175L-Flag, and UltraPure DNase/RNase-Free distilled water (Invitrogen) as required. Microcentrifuge tubes were then incubated for 2 h at 37°C on a rotter. Thereupon, the samples’ volume was increased from 50 μL to 800 µL by adding RIPA (50 mM Tris–HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 1% IGEPAL). To pull-down protein/2′3′-cGAMP-Biotin or protein/2′3′-cGAMP-Cy5 conjugate reactions, 100 µL of Dynabeads M-280 Streptavidin (Invitrogen; 11205D) was added to relevant microcentrifuge tubes with biotinylated 2′3′-cGAMP and 2 µL of anti-Strep antibody for protein/2′3′-cGAMP-Cy5 conjugate. After overnight incubation at 4°C on a rotter, Dynabeads were separated with 4,000 rpm centrifugation for 5 min and washed with TBST (gentle vortex for 1 min, three cycles). On the other hand, samples containing protein-2′3′-cGAMP-Cy5 interactions were isolated from protein A/G PLUS‐Agarose. Finally, all bound proteins were purified by performing protein elution using a 100 mM glycine buffer solution at pH 2 to 2.5 (Santa Cruz Biotechnology). The resulting eluted fraction was subsequently neutralized using a 500 mM NH4HCO3 solution, and the eluted fractions were then subjected to SDS-PAGE.
Mass spectrometry
The sample preparation was done as previously optimized (
51). Briefly, HEK293T cells transfected with B175L-Strep or control were harvested at 36 h post-transfection. Cell lysates were pulled down with Strep-Tactin Sepharose resin overnight at 4°C. After washing the resin with PBS and lysis buffer, the resin-bound proteins were separated with elution buffer (100 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, and 2.5 mM desthiobiotin); the eluted proteins were further concentrated using an Amicon Ultra-0.5 (10K cutoff) centrifugal filter (Merck Millipore; UFC501096). Four to fifteen percent of NuPAGE gels (Invitrogen; NP0323PK2) were used for broad molecular weight protein separation, followed by silver staining (
52). Next, the protein bands present in the gel were subjected to mass spectrometry analysis. Finally, the B175L-unique mass spectrometry results were filtered against the proteins of the control and checked for specific bound proteins whose importance is associated with IFN-I pathway. Among thousands of proteins, the STING (UniProt: Q86WV6) was selected for further studies, and that selection was dependent upon the biological relevance in the context of immunology, STING-related previous findings, and experimental feasibility.
Statistical analysis
All graphs and statistical analyses were carried out with the GraphPad Prism software, version 6, for Windows. The data are presented as means and standard deviations (SD) for at least three independent experiments. At each time point, an unpaired t-test was used to compare the control and treatment groups. P values of <0.05, <0.01, <0.001, or <0.0001 were regarded as significant.