key: cord-0908682-6i750bco
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: 2021-05-07
journal: iScience
DOI: 10.1016/j.isci.2021.102519
sha: 5036b6a9a3a5e7dae37e9ea26d567f1260af2d41
doc_id: 908682
cord_uid: 6i750bco

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. Although mice deficient for MyD88, TRIF, and MAVS still moderately control NiV infection when compared with wild-type 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 represents attractive targets to develop countermeasures against severe disease induced by these pathogens.

RNA sensors are insufficient for the effective control of paramyxovirus infection STING adaptor protein is involved in controlling Nipah virus infection in mice cGAS/STING axis is primordial for optimal production of IFN-I against NiV and MeV STING protein can be activated during infections by RNA viruses

The innate immunity represents the first line of host defense against invading pathogens (Akira et al., 2006) . Exogenous motifs associated with viral infections involved in stimulating innate responses include pathogen-derived nucleic acids, DNA, or RNA (Akira et al., 2006; Mogensen, 2009 ). Its ensuing detection activates pattern recognition receptor (PRR)-associated adaptor molecules that are responsible for subsequent expression of type I and III interferons (IFNs) (Park and Iwasaki, 2020) and the induction of IFN-related genes, which are important for the control of virus infection (Borden et al., 2007; Der et al., 1998; Sen and Peters, 2007) . Four major axes, defined by their nodal adaptor, are able to induce strong innate immune responses upon sensing of pathogen-related nucleic acids (Baccala et al., 2009) . Three of them, Toll-like receptor (TLR)-associated adaptor molecules: myeloid differentiation primary response 88 (MyD88) (Wesche et al., 1997) , Toll/interleukin-1 receptor/resistance [TIR] domain-containing adaptor-inducing IFN-b (TRIF) (Yamamoto et al., 2003) , and RIG-I-like receptor (RLR)-associated mitochondrial antiviral signaling protein (MAVS), (Seth et al., 2005) are dedicated to sense DNA and/or RNA. The cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)/stimulator of interferon genes (STING also known as ERIS, MITA, or TMEM173) pathway is the leading sensor for the detection of cytosolic DNA (Ishikawa and Barber, 2008; Sun et al., 2013) . The cGAS/STING axis seems to be involved in the sensing of various different RNA viruses, which may express viral proteins that counteract cGAS/STING activity at different levels. Thus the cGAS/STING pathway may also contribute to the control of RNA viruses (Ni et al., 2018) . Recently, activation of the cGAS/STING pathway has been observed following infection by the positive-strand RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Neufeldt et al., 2020) .

In recent years, members of the Paramyxoviridae family have caused numerous emerging zoonoses and/or epidemics (Thibault et al., 2017) . This viral family contains both old and new human and zoonotic viral pathogens such as measles virus (MeV) and Nipah virus (NiV) . Although MeV has been almost eradicated from most developed countries through vaccination campaigns, the number of cases and deaths significantly increased within the last decade killing more than 100.000 people every year (Ferren et al., 2019) . Moreover, as NiV is the most virulent paramyxovirus with mortality rates between 40% and 100% during epidemics and Our previous work evaluating innate sensors involved in the protection of mice following NiV infection suggests control of the virus through the activation of MyD88 and MAVS pathways, whereas TRIF seems dispensable (Iampietro et al., 2020) . Although, both MyD88 and MAVS are important to produce high levels of type-I IFNs (IFN-I), double knockout (KO) mice still exhibit some resistance against NiV infection. This is in contrast to the interferon-a/b receptor (IFNAR) KO mice that lack any IFN-I-related responses and are unable to control NiV infection (Dhondt et al., 2013; Iampietro et al., 2020) . We thus hypothesized that the cGAS/STING axis of the innate immunity could also contribute to the control of NiV infection.

Once activated in the cytoplasm of infected cells, cGAS uses adenosine triphosphate (ATP) and guanosine triphosphate (GTP) as substrates for cyclization into cGAMP. cGAMP triggers STING and further results in IFN-I expression (Ishikawa and Barber, 2008; Sun et al., 2013) . Successive conformational changes ensure the activation of cGAS and STING in cascade. Briefly, cGAS in ''resting'' state is a monomer containing a conserved zinc-ion-binding domain as DNA-binding module (Civril et al., 2013; Kranzusch et al., 2014) . Upon DNA binding in the cytoplasm, cGAS dimerizes and further oligomerizes to induce optimal cGAMP production (Gao et al., 2013; Zhang et al., 2014) . Thereafter, cGAMP binds to STING in its ligand-binding domain and induces inward rotations leading to dimerization and later oligomerization of STING (Cong et al., 2019; Ergun et al., 2019; Shang et al., 2012) . In addition, upon binding of cGAMP, STING exits the endoplasmic reticulum to the Golgi apparatus. There, it transduces a downstream signaling pathway by recruiting TANK-binding kinase 1 (TBK1) and IRF-3 (but not IRF-7) transactivator and activating the nuclear factor (NF)-kB pathway that results in the activation of type I IFN and cytokine genes (Dobbs et al., 2015; Gui et al., 2019; Liu et al., 2015) . The recruitment of IRF-3 relies on the phosphorylation of STING on its Ser366 (Ser365 in murine) by TBK1 (Tsuchiya et al., 2016) . In addition, the E3 ubiquitin ligases TRIM32 and TRIM56 promote the non-degradative K-63-linked ubiquitination of Lys224 of STING to trigger a cytokine response (Ni et al., 2017; Tanaka and Chen, 2012) . As a third type of post-translational modifications (PTMs) STING palmitoylation governs its trafficking to the Golgi (Mukai et al., 2016) .

We report here that both cGAS and STING are required for mounting an efficient innate immune response upon NiV and MeV infection. In infected cells, STING is phosphorylated on S366 (S365 in mouse) and is K63 linked ubiquitinated, confirming the presence of its activated form at later time points following RNA virus infection, similarly to what has been observed by others after infection with DNA virus (Chiang and Gack, 2017).

Our previous study revealed a complementary role of MyD88 and MAVS in the partial containment of NiV, suggesting that the complete resistance of mice against NiV involves an additional activation pathway of the IFN-I response (Iampietro et al., 2020) . The cGAS/STING axis has recently emerged as critical in the cross-talk between innate sensing of cytosolic DNA and RNA viruses (Ni et al., 2018) . We therefore analyzed the susceptibility of mice bearing gene deletions in both single TLR and IL-1R (MyD88 KO) pathway, or in combination with TRIF (MyD88/TRIF KO), MAVS (MyD88/TRIF/MAVS KO), and STING (MyD88/TRIF/MAVS/ STING KO) signaling platforms (Figures 1 and S1 ). The animals were infected intraperitoneally and monitored during 24 days for clinical signs and/or death. Infected wild-type (WT), MyD88 KO, and MyD88/TRIF KO mice did not manifest any clinical sign of disease, whereas triple MyD88/TRIF/MAVS KO and quadruple MyD88/TRIF/MAVS/STING KO mice exhibited symptoms of neurological disorders similar to those observed in IFNAR KO mice ( Figure 1A ). Moreover, whereas 60% triple KO mice survived NiV challenge, all mice bearing the quadruple deficiency succumbed by day 11 post-infection, indicating a crucial and non-redundant role for STING in the protection of mice ( Figure 1A ). NiV nucleoprotein (NiV-N) protein levels in the brain of autopsied animals deficient for MyD88/TRIF/MAVS and MyD88/TRIF/MAVS/STING were comparable to those observed in the brain of IFNAR KO mice at the time of death ( Figure 1B ). Furthermore, whereas analysis of NiV load in murine brain determined equivalent NiV-N RNA levels within these three deficient murine models ( Figure 1C ), evaluation in the spleen showed higher viral loads in mice bearing the quadruple deficiency compared with the triple KO mice ( Figure S1A ). In parallel, the lower production of IFNb (Figures 1D and S1B) and less efficient expression of IFNa (Figures 1E and S1C) in the brains and spleens of MyD88/TRIF/MAVS KO and MyD88/TRIF/MAVS/STING KO mice compared with WT were

To further evaluate the role of STING during NiV infection in mice, we analyzed the impact of the gene deletion of various nodal adaptors on the in vitro NiV replication in primary murine embryonic fibroblasts (pMEFs). The pMEFs were generated from mice bearing the corresponding deleted genes as described previously (Brune et al., 2001) . They were infected with rNiV-eGFP to allow imaging of viral infection by fluorescent microscopy. In agreement with above in vivo observations, MyD88 KO and MyD88/TRIF KO pMEFs were able to control NiV replication as well as WT pMEFs, with only few observed infected cells. In contrast, NiV rapidly spread within the culture of MyD88/TRIF/MAVS KO and MyD88/TRIF/MAVS/STING KO pMEFs, although not as extensively as in IFNAR KO cells ( Figure 2A) . Moreover, single STING KO pMEFs were unable to control viral spread, highlighting a major role of STING into mouse innate defense against NiV infection ( Figure 2A ). The poor permissiveness of WT, MyD88 KO, and MyD88/TRIF KO cells was confirmed by a low amount of viral RNA in these cells ( Figure 2B ) and limited release of viral RNA in the supernatant ( Figure 2C ). These three cell types exhibited comparable amounts of IFNb mRNA ( Figure 2D ), whereas this mRNA was nearly undetectable in non-infected WT cells. However, contrary to WT pMEFs, which exhibit a (B) Immunohistochemistry of murine brains following NiV infection. Brains of WT mouse, IFNAR KO mouse, MyD88/TRIF/ MAVS KO, and MyD88/TRIF/MAVS/STING KO were collected on days 2, 6, 13, and 11, respectively. Scale bars, 100 mm. (C-E) Expression of NiV nucleoprotein (NiV-N) in the brain of NiV-infected mice, harvested on the day of death or euthanized at the end of protocol for different genotypes, was determined by RT-qPCR. Data are represented as mean G SEM. Analysis of IFNb and IFNa expression by RT-qPCR in organs harvested 2-13 days after infection. All samples were analyzed using one-way analysis of variance, followed by the Tukey multiple comparisons test, *p < 0.05 compared with WT condition. Figure 2B ). This intermediate phenotype of STING KO cells was associated with no significant IFNb mRNA induction, but high levels of IFNa mRNA ( Figures 2D and 2E ). Altogether, these results indicate that in murine pMEFs (1) the control of NiV infection is mediated mainly by the IFNb rather than the IFNa response;

(2) STING is necessary for the induction of IFNb, but not IFNa; and (3) MAVS, in combination with MyD88 or not, is important for the activation of IFN-I genes confirming our previous observations (Iampietro et al., 2020) .

The role of STING and its upstream activator cGAS in the innate response to NiV was further explored in the human monocytic THP-1 cell line that was either WT or deficient in cGAS (cGAS KO) or STING (STING KO). WT THP-1 cells have the advantage of exhibiting a limited permissiveness to infections with both NiV and the WT-derived recombinant MeV strain rMeV-EdmH-eGFP, another member of the Paramyxoviridae family belonging to the closely related Morbillivirus genus. This virus expresses the Edmonston vaccinederived H glycoprotein allowing the use of ubiquitously expressed CD46 as cellular receptor (Naniche et al., 1993) and hence entry into THP-1 cells that do not or minimally express the known physiological Figure 3C ) and cell supernatants ( Figure 3D ). Correlatively, and in agreement with observations made in pMEFs infected with NiV, the higher permissiveness of cGAS KO and STING KO THP-1 cells was associated with the abolition of IFNb and/or IFNa mRNA accumulations ( Figures 3E  and 3F ). Comparable results were obtained after infection by MeV, although a reduced but significant accumulation of both IFNb and IFNa mRNA was still observed in cGAS KO and STING KO cells ( Figures 3G-3J ).

As an alternative and complementary approach, we analyzed the paramyxoviral propagation within WT THP-1 cells treated with selective inhibitors of cGAS and STING, namely, RU-521 and H151, respectively. RU.521 is a competitive inhibitor of ATP and GTP substrates for binding to the cGAS catalytic pocket that prevents their cyclization into cGAMP (Vincent et al., 2017; Xie et al., 2019) . H151 covalently binds to STING Cys91 and inhibits palmitoylation of STING and consequently the activation of IFN-I production (Haag et al., 2018) . Addition of RU-521 or H151 1 h before infection resulted in an improved propagation of both NiV and MeV as evidenced by fluorescence microscopy (Figures 3K and 3L ) and flow cytometry ( Figures S2C-S2E) .

Thus, the cGAS/STING axis appears to play a critical role in human cells to control paramyxovirus infections by allowing expression of IFNb and to a lower extent of IFNa.

The increased viral infection observed upon abolition of either cGAS or STING suggested that paramyxoviruses could activate STING. This was investigated by analyzing specific phosphorylation and/or ubiquitination of activated STING in pMEFs, THP-1, and human pulmonary microvascular endothelial cells (HPMEC) as representative of primary targets of NiV infection in humans (Figures 4 and S3) . A time course follow-up of the activation of cGAS/STING axis induced by viral infection was performed at 6, 24, and 48 h post-infection (hpi) by western blot using antibodies against STING-S366 P (Chiang and Gack, 2017). The expression level of STING remained unchanged throughout the 48h observation, whereas moderate amounts of STING-S366 P became detectable at 24 hpi and further increased at 48 hpi after NiV or MeV infection of HPMECs (Figures 4A and 4B ) and THP-1 cells ( Figures 4C and S3A) . Importantly, STING-S366 P was not detected in NiV-or MeV-infected cGAS KO THP-1 cells at 24 and 48 hpi, and as expected in STING KO THP-1 cells ( Figures 4C and S3A) . Accordingly, STING-S366 P was also not detected in THP-1 cells infected and treated with either RU-521 or H151 ( Figure S3A ).

Accordingly, whereas uninfected WT pMEFs, NiV-infected STING KO, and MyD88/TRIF/MAVS/STING KO cells did not elicit any mouse STING-S365 P band (Tsuchiya et al., 2016) , NiV-infected WT and MyD88/TRIF/MAVS KO pMEFs displayed activation of STING protein ( Figure S3B ). Importantly, the detection of the STING-S365 P band in MyD88/TRIF/MAVS KO pMEFs indicates that NiV infection activates the STING signaling pathway independently from the TLR-MyD88/TRIF and RLR/MAVS pathways of innate immunity. As K63-linked ubiquitination (Ub-K63) is another hallmark of STING activation, mainly associated with the activation of NF-kB (Chiang and Gack, 2017), STING ubiquitination was also evaluated in HPMECs infected for 48 h with NiV or MeV. Immunoprecipitated STING using anti-STING antibodies was found to be labeled by either anti-STING-S366P and anti-Ub-K63 antibodies through single phosphorylation or ubiquitination ( Figures 4D and 4E ) or K63 polyubiquitinations ( Figure S3C ) as described previously (Prabakaran et al., 2018; Zhang et al., 2012) . As an apoptotic environment could activate STING in bystander cells (Ahn et al., 2012) , a potential effect of virus-induced cell death in infected cultures was evaluated by analyzing caspase 3 activity. The levels of cleaved caspase 3 were minimally increased between uninfected and NiV-or MeV-infected cells, indicating that cell death had likely a minor impact on STING activation until 48 h post-infection ( Figures 4A-4C , S3A, S3D, and S3E).

We conclude that negative-strand RNA paramyxoviruses activate the cGAS/STING axis to trigger the innate immune responses.

Although its role in response to DNA virus infection has been well deciphered (Eaglesham and Kranzusch, 2020) , the role of cGAS/STING axis against RNA viruses such as paramyxoviruses is poorly understood. While cGAS was reported to interact with dsRNA, no production of cGAMP was detected, thus cGAS could not canonically activate STING directly (Civril et al., 2013) . However, previous studies have shown interactions between RNA viruses and cGAS/STING in (1) their control by innate immunity or (2) iScience Article disrupt the cGAS/STING pathway, reflecting a likely role of STING in host response (Aguirre et al., 2017; Franz et al., 2018; Ishikawa et al., 2009; Schoggins et al., 2014) .

Paramyxoviruses replicate within the cytoplasm using their own RNA-dependent RNA polymerase, i.e., their replicative cycle does not rely on a DNA intermediate step (Gerlier and Lyles, 2011) . As cGAS is exclusively activated upon binding to dsDNA (Kranzusch et al., 2013; Sun et al., 2013) or DNA/RNA hybrid (Mankan et al., 2014) , we have to speculate about the identity of the cGAS agonist during infection by paramyxoviruses. Our experiments suggest that apoptosis is not involved in the mechanism of activation of the cGAS/STING pathway as caspase 3 activity remains undetected following NiV and MeV infections. As apoptotic caspases have been described to suppress mitochondrial-induced STING-mediated IFN-I production (White et al., 2014) , we cannot exclude additional unknown mechanisms leading to the release of the mitochondrial DNA at some stage of viral infection, as evidenced after infection with dengue virus, a positive-stranded RNA virus (Aguirre et al., 2017; Sun et al., 2017) . Furthermore, from observations made with Sendai virus, another paramyxovirus, possible virus-induced necroptosis appears unlikely to provoke STING activation (Schock et al., 2017) . Finally, the role of viral-induced formation of syncytia remains to be explored because mechanical and bacterial induced cell-cell fusion has been reported to activate cGAS/ STING pathway (Ku et al., 2020) . Although the coexpression of MeV H and F glycoproteins did not result in the activation of IFN-I per se (Herschke et al., 2007) , we cannot exclude a contribution of cell-cell fusion as a potential amplifier of cGAS/STING activation.

Collectively, the obtained data fit with a model where the infection by NiV or MeV activates cGAS/STING pathway. This activation results in the induction of the IFNb and possibly the IFNa genes (see below) that meyer et al., 2019) . Notably, the comparison of the triple and quadruple KO pMEFs indicate that cGAS/ STING pathway strongly reinforces (or even conditions) the activation of type I IFN responses via the TLR8/MyD88 and/or the RLR/MAVS pathways by paramyxoviruses, including MeV (Ikegame et al., 2010; Runge et al., 2014; Seth et al., 2005) .

STING can bind to RIG-I and MAVS (Sun et al., 2012) and is thought to potentiate the RLR/MAVS signaling leading to the activation of type I IFN (Ishikawa et al., 2009; Zevini et al., 2017; Zhong et al., 2008) . The present data fit with this model. The defect of cGAS/STING pathway strongly facilitates the replication of both NiV and MeV as expected from the observed loss in activating IFN-I response. We cannot exclude that this facilitation results also from the concomitant disappearance of a STING-dependent inhibition of the translation of viral proteins as recently reported (Franz et al., 2018) . However, they reported that the absence of STING modestly affects IFN response by negative-stranded RNA viruses infections compared with the present work.

Upon infection of MeV and/or NiV in mouse and/or human cells, the absence of cGAS/STING pathway affects IFNb response and to a lower and more variable extent IFNa response. Owing to cell-dependent ability to produce IFNa, a different outcome may reflect higher expression of IRF-7 in pMEFs (Sharma et al., 2019) compared with THP-1 cells (Green et al., 2020) . Indeed, whereas activation of the IFNb gene optimally relies on IRF3 homodimers or IRF3/IRF7 heterodimers and NF-kB (Honda et al., 2005; Wathelet et al., 1998) , the one for IFNa genes relies mostly relies on IRF7 homodimers (Yeow et al., 2000) . Interestingly, STING mostly targets IRF-3 (Zhong et al., 2008) and NF-kB (Stempel et al., 2019) and consequently preferentially favors IFNb.

Our study demonstrates that STING is activated against RNA viruses as also recently reported with SARS-CoV-2 (Neufeldt et al., 2020) and highlights that STING is modified and activated through S366 phosphorylation and/or K-63-linked ubiquitination during both NiV and MeV infections. Additional studies will uncover the occurrence of potential additional PTMs, STING subcellular location, and the source of the cognate DNA that activate cGAS during RNA virus infection.

In conclusion, cGAS/STING activation occurs during paramyxovirus infections, both in vitro and in vivo. This highlights an undefined aspect of the immune regulation against negative-strand viruses and reveals cGAS/STING as potential targets in the development of novel antiviral strategies.

Although mice are not the natural host of NiV and MeV infections, they provide numerous genetic tools to study their respective innate immune control. Further validation of the obtained results in human cell types or in a nonhuman primate model may allow overcoming the limitation of the murine model. Additional analysis of the role of cGAS/STING axis in the production of inflammatory cytokines will have to be performed. Furthermore, as the study of NiV requires the strict application of the BSL4 biosafety rules from the P4 Jean Mé rieux laboratory in handling and inactivation of the samples, resulting in over-dilutions of the biological material in our co-immunoprecipitation and western blot experiments, there is a certain limit in the accurate detection of specific cellular or viral processes. Additional development of methodology and adjustments may be needed to study further the mechanisms, and the reproduction of similar results in MeV infection, requiring only BSL2 conditions, could overcome this limitation.

Detailed methods are provided in the online version of this paper and include the following: 

The authors declare no competing interests. Several lines of transgenic mice, all in C57BL/6 genetic background, were used: wild type (WT) C57BL/6J mice and following knockout (KO) models: mice deleted for IFN-I receptor (IFNAR-KO) (Muller et al., 1994) , TLR adaptor protein MyD88 (MyD88-KO) (Adachi et al., 1998) , and mice crossed to bear several deletions, including MyD88/TRIF-KO (Waibler et al., 2007) , MyD88/TRIF/MAVS-KO (Spanier et al., 2014) and MyD88/ TRIF/MAVS/STING KO (Tegtmeyer et al., 2019) .

Primary murine embryonic fibroblasts (pMEFs) were isolated from murine embryos obtained from female pregnant mice 13 days after conception (Brune et al., 2001) and cultured in Dulbecco's modified Eagle's medium (DMEM) GlutaMAX supplemented with 10% heat-inactivated fetal bovine serum (FBS), 0.2% 2-mercaptoethanol, 1% HEPES, 1% nonessential amino acids, 1% sodium pyruvate, and 2% penicillin-streptomycin mix. For infections, pMEFs were plated in 12-well plates at 2.10 5 cells per well and cultured with rNiV-eGFP at a multiplicity of infection (MOI) of 0.3 plaque-forming units (PFUs)/cell for 1 h at 37 C. Virus-containing medium was then removed, and cells were washed once with 13 phosphate-buffered saline. Finally, fresh DMEM was added to cells that were incubated for 24 h at 37 C. Human male monocytic THP-1 cell lines WT, cGAS KO or STING KO were obtained (Mankan et al., 2014) and cultured in RPMI 1640 GlutaMAX supplemented with 10% heat-inactivated FBS, 1% HEPES and 2% of penicillin-streptomycin mix. Human pulmonary microvascular endothelial cells (HPMEC) (Krump-Konvalinkova et al., 2001) were from a male donor and cultured in Endothelial Cell Growth Medium, in flasks coated with 0,1% bovine gelatine in PBS. For infection, HPMECs were plated in 12-well plates at 2.10 5 cells per well and cultured with rNiV-eGFP or rMeV-edmH-eGFP at a MOI of 1 PFUs/cell for 24 or 48 hours at 37 C. All cell types were incubated at 37 C with 5% CO2 and were tested negative for Mycoplasma spp.

Animals were handled in strict accordance with good animal practice as defined by the French national charter on the ethics of animal experimentation and all efforts were made to minimize suffering. Animal work was approved by the Regional ethical committee and French Ministry of High Education and Research and experiments were performed in the INSERM Jean Mé rieux BSL-4 laboratory in Lyon, France (French Animal regulation committee N 00962.01).

Groups (male and female) of 5-10 mice from each line, 4-6 weeks of age, were anesthetized with isoflurane and infected intraperitoneally with 10 6 PFUs of Nipah virus Malaysia strain, contained in a 200 mL volume. Animals were monitored for 24 days after infection and manipulated in accordance to good experimental practice and approved by the regional ethics committee CECCAPP ( Drugs H151, a specific inhibitor for STING and RU.521, a specific inhibitor for cGAS were added 1 h previous infection of THP-1 cells at 10 mM and 10 mg/ml, respectively, selected according to the previously published results (Chang et al., 2020; Hayden et al., 2020) . Then, cells were infected with rNiV-eGFP or rMeV-edmH-eGFP and incubated for 24 or 48 h at 37 C with 5% CO2.

NiV Malaysia (isolate UMMC1; GeneBak AY029767, recombinant NiV (rNiv)-enhanced green fluorescent protein (eGFP) (Yoneda et al., 2006) and recombinant MV IC323 vaccine strain, expressing Edmonston H and eGFP (Hashimoto et al., 2002) , kindly provided by Dr Y. Yanagi (Kyushu University, Japan) and were prepared by infecting Vero-E6 cells, in the INSERM Jean Mé rieux biosafety level 4 (BSL-4) and BSL-2 laboratories at CIRI in Lyon, France respectively. iScience Article Immunofluorescence For PMEFs and THP-1 infections, 3310 5 and 5310 5 cells were seeded in 12-well plates, respectively, before being infected with rNiV-eGFP and rMeV-edmH-eGFP at a MOI of 0.3 or 0.1 and cultured for 24 or 48 h at 37 C with 5% CO2. Then, cells were evaluated for eGFP expression using a Zeiss Axiovert 100M microscope in the BSL-4 or a NIKON Eclipse Ts2R in the BSL-2, and photographs were taken 24 and 48 h after infection and treated using ImageJ software version Java 1.8.0_112.

THP-1 cells were seeded in 12-well plates at 3310 5 and 5310 5 per well before being infected with 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. Then, cells were washed, reconstituted in PBS 1X and evaluated for eGFP expression using a Gallios flow cytometer in the BSL-4 or a 4L Fortessa flow cytometer. Analyses were performed 24 and 48 h after infection.

The results are presented in the form of histograms which represent the mean eGFP positive cells for each conditions and error bars represent the standard errors (SE) for n=3 experimental replicates. The different conditions were compared to the control (WT+). Statistical significance was assessed by a one-way ANOVA, followed by a Tukey's multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 (threshold of significance of 5%).

The results are presented in the form of histograms, which represent the mean of copies of mRNA for a gene for each condition and error bars represent the standard errors (SE) for n=3 experimental replicates. The different conditions were compared to the control (WT+). Statistical significance was assessed by a one-way ANOVA, followed by a Tukey's multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 (threshold of significance of 5%).

Densitometric analyses of cleaved caspase 3 immunoblots from three independent experiments were performed using the VersaDoc Imaging System (Bio-Rad) and analyzed with ImageJ 1.52p Fiji package software (https://imagej.net/Fiji). GAPDH expression was used for normalization. The results are presented in the form of histograms and error bars represent the standard errors (SE) for n=3 independent experiments.

iScience 24, 102519, June 25, 2021 iScience Article

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Ligand-induced ordering of the C-terminal tail primes STING for phosphorylation by TBK1

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

Modified vaccinia virus Ankara induces Toll-like receptor-independent type I interferon responses

Virus infection induces the assembly of coordinately activated transcription factors on the IFN-b enhancer in vivo

Fatal measles virus infection prevented by brainpenetrant fusion inhibitors

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

Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production

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

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

Reconstitution of virus-mediated expression of interferon a genes in human fibroblast cells by ectopic interferon regulatory factor-7

Establishment of a Nipah virus rescue system

Crosstalk between cytoplasmic RIG-I and STING sensing pathways

TRIM32 protein modulates type I interferon induction and cellular antiviral response by targeting MITA/STING protein for K63-linked ubiquitination

The cytosolic DNA sensor cGAS forms an oligomeric complex with DNA and undergoes switch-like conformational changes in the activation loop

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

Slides were deparaffinated and rehydrated in three Xylene baths for 5 min each, followed by two 100% alcohol baths for 5 min, and then succeeded with multiple baths using decreasing level of alcohol for 3 min each. After deparaffination, slides were put in a sodium citrate solution in a boiling water bath for 20 min for heat-induced epitope retrieval and washed 3 times in PBS for 3 min afterwards. Activity of endogenous peroxydase was blocked using a H2O2 0.3% solution. Blocking of non-specific epitopes is done using PBS-2.5% decomplemented Normal Horse Serum + 0.15% Triton X-100 for 30 min. Then, primary rabbit anti-NiV N antibody was used at 1/10000 dilution and incubated overnight at 4 C in the blocking buffer. For secondary antibody and revealing steps, ImmPress system (anti-rabbit ig/peroxydase) was used

Supernatants were incubated with a rabbit anti-STING antibody for 2 h at 4 C. Then, protein A/G agarose beads were added to the mix overnight at 4 C. Beads were then washed three times in washing buffer (RIPA buffer, supplemented with a cocktail of protease-phosphatase inhibitor), and proteins were eluted in 100 ml of elution buffer (Reducing agent 10X, Laemmli 4X, RIPA buffer, supplemented with a cocktail of protease-phosphatase inhibitors) for 15 min at 96 C. Then the eluate and a sample of input of the cell

TBS-T) + 5% milk for 1 h and then incubated overnight with primary antibodies, mouse anti-GAPDH, rabbit anti-STING, rabbit anti-human S366 p-STING, rabbit anti-mouse S365 p-STING, rabbit anti-Caspase 3, rabbit anti-cleaved Caspase 3 and rabbit anti-Ub-K63 antibodies, diluted 1:1000 in TBS-T + 0.2% milk. Membranes were then washed 3 times using TBS-T and incubated on an additional 1 h with horseradish peroxydase conjugated anti-mouse or anti-rabbit IgG antibodies (diluted 1:5000 in TBS-T + 5% milk). Membranes were then washed 5 times in TBS-T, incubated in Super Signal West Dura to stain cell lysates or in Super Signal West Femto reagent to stain bead eluates

2009) were used for all validations and calculations. To validate the efficacy of our primers, a PCR on a positive control (cDNA from corresponding stimulated cells) has been performed before dosing several dilutions of our PCR products using the Denovix DS-11-FX spectrophotometer to evaluate the quantity of DNA in each dilution. Then, the PCR products were 10-fold serially diluted with a range from 10 -3 to 10 -12 to validate efficacy, specificity and sensitivity of each couple of primers used for qPCR. Moreover, the exact number of copies in each qPCR reaction was obtained by calculating the ''N0 Samples = N0 Standard * Efficacy -DCt '' before being converted using molecular weight of each targeted gene and Avogadro number. Results obtained were either kept as ''copies/ml'' for supernatants or converted to ''copies/mg of RNA'' for cell lysates. Finally, normalization was performed by dividing obtained numbers of RNA copies of the target genes with the deviation of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as house-keeping gene. Specific set of primers were designed and validated for the detection of human hGAPDH

This study did not generate new unique reagents.

No new data/code was generated in this study