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MITA oligomerization upon viral infection is dependent on its N-glycosylation mediated by DDOST [1]

['Yi Tu', 'State Key Laboratory Of Virology', 'Frontier Science Center For Immunology', 'Metabolism', 'College Of Life Sciences', 'Wuhan University', 'Wuhan', 'Xiu-Juan Yin', 'Qian Liu', 'Shan Zhang']

Date: 2022-12 

The mediator of IRF3 activation (MITA, also named STING) is critical for immune responses to abnormal cytosolic DNA and has been considered an important drug target in the clinical therapy of tumors and autoimmune diseases. In the present study, we report that MITA undergoes DDOST-mediated N-glycosylation in the endoplasmic reticulum (ER) upon DNA viral infection. Selective mutation of DDOST-dependent N-glycosylated residues abolished MITA oligomerization and thereby its immune functions. Moreover, increasing the expression of Ddost in the mouse brain effectively strengthens the local immune response to herpes simplex virus-1 (HSV-1) and prolongs the survival time of mice with HSV encephalitis (HSE). Our findings reveal the dependence of N-glycosylation on MITA activation and provide a new perspective on the pathogenesis of HSE.

Interferons (IFNs) play critical roles in controlling viral infection. Insufficient production of IFNs leads to chronic infection and disease progression. Here, we report that DDOST is an important regulator of the MITA/STING-mediated production of antiviral type I IFNs. DDOST knockdown impairs the MITA-mediated IFN response to HSV-1 and double-stranded DNA (dsDNA). Mechanistically, DDOST mediates HSV-1-induced N-glycosylation of MITA at certain residues, which is essential to promote MITA oligomerization and consequent immune functions. Moreover, the expression of endogenous DDOST was specifically low in the brain. Increasing the expression of Ddost in the mouse brain strengthens the local immune functions and prolongs the survival time of mice with HSE. These findings reveal a regulatory mechanism of MITA oligomerization and provide one explanation for the pathogenesis of HSE, which provides a clue for developing a new antiviral drug.

Funding: This work was supported by grants from the National Key Research and Development Program of China (2018YFA0800700 (YL), http://most.gov.cn/ ) and the National Natural Science Foundation of China (32170723 (YL), 31970894 (YL), https://www.nsfc.gov.cn/ ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In the present study, we reported DDOST as a new mediator of MITA posttranslational modification upon DNA viral infection. Selective mutation of DDOST-dependent N-glycosylated residues abolished MITA oligomerization and thereby its immune functions. Furthermore, Ddost is expressed at much lower levels in the brain than in most peripheral tissues. Overexpression of Ddost in the brain effectively improves immune responses, limits the replication of HSV-1, and prolongs the survival time of mice with HSE.

Posttranslational modification of MITA regulates its functions in various aspects. As mentioned above, TBK1-catalyzed phosphorylation of MITA leads to its degradation and attenuated IFN production. Degradation of MITA is also mediated by K48-linked polyubiquitination, catalyzed by RNF5, TRIM30α, and TRIM29. SUMOylation and deSUMOylation of MITA, catalyzed by TRIM38 and SENP2, respectively, at different stages delicately regulate the protein level of MITA to maintain proper immune responses [ 13 – 17 ]. The translocation of MITA from the ER to the ERGIC is also regulated by posttranslational modification. K63-linked polyubiquitination of MITA, catalyzed by RNF115 and mitochondrial E3 ubiquitin protein ligase 1 (MUL1), potentiates its translocation for further signaling [ 18 , 19 ]. Palmitoylation of MITA at the Golgi apparatus, catalyzed by protein palmitoyltransferases, is critical for MITA recruitment of downstream TBK1 and IRF3 [ 20 , 21 ]. Recently, Golgi apparatus-synthesized O-linked sulfation of glycosaminoglycans (sGAGs) was reported to be critical for MITA oligomerization and immune signal transduction [ 22 ]. Because of its critical role in multiple diseases, how MITA is regulated has attracted much research.

As the main cytosolic DNA sensor, cyclic GMP-AMP (cGAMP) synthase (cGAS) initiates the synthesis of the second messenger cGAMP, which subsequently binds to endoplasmic reticulum (ER)-located MITA [ 5 , 6 ]. MITA then undergoes oligomerization and translocates from the ER via ER-Golgi intermediate compartments (ERGIC) to perinuclear punctate structures with the help of coat protein complex II vesicles [ 2 , 7 – 9 ]. In this process, MITA recruits the kinase TBK1, which in turn phosphorylates MITA and leads to MITA degradation [ 10 – 12 ]. The transcription factor IRF3 is also recruited by MITA and phosphorylated by TBK1. Subsequently, activated interferon regulatory factor 3 (IRF3) enters the nucleus and initiates the transcription of IFN-Is, including IFNα and IFNβ. Secreted IFN-I functions as an autocrine/paracrine factor to further bind with IFN receptor (IFNR) on the cell surface and activate the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. Eventually, the transcription factor STATs lead to the production of multiple IFN-stimulated genes (ISGs), which perform antiviral immune responses. In parallel, MITA also recruits IKK to activate the NF-κB-driven production of proinflammatory cytokines [ 11 ].

The mediator of IRF3 activation (MITA, also known as STING, MPYS, or ERIS) is an essential adaptor protein in innate immune signaling leading to the production of type I interferon (IFN-I) and proinflammatory cytokines in response to cytosolic double-stranded DNA (dsDNA) [ 1 – 3 ]. Because cytosolic dsDNA can either invade pathogenic DNA or abnormally release mitochondrial DNA, the immune responses initiated to eliminate these abnormal dsDNA have been extensively studied in multiple diseases, such as viral infection, tumors, and radiotherapeutic emergency [ 4 ].

Results

The OST complex is indispensable for antiviral IFN production To identify potential regulators of antiviral signaling, we searched for MITA-interacting candidates in the open access shotgun mass spectrometry database (https://www.imexconsortium.org/) [23]. We found several members of the oligosaccharyltransferase (OST) complex in the database, including dolichyl-diphosphooligosaccharide-protein glycosyltransferase noncatalytic subunit (DDOST), STT3 oligosaccharyltransferase complex catalytic subunit A (STT3A), and ribophorin I (RPN1). The OST complex, which is responsible for protein glycosylation, consists of a catalytic subunit (STT3A or STT3B) and several noncatalytic subunits, including DDOST, RPN1, RPN2, defender against cell death 1 (DAD1), magnesium transporter 1 (MAGT1), oligosaccharyltransferase complex subunit 4 (OST4), transmembrane protein 258 (TMEM258), and tumor suppressor candidate 3 (TUSC3) [24–26]. We then performed endogenous coimmunoprecipitation (Co-IP) with an anti-MITA antibody and identified MITA-associated OST subunits in human monocytic THP-1 cells upon HSV-1 infection. The marked interaction between MITA and RPN1 or DDOST in the steady state was observed by immunoblotting (Fig 1A). HSV-1 infection induced the marked recruitment of STT3B to MITA and strengthened the interaction between endogenous MITA and DDOST or RPN1 (Fig 1A). However, no marked interaction between MITA and RPN2, MAGT1, or DAD1 was found. Additionally, the expression of STT3B was elevated upon HSV-1 infection, but the expression of DDOST remained at a constant level (Fig 1A). Because of the failure to detect endogenous STT3A, we overexpressed Flag-MITA and HA-STT3A plasmids in HEK293T cells and detected the interaction between MITA and STT3A by Co-IP (S1A Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 1. The OST complex is indispensable for antiviral IFN production. (A) THP-1 cells were infected with HSV-1 (MOI = 2) for the indicated time, and endogenous MITA was then immunoprecipitated with the indicated antibodies before immunoblotting analysis. ACTB: β-actin. (B) Consensus transcript levels of the indicated genes in human tissues were adopted from The Human Protein Atlas version 21.0 (www.proteinatlas.org) and are shown as the relative value to the transcript level of each gene in the brain. The values in the brackets represent the relative value to the transcript level of DDOST in the brain. (C) Different mouse organs were obtained and analyzed for the expression of Ddost by immunoblots. We discriminated proteins from humans or other species by a certain naming rule, for example, DDOST, as all letters capitalized to describe human proteins, and Ddost, as the first letter capitalized to describe mouse proteins. (D) HEK293T cells in 24-well plates were transfected with DDOST-RNAi NO.1 (Di#1), NO.2 (Di#2), RPN1-RNAi, RPN2-RNAi, STT3B-RNAi, STT3A-RNAi or empty vector control (Coni) by calcium phosphate transfection for 24 hours and then retransfected with pRK-HA-cGAS and pRK-HA-MITA together with the IFNβ firefly luciferase reporter gene (50 ng/well) and TK Renilla luciferase reporter gene (5 ng/well) by Lipofectamine 2000. A dual luciferase reporter assay was performed after 24 hours. Data displayed are the mean ± SD of each technical repeat (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t test). (E-I) Stable DDOST-, RPN1-, RPN2-, STT3A- or STT3B-knockdown THP-1 cells were constructed and infected with HSV-1 (MOI = 2) for the indicated time before immunoblotting analysis. The relative ratio (Rel. ratio) of phosphorylated TBK1 (pTBK1) to TBK1 or phosphorylated IRF3 (pIRF3) to IRF3 was calculated by measuring the grayscale values of the bands and then normalized by the value at the time of the activation peak in the control group. One representative result from at least three independent experiments is shown. (J) DDOST knockdown THP-1 cells and control cells were transfected with ISD45 (2 μg/mL) by Lipofectamine 2000 for the indicated time before immunoblotting analysis. The relative ratio (Rel. ratio) of pTBK1 to TBK1 or pIRF3 to IRF3 was calculated by measuring the grayscale values of the bands and then normalized by the value at the time of the activation peak in the control group. One representative result from at least three independent experiments is shown. https://doi.org/10.1371/journal.ppat.1010989.g001 We then examined the expression level of OST subunits in different organs using the online database (https://www.proteinatlas.org/). The transcript abundance of DDOST in the brain was much lower than that in other organs, and a similar phenomenon was found in OST subunits other than DAD1 (Fig 1B). Choosing DDOST as a representative subunit, we detected the protein levels of Ddost in different mouse organs. As shown in Fig 1C, the expression of Ddost in the brain was indeed much lower than that in the lung, liver, spleen, skin, intestine, colorectum or kidney. Then, we sought to identify whether the low expression of the OST subunit affects MITA-mediated antiviral signaling. Specific RNA interference (RNAi) plasmids for the OST subunit were constructed, and their knockdown efficiency was confirmed by immunoblotting or qPCR (S1B–S1E Fig). Two RNAi plasmids specific to DDOST were named DDOST-RNAi No. 1 (Di#1) and No. 2 (Di#2). Quantified by qPCR, Di#2 inhibited the transcription level of DDOST to 11% of the control cells, better than Di#1 (48%) (S1B Fig). Then, IFN-β luciferase reporter assays were performed in HEK293T cells with transient transfection of RNAi plasmids. As shown in Fig 1D, knockdown of DDOST, RPN1, RPN2 or STT3B markedly inhibited cGAS-MITA-mediated activation of the IFN-β promoter, although no marked interaction between MITA and RPN2 was observed. In contrast, STT3A knockdown potentiated IFN-β promoter activation (Fig 1D), suggesting that the role of STT3A in MITA signaling differs from that of other detected subunits. Then, we prepared stable OST subunit knockdown THP-1-cell lines and infected cells with HSV-1. Consistently, DDOST, RPN1, RPN2, and STT3B knockdown inhibited HSV-1 infection-induced phosphorylation of TBK1 and IRF3 (Fig 1E–1H). In particular, both the Di#1 and Di#2 cell lines showed reduced activation of MITA signaling in response to HSV-1 compared with the control cells, and the suppression degree correlated with the knockdown efficiency of the corresponding RNAi plasmid. (Fig 1E). However, STT3A knockdown slightly promoted the phosphorylation of TBK1 and IRF3 (Fig 1I). To identify whether DDOST affects MITA signaling or virus entry directly, we transfected DDOST-knockdown THP-1 cells with double-stranded 45-mer IFN stimulatory DNA (ISD45) and observed that dsDNA-induced phosphorylation of TBK1 and IRF3 was also attenuated by DDOST knockdown (Fig 1J). In addition, DDOST knockdown impaired gradient viral infection (multiplicity of infection (MOI) = 1, 2, 4)-induced phosphorylation of TBK1 and IRF3 (S1F Fig). These data suggest that the OST complex is required for regulating MITA-mediated antiviral signaling, and the low expression of OST subunits in the brain is possibly one of the reasons for HSE. We then chose DDOST as a representative subunit to explore its antiviral functions.

DDOST potentiates the function of MITA at the ER Under fluorescence microscopy, we observed that cherry-DDOST colocalized with GFP-MITA and the ER marker CLAR but not the Golgi matrix protein GM130, mitochondrial BID, lysosomal LAMP1, or endosomal Rab5 (Fig 3A and 3B). It has been demonstrated that the diffuse distribution of MITA at the ER in the steady state aggregates to perinuclear vesicles upon DNA viral infection [2,27]. However, HSV-1-induced perinuclear aggregation of MITA was dramatically inhibited by DDOST knockdown (Fig 3C), suggesting that DDOST promotes the translocation of MITA from the ER to the perinuclear vesicles. By Co-IP assays, we confirmed that DDOST knockdown inhibited MITA recruitment of downstream TBK1 upon HSV-1 infection (Fig 3D). Consistently, HSV-1-induced phosphorylation of MITA, mainly catalyzed by TBK1, was also inhibited by DDOST knockdown (Fig 3E). These results suggest that DDOST facilitates MITA trafficking from the ER to perinuclear vesicles and thereby promotes MITA activation by recruiting TBK1. PPT PowerPoint slide

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TIFF original image Download: Fig 3. DDOST potentiates the function of MITA at the ER. (A, B) HeLa cells were transfected with Cherry-DDOST and GFP-MITA (A) or GFP-tagged organelle markers (B) and visualized under confocal microscopy 24 hours later. Vec: empty vector as a control; Scale bar: 10 μm. (C) DDOST knockdown HaCaT cells and control cells were infected with HSV-1 (MOI = 2) for 3 hours before staining with rabbit anti-MITA and Cy3 goat anti-rabbit IgG. Scale bar: 10 μm. (D) DDOST knockdown THP-1 cells and control cells were infected with HSV-1 (MOI = 2) for the indicated time. Cell lysates were subjected to Co-IP and immunoblotting analysis using the indicated antibody. (E) DDOST knockdown THP-1 cells and control cells were infected with HSV-1 (MOI = 2) for the indicated time before immunoblotting analysis. One representative result from at least three independent experiments is shown. (F, G) HEK293T cells were transfected with HA-MITA plus full length Flag-DDOST or its truncated mutants (F) or Flag-DDOST plus full length HA-MITA or its truncated mutants (G). Then, Co-IP and immunoblotting analysis were sequentially performed using the indicated antibodies 24 hours posttransfection. The related interaction diagrams are shown in the lower panels. FL: full length; TM: transmembrane domain; IgL: immunoglobin light chain. https://doi.org/10.1371/journal.ppat.1010989.g003 To delineate the minimal domains responsible for DDOST-MITA interaction at the ER, we constructed a series of truncations and performed Co-IP. The results showed that DDOST and MITA interacted through their respective transmembrane domains (TM) (Fig 3F and 3G). GST pulldown assays again verified the association between DDOST and MITA (S3 Fig). Collectively, these results suggest that DDOST interacts with MITA at the ER through the TM domain and facilitates MITA trafficking from the ER to perinuclear vesicles upon viral infection.

DDOST mediates the N-glycosylation of MITA upon viral infection DDOST is a noncatalytic subunit of the OST complex [28,29]. We then sought to identify whether N-glycosylation of MITA is involved in antiviral immune responses. PNGase F, as a glycosidase, catalyzes the deamidation of Asn and thereby removes the glycans attached to Asn residues in certain N-glycosylated motifs of proteins. The consequent deglycosylated proteins can be recognized by lower shifted bands in immunoblots. We immunoprecipitated ectopically expressed HA-MITA with an anti-HA antibody. The beads conjugated with HA-MITA were then incubated with PNGase F, which can catalyze the deamidation of N-glycosylated Asn. The result showed that partial MITA was deglycosylated, shifting to a lower position (Fig 4A). THP-1 cells were then infected with HSV-1 and treated with cycloheximide (CHX) to inhibit the new synthesis of proteins. Endogenous MITA was immunoprecipitated and further digested by PNGase F. The results showed that HSV-1 infection enhanced N-glycosylation of MITA, which could be mostly inhibited by CHX treatment, suggesting that virus-induced N-glycosylation of MITA mainly belongs to newly synthesized MITA (Fig 4B). Deglycosylation assays were then performed in DDOST knockdown cells. DDOST knockdown markedly reduced HSV-1-induced N-glycosylation of MITA, suggesting that DDOST is responsible for the glycosylation of newly synthesized MITA (Fig 4C). PPT PowerPoint slide

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TIFF original image Download: Fig 4. DDOST mediates the N-glycosylation of MITA upon viral infection. (A) Cell lysates of HA-MITA-transfected HEK293T cells were immunoprecipitated with an anti-HA antibody. The beads coupled with HA-MITA were incubated with PNGase F or control buffer at 37°C overnight before the immunoblotting analysis. (B) THP-1 cells were treated with or without 20 μM CHX and infected with HSV-1 (MOI = 2) for the indicated times. Then, immunoprecipitation assays were performed with an anti-MITA antibody (ABclonal, A3575) followed by digestion with PNGase F and immunoblotting. One representative result from at least three independent experiments is shown. (C) DDOST knockdown THP-1 cells and control cells were infected with HSV-1 (MOI = 2) before immunoprecipitation with anti-MITA antibody (ABclonal, A3575) and digestion with PNGase F. One representative result from at least three independent experiments is shown. (D, E) Immunoprecipitated endogenous MITA in DDOST knockdown THP-1 cells and control cells untreated or treated with HSV-1 (MOI = 2) for 6 hours was digested with PNGase F and then analyzed by LC–MS/MS to identify N-glycosylation sites of MITA. PNGase F cleaves the amide bond of N-glycosylated Asn and converts Asn to Asp. The b- and y-series fragment ions derived from a certain N-glycopeptide moiety are displayed (D). The abundance of N-glycopeptides to total peptides was calculated and displayed (E). (F) Sequence alignment of MITA homologs in humans, mice, pigs, green monkeys, and bovines. https://doi.org/10.1371/journal.ppat.1010989.g004 N-glycosylation occurs on asparagine in the consensus motif Asn-X-Ser/Thr (where X is not proline) or other rare modification motifs, such as Asn-X-Cys/Val, Asn-Gly, (Ser/Thr)-X-Asn motifs and nonconsensus sequences [30,31]. We then identified the N-glycosylation sites of MITA with or without viral infection by liquid chromatography with tandem mass spectrometry (LC–MS/MS). Three residues, N183 in the Thr-X-Asn motif, N211 in the Asn-X-Ser motif, and N242 in the nonconsensus motif, were convincingly identified through three independent assays (Fig 4D). Because the total abundance of digested peptides for MS identification in DDOST knockdown cells was comparable with that in control cells, we calculated the ratio of N-glycosylated MITA peptide abundance to the total peptide abundance and found that HSV-1 infection potentiated N-glycosylation of MITA at N183, N211 and N242 (Fig 4E). However, in DDOST knockdown cells, virus-induced glycosylation at N183, N211, and N242 was inhibited, suggesting that DDOST mediates the glycosylation of MITA at these three residues (Fig 4E). Sequence alignment demonstrated that N183 and N242 of MITA were highly conserved in humans, mice, green monkeys, pigs, and bovines (Fig 4F).

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010989

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