key: cord-0314091-1x7z0qsi
authors: Iampietro, Mathieu; Dumont, Claire; Mathieu, Cyrille; Spanier, Julia; Robert, Jonathan; Charpenay, Aude; Dupichaud, Sébastien; Dhondt, Kévin P.; Aurine, Noémie; Pelissier, Rodolphe; Ferren, Marion; Mély, Stéphane; Gerlier, Denis; Kalinke, Ulrich; Horvat, Branka
title: Activation of cGAS/STING pathway upon paramyxovirus infection
date: 2020-12-26
journal: bioRxiv
DOI: 10.1101/2020.12.26.424443
sha: 006d94acf6a0b6274802a5cc55103a458a36b11e
doc_id: 314091
cord_uid: 1x7z0qsi

During inflammatory diseases, cancer and infection, the cGAS/STING pathway is known to recognize foreign or self-DNA in the cytosol and activate an innate immune response. Here, we report that negative-strand RNA paramyxoviruses, Nipah virus (NiV) and Measles virus (MeV), can also trigger the cGAS/STING axis. While mice deficient for MyD88, TRIF and MAVS still moderately control NiV infection when compared to WT mice, additional STING deficiency resulted in 100% lethality, suggesting synergistic roles of these pathways in host protection. Moreover, deletion of cGAS or STING resulted in decreased type-I interferon production with enhanced paramyxoviral infection in both human and murine cells. Finally, the phosphorylation and ubiquitination of STING, observed during viral infections, confirmed the activation of cGAS/STING pathway by NiV and MeV. Our data suggest that cGAS/STING activation is critical in controlling paramyxovirus infection, and possibly represent attractive targets to develop countermeasures against severe disease induced by these pathogens.

The innate immunity represents the first line of host defense against invading pathogens 40 (Akira et al., 2006) . Exogenous motifs associated with viral infections involved in stimulating innate 41 responses include pathogen-derived nucleic acids, DNA or RNA (Akira et al., 2006; Mogensen, 2009 ). 42

Its ensuing detection activates pattern recognition receptor (PRR)-associated adaptor molecules 43 that are responsible for subsequent expression of type I and III interferons (IFNs) (Park and Iwasaki, 44 2020) and the induction of IFN-related genes, which are important for the control of virus infection 45 (Borden et al., 2007; Der et al., 1998; Sen and Peters, 2007) . Four major axes, defined by their nodal 46 adaptor, are able to induce strong innate immune responses upon sensing of pathogen-related 47 nucleic acids (Baccala et al., 2009 ). Three of them, Toll-like receptor (TLR)-associated adaptor 48 molecules: myeloid differentiation primary response 88 (MyD88) (Wesche et al., 1997) , 49

Toll/interleukin-1 receptor/resistance [TIR] domain-containing adaptor-inducing IFN-β (TRIF) 50 (Yamamoto et al., 2003) and RIG-I-like receptor (RLR)-associated mitochondrial antiviral signaling 51 protein (MAVS) (Seth et al., 2005) are dedicated to sense DNA and/or RNA. The cyclic guanosine 52 monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)/stimulator of interferon 53

genes (STING also known as ERIS, MITA or TMEM173) pathway is the leading sensor for the 54 detection of cytosolic DNA (Ishikawa and Barber, 2008; Sun et al., 2013) . The cGAS/STING axis seems 55

to be involved in the sensing of various different RNA viruses, which may express viral proteins that 56 counteract cGAS/STING activity at different levels. Thus, the cGAS/STING pathway may also 57 contribute to the control of RNA viruses (Ni et al., 2018) . Recently, activation of the cGAS/STING 58 pathway has been observed following infection by the positive strand RNA virus SARS-CoV-2 59 (Neufeldt et al., 2020) . 60

In recent years, members of the Paramyxoviridae family have caused numerous emerging 61 zoonoses and/or epidemics (Thibault et al., 2017) . This viral family contains both old and new human 62

and zoonotic viral pathogens such as measles virus (MeV) and Nipah virus (NiV). While MeV has 63 been almost eradicated from most developed countries through vaccination campaigns, the 64 number of cases and deaths significantly increased within the last decade killing more than 100.000 65

people every year (Ferren et al., 2019) . Moreover, as NiV is the most virulent paramyxovirus with 66 mortality rates between 40-100% during epidemics and remains without any licensed treatment or 67

vaccine (Pelissier et Figure 1A) . Moreover, while 60% triple KO mice survived NiV challenge, 116

all mice bearing the quadruple deficiency succumbed by day 11 post-infection, indicating a crucial 117

and non-redundant role for STING in the protection of mice ( Figure 1A ). NiV nucleoprotein (NiV-N) 118

protein levels in the brain of autopsied animals deficient for MyD88/TRIF/MAVS and 119

MyD88/TRIF/MAVS/STING were comparable to those observed in the brain of IFNAR KO mice at 120 time of death ( Figure 1B) . Furthermore, while analysis of NiV load in murine brain determined 121 equivalent NiV-N RNA levels within these three deficient murine models, evaluation in the spleen 122

showed higher viral loads in mice bearing the quadruple deficiency compared to the triple KO mice 123

( Figure 1C and S1A). In parallel, the lower production of IFNβ ( Figure 1D and S1B) and IFNα ( Figure  124 1E and S1C 

STING controls NiV replication in primary murine embryonic fibroblasts (pMEFs). 142

To further evaluate the role of STING during NiV infection in mice, we analyzed the impact 143

of the gene deletion of various nodal adaptors on the in vitro NiV replication in pMEFs. The pMEFs 144

were generated from mice bearing the corresponding deleted genes as described previously (Brune 145 et al., 2001). They were infected with rNiV-eGFP to allow imaging of the viral infection by fluorescent 146 microscopy. In agreement with the above in vivo observations, MyD88 KO and MyD88/TRIF KO 147 pMEFs were able to control NiV replication as well as WT pMEFs, with only few observed infected 148 cells. In contrast, NiV rapidly spreads within the culture of MyD88/TRIF/MAVS KO and 149

MyD88/TRIF/MAVS/STING KO pMEFs although not as extensively as in IFNAR KO cells ( Figure 2A ). 150

Moreover, single STING KO pMEFs were also unable to control the viral spread, highlighting a major 151 role of STING in the mouse innate defense against NiV infection ( was analyzed using 1-way analysis of variance, followed by the Tukey multiple comparisons test. *P< 0.05; **P < 0.01; ***P< 0.001; 211 ****P < 0.0001 compared to infected WT condition.

As an alternative and complementary approach, we analyzed the paramyxoviral propagation 214

within microscopy ( Figure 3K and 3L) and flow cytometry ( Figure S2C, S2D, S2E ). 221

Thus, the cGAS/STING axis also appears to play a critical role in human cells to control 222 paramyxovirus infections by allowing the expression of IFNβ and to a lower extent that of IFNα.

Paramyxovirus infection activates the cGAS/STING pathway in both murine and human cells.

The increased viral infection observed upon abolition of either cGAS or STING suggested that 226

paramyxoviruses could activate STING. This was investigated by analyzing specific phosphorylation 227

and/or ubiquitination of activated STING in pMEFs, THP-1 and human pulmonary microvascular ( Figure S3B) . Importantly, the detection of the STING-S365 P band in MyD88/TRIF/MAVS KO pMEFs 242

indicates that NiV infection activates the STING signaling pathway independently from the TLR-243

MyD88/TRIF and RLR/MAVS pathways of innate immunity. Since K63-linked ubiquitination (Ub-K63) 244

is another hallmark of STING activation, mainly associated to the activation of NF-κB (Chiang and 245

Gack, 2017), STING ubiquitination was also evaluated in HPMECs infected for 48 h with NiV or MeV.

STING immunoprecitated using anti-STING antibodies was found to be labelled by both anti-STING-247 S366 P and anti-Ub-K63 antibodies ( Figure 4D and 4E) . Our study demonstrates that STING is activated against RNA viruses as also recently reported 309

with SARS-CoV-2 (Neufeldt et al., 2020) and highlights that STING is modified and activated through 310 S366 phosphorylation and/or K-63 linked ubiquitination during NiV and MeV infection. Additional 311 studies will uncover the occurrence of others PMTs modification, STING subcellular location and the 312 source of the cognate DNA that activate cGAS during RNA virus infection. 313

In conclusion, cGAS/STING activation occurs during paramyxovirus infections, both in vitro 314

and in vivo. This highlights an undefined aspect of the immune regulation against negative strand 315 viruses and reveals cGAS/STING as potential targets in the development of novel antiviral strategies. Mérieux BSL-4 laboratory in Lyon, France.

Cell Lines 338

Primary murine embryonic fibroblasts (pMEFs) were isolated from murine embryos obtained from 339 pregnant mice 13 days after conception, as described elsewhere (Brune et al., 2001) Brains from mice were embedded in paraffin wax and sectioned at 7 µm. Slides were deparaffinated 367 and rehydrated in three Xylene baths for 5 min each, followed by two 100% alcohol baths for 5 min, 368

and then succeeded with multiple baths using decreasing level of alcohol for 3 min each. After 369 deparaffination, slides were put in a sodium citrate solution in a boiling water bath for 20 min for 370 heat-induced epitope retrieval and washed 3 times in PBS for 3 min afterwards. Activity of 371 endogenous peroxydase was blocked using a H2O2 0.3% solution. Blocking of non-specific epitopes 372 is done using PBS-2.5% decomplemented Normal Horse Serum + 0.15% Triton X-100 for 30 min. 373

Then, primary rabbit anti-NiV N antibody was used at 1/10000 dilution and incubated overnight at 374 4°C in the blocking buffer. For secondary antibody and revealing steps, ImmPress system (anti-rabbit 375 ig/peroxydase) was used. Counterstaining was performed using Harris solution and photographs 376

were taken with a microscope Zeiss Axiovert 100M. 377 378

Co-immunoprecipitations 379 HPMEC cells (5×10 5 ) were seeded in 6-well plates. 16 h after seeding, cells were infected with the 380 appropriate dilution of rNiV-eGFP or rMeV-edmH-eGFP at a MOI of 1 in RPMI described above. 381

Forty-eight hours post-infection, cells were lysed in RIPA buffer, supplemented with a cocktail of 382

protease-phosphatase inhibitors for 30 min on ice, and centrifuged for 10 min at 4°C at 15,000 g. 383

Supernatants were incubated with a rabbit anti-STING antibody for 2 h at 4°C. Then, protein A/G 384 agarose beads were added to the mix overnight at 4°C. Beads were then washed three times in 385

washing buffer (RIPA buffer, supplemented with a cocktail of protease-phosphatase inhibitor), and 386

proteins were eluted in 100 µl of elution buffer (Reducing agent 10X, Laemmli 4X, RIPA buffer, 387

supplemented with a cocktail of protease-phosphatase inhibitors) for 15 min at 96°C. Then the 388 eluate and a sample of input of the cell extract were run on polyacrylamide gel electrophoresis (SDS-389 PAGE) and analyzed by western-blotting.

Immunoblot Analysis 392

Heated protein lysates were separated by 4-15% SDS-PAGE and electro transferred for 1 h onto 393 polyvinylidene difluoride (PVDF) membranes at 4°C. PVDF membranes were blocked in Tris-buffered 394 saline containing 0.05% Tween 20 (TBS-T) + 5% milk for 1 h and then incubated overnight with 395 primary antibodies, mouse anti-GAPDH, rabbit anti-STING, rabbit anti-human S366 p-STING, rabbit 396

anti-mouse S365 p-STING, rabbit anti-Caspase 3, rabbit anti-cleaved Caspase 3 and rabbit anti-Ub-397

K63 antibodies, diluted 1:1000 in TBS-T + 0.2% milk. Membranes were then washed 3 times using 398

TBS-T and incubated on an additional 1 h with horseradish peroxydase conjugated anti-mouse or 399

anti-rabbit IgG antibodies (diluted 1:5000 in TBS-T + 5% milk were analyzed using StepOne software and calculations were done using the 2 ΔΔCT method.

Expression was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 412 expressed as copies of mRNA. Specific set of primers were designed and validated for the detection 413 of human hGAPDH, hIFNα and hIFNβ, murine mGAPDH, murine mIFNα and mIFNβ and viral NiV N 414

and MeV N.

Immunofluorescence 417

For PMEFs and THP-1 infections, 3×10 5 and 5×10 5 cells were seeded in 12-well plates, respectively, 418 before being infected with rNiV-eGFP and rMeV-edmH-eGFP at a MOI of 0.3 or 0.1 and cultured for 419 24 or 48 h at 37°C with 5% CO2. Then, cells were evaluated for eGFP expression using a Zeiss 420

Axiovert 100M microscope in the BSL-4 or a NIKON Eclipse Ts2R in the BSL-2, and photographs were 421 taken 24 and 48 h after infection and treated using ImageJ software version Java 1.8.0_112.

Flow cytometry 424

THP-1 cells were seeded in 12-well plates at 3×10 5 and 5×10 5 per well before being infected with 425

rNiV-eGFP and rMeV-edmH-eGFP at a MOI of 0.1 and cultured for 24 or 48 h at 37°C with 5% CO2. 426

Then, cells were washed, reconstituted in PBS 1X and evaluated for eGFP expression using a Gallios 427 flow cytometer in the BSL-4 or a 4L Fortessa flow cytometer. Analyses were performed 24 and 48 h 428 after infection.

Ethical statement 431

Animals were handled in strict accordance with good animal practice as defined by the French 432 national charter on the ethics of animal experimentation and all efforts were made to minimize 433

suffering. Animal work was approved by the Regional ethical committee and French Ministry of High 434

Education and Research and experiments were performed in the INSERM Jean Mérieux BSL-4 435 laboratory in Lyon, France (French Animal regulation committee N° 00962.01).

Analysis of eGFP quantification in THP-1 cells 438

The results are presented in the form of histograms which represent the mean eGFP positive cells 439

for each conditions and error bars represent the standard errors (SE) for n=3 experimental 440

replicates. The different conditions were compared to the control (WT+). Statistical significance was 441 assessed by a one-way ANOVA, followed by a Tukey's multiple comparisons test; *p < 0.05, **p < 442 0.01, ***p < 0.001 and ****p < 0.0001 (threshold of significance of 5%). 443 444 qPCR analysis 445

The results are presented in the form of histograms, which represent the mean of copies of mRNA 446 for a gene for each condition and error bars represent the standard errors (SE) for n=3 experimental 447

replicates. The different conditions were compared to the control (WT+). Statistical significance was 448 assessed by a one-way ANOVA, followed by a Tukey's multiple comparisons test; *p < 0.05, **p < 449 0.01, ***p < 0.001 and ****p < 0.0001 (threshold of significance of 5%).

Densitometry 452

Densitometric analysis of cleaved caspase 3 immunoblots from three independent experiments 453

were performed using the VersaDoc Imaging System (Bio-Rad) and analyzed with ImageJ 1.52p Fiji 454 package software (https://imagej.net/Fiji). GAPDH expression was used for normalization. 455 456

Dengue virus NS2B protein targets cGAS 477 for degradation and prevents mitochondrial DNA sensing during infection

STING manifests self DNA-dependent 479 inflammatory disease

Pathogen recognition and innate immunity

Sensors of the innate immune system: their mode of action

Interferons at age 50: past, current and future impact on biomedicine

A mouse model for cytomegalovirus 489 infection

Post-translational Control of Intracellular Pathogen Sensing 491 Pathways

Structural mechanism of cytosolic DNA sensing by cGAS

Crystal structures of porcine STING CBD -CDN complexes reveal the mechanism of ligand 497 recognition and discrimination of STING proteins

Ligand binding determines whether CD46 is 500 internalized by clathrin-coated pits or macropinocytosis

Identification of genes 502 differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays

Type I interferon signaling protects mice from lethal henipavirus infection

STING 508 Activation by Translocation from the ER Is Associated with Infection and Autoinflammatory 509 Disease

Conserved strategies for pathogen evasion of cGAS-511 STING immunity

STING Polymer Structure Reveals 513 Mechanisms for Activation, Hyperactivation, and Inhibition

Measles Encephalitis: Towards New Therapeutics. 515 Viruses 11

STING-517 dependent translation inhibition restricts RNA virus replication

Cyclic [G(2′,5′)pA(3′,5′)p] Is the Metazoan Second 521 Messenger Produced by DNA-Activated Cyclic GMP-AMP Synthase

Interplay between innate immunity and negative-strand RNA 523 viruses: towards a rational model

Macrophage development and activation involve coordinated 527 intron retention in key inflammatory regulators

Autophagy 529 induction via STING trafficking is a primordial function of the cGAS pathway

Targeting STING with covalent small-molecule 533 inhibitors

IRF-7 is the master regulator of type-I interferon-dependent 536 immune responses

Control of Nipah Virus

Adaptors Mitochondrial Antiviral Signaling Protein (MAVS) and Myeloid Differentiation Primary 540 Response 88 (MyD88)

Both RIG-I 542 and MDA5 RNA Helicases Contribute to the Induction of Alpha/Beta Interferon in Measles Virus-543 Infected Human Cells

STING is an endoplasmic reticulum adaptor that facilitates 545 innate immune signalling

STING regulates intracellular DNA-mediated, type I 547 interferon-dependent innate immunity

Structure of Human cGAS 549 Reveals a Conserved Family of Second-Messenger Enzymes in Innate Immunity

Structure-Guided Reprogramming of Human cGAS Dinucleotide Linkage 553 Specificity

Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces 556 IRF3 activation

Cytosolic RNA:DNA hybrids activate the 559 CGAS -STING axis

561 review of emerging infectious diseases requiring urgent research and development efforts

Pathogen recognition and inflammatory signaling in innate immune 564 defenses

Activation of STING requires palmitoylation at the Golgi

Human membrane cofactor protein (CD46) acts as a cellular receptor for 570 measles virus

SARS-CoV-2 infection induces a pro-573 inflammatory cytokine response through cGAS-STING and NF-κB (Microbiology)

Ubiquitination of STING at lysine 224 controls IRF3 576 activation

cGAS and STING: At the intersection of DNA and RNA 578 virus-sensing networks

Tumor cell marker PVRL4 (nectin 4) is an epithelial cell receptor for measles virus

Type I and Type III Interferons -Induction, Signaling, Evasion, 583 and Application to Combat COVID-19

Recent advances in the understanding of Nipah 585 virus immunopathogenesis and anti-viral approaches

In Vivo Ligands of MDA5 and RIG-I in Measles 588 Virus-Infected Cells

Pan-viral specificity of IFN-induced 591 genes reveals new roles for cGAS in innate immunity

Viral stress-inducible genes

Identification and characterization of MAVS, 594 a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3

Crystal structures of STING protein reveal basis for recognition of cyclic di-GMP

Quantitative Proteome Analysis of Atg5 -Deficient Mouse Embryonic Fibroblasts Reveals 600 the Range of the Autophagy-Modulated Basal Cellular Proteome

Nipah Virus: Past Outbreaks and 603 Future Containment

The herpesviral antagonist m152 reveals 606 differential activation of STING -dependent IRF and NF -κB signaling and STING 's dual role 607 during MCMV infection

Dengue virus activates cGAS through the release of mitochondrial 610 DNA

Coronavirus Papain-like Proteases Negatively Regulate Antiviral 613

Innate Immune Response through Disruption of STING-Mediated Signaling

Cyclic GMP-AMP synthase is a cytosolic 615 DNA sensor that activates the type I interferon pathway

STING specifies IRF3 phosphorylation by TBK1 in the 617 cytosolic DNA signaling pathway

SLAM (CDw150) is a cellular receptor for 619 measles virus

STING induces early IFN-β in the liver and constrains 622 myeloid cell-mediated dissemination of murine cytomegalovirus

Zoonotic 624 Potential of Emerging Paramyxoviruses: Knowns and Unknowns

Ligand-induced 626 Ordering of the C-terminal Tail Primes STING for Phosphorylation by TBK1

Small molecule inhibition of cGAS reduces interferon expression in 630 primary macrophages from autoimmune mice

Virus Infection Induces the Assembly of Coordinately Activated Transcription Factors on the IFN-β 633

MyD88: an adapter that 635 recruits IRAK to the IL-1 receptor complex

Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic 638 activity and liquid-phase condensation

Role of adaptor TRIF in the MyD88-independent toll-like 641 receptor signaling pathway

Reconstitution of Virus-mediated Expression of Interferon α Genes in Human Fibroblast 644 Cells by Ectopic Interferon Regulatory Factor-7

Crosstalk between Cytoplasmic RIG-I and STING 646 Sensing Pathways

The Cytosolic DNA Sensor cGAS Forms an Oligomeric Complex with DNA and 649 Undergoes Switch-like Conformational Changes in the Activation Loop

The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor 652 activation

The work was supported by INSERM, LABEX ECOFECT (ANR-11-LABX-0048) of Lyon University, within 458 the program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National 459Research Agency (ANR), by ANR-