In our previous study, we occasionally observed increased RTN3 expression upon vesicular stomatitis virus (VSV) infection, leading us to investigate why RNA viral infection increases RTN3 expression. Wild-type (WT) HEK293T cells were infected with enhanced GFP-tagged vesicular stomatitis virus (VSV-eGFP) or treated with the dsRNA synthetic analog poly(I:C) at different time points. We observed that RTN3 protein levels were dramatically increased during VSV-eGFP infection (Figure 1A, top) or under poly(I:C) stimulation (Figure 1A, bottom), with both conditions showing the same pattern of RTN3 mRNA upregulation (Figure 1B and C, top) accompanied by increased TNF mRNA levels that were used to indicate the effectiveness of VSV-eGFP or poly(I:C) treatment toward inflammatory induction in stimulated cells (Figure 1B and C, bottom). Interestingly, we observed that both TNF-α and IFN-β could upregulate RTN3 expression at both the protein (Figure 1D) and mRNA levels (Figure 1E). In addition, human PBMCs from healthy donors infected with VSV exhibited an accordant upregulation of RTN3 expression (Figure 1F). These results suggest that RTN3 levels are increased during RNA viral infection.

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Since RTN3 is conserved and ubiquitously expressed in various tissues, we further assessed the pattern of RTN3 upregulation in multiple cell lines, including A549 and THP-1 cells. As expected, the protein (Figure 1—figure supplement 1A and B) and mRNA (Figure 1—figure supplement 1C) levels of RTN3 were both significantly increased by poly(I:C) stimulation or VSV-eGFP infection in A549 cells, with a similar phenomenon observed in THP-1 cells (Figure 1—figure supplement 1D and E). The upregulation of RTN3 expression was greatly attenuated in CHOP knockout cells compared with wild-type cells under VSV infection (Figure 1—figure supplement 1F). Notably, confocal microscopy analysis of HeLa cells transfected with mCherry-tagged RTN3 followed by challenge with poly(I:C) showed that RTN3 was localized on the endoplasmic reticulum (ER), while under poly(I:C) stimulation, RTN3 proteins converged and formed many aggregated bodies of varying sizes (Figure 1—figure supplement 1G). The coimmunoprecipitation and immunoblot analysis with GFP-tagged RTN3 and HA-tagged RTN3 further proved the existence of this aggregated body (Figure 1—figure supplement 1H). It was further confirmed that RTN3 was colocalized with calnexin on the ER as well as their tight colocalization within the aggregation under poly(I:C) stimulation, which substantiated that RTN3 aggregation was localized on the endoplasmic reticulum (ER) (Figure 1—figure supplement 1I). Taken together, these data suggest that RTN3 expression is induced by RNA viral infection and its observed upregulation and self-aggregation indicate that RTN3 may be involved in regulating innate immune and inflammatory responses.

To investigate the function of RTN3 in regulating innate immune responses, we performed luciferase assays using ISRE-luc, IFNβ-Luc, and NF-κB-Luc reporters and observed that RTN3 markedly inhibited all activities induced by RIG-I overexpression (Figure 2A). In addition, poly(I:C)- or Sendai virus (SeV)-stimulated ISRE-luc activities were both restrained by RTN3 overexpression in a dose-dependent manner (Figure 2B). However, RTN3 overexpression had a slightly negative effect on MDA5-induced ISRE-luc activities (Figure 2—figure supplement 1A) and barely inhibited TLR3-induced ISRE-luc activities (Figure 2—figure supplement 1B). Constant cGAS-STING or IRF3-induced ISRE-luc activities were observed upon RTN3 overexpression, excluding the possibility that RTN3 inhibits antiviral responses by targeting cGAS-STING-IRF3-axis (Liu et al., 2012; Figure 2—figure supplement 1C and D). These data reveal that RTN3 primarily suppresses RIG-I-mediated antiviral immune responses.

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To further demonstrate the attenuation of antiviral activities by RTN3, HEK293T cells were transfected with an RTN3-encoding plasmid and subsequently infected with VSV-eGFP. Fluorescence microscopy and flow cytometry results showed that RTN3 overexpression rendered the cells highly susceptible to viral infection and augmented virus amplification compared to the empty vector-transfected cells, which displayed a less sensitive phenotype (Figure 2C and D), and the MTS assay performed with control cells and RTN3-overexpressing HEK293T cells for over 36 hr showed that the cell viability was not affected by RTN3 overexpression (Figure 2—figure supplement 1E). A consistent result was also observed in long-term puromycin-selected, stable HA-tagged RTN3 vs. HA-tagged empty vector-expressing THP-1 cells (THP-1^HA-RTN3 vs. THP-1^HA-Ev) (Figure 2—figure supplement 1F), which excluded the possibility that ectopic RTN3 expression may transiently overload the endoplasmic reticulum (ER) and impair the translation of antiviral proteins and cell survival.

To further assess the role of RTN3 in attenuating antiviral activities, we transfected HEK293T cells with RTN3 or empty vector and followed by infection with VSV-eGFP. The phosphorylation of IKKα/β, TBK1, p65, and IRF3 was dramatically decreased in RTN3-overexpressing cells compared to that observed in the control groups (Figure 2E, left). Quantitative analysis by gray intensity scanning of immunoblot bands further demonstrated the significant decreases in the phosphorylation of the two kinases and two transcription factors (Figure 2E, right).

Accordingly, we investigated whether RTN3 suppresses type I IFN- and interferon-stimulated gene (ISG) expression. RT-PCR results showed that RTN3-overexpressing cells exhibited a deficiency in IFNB1, IFIT2, IFIT1, TNF and the proinflammatory cytokines CCL20 and IL6 during VSV infection (Figure 2F). Similarly, the mRNA levels of IFNB1, IFIT2, and IFIT1 were downregulated to a great extent in THP-1^HA-RTN3 cells compared to THP-1^HA-Ev cells after VSV infection (Figure 2—figure supplement 1G). Because the amounts of the proinflammatory cytokines CCL20 and IL6 were both markedly decreased by RTN3 overexpression under dsRNA stimulation [VSV or poly(I:C)], we assessed whether any other inflammatory cytokines or related genes were altered by RTN3 overexpression. To this end, we performed RT-PCR array analysis and observed that many inflammatory factors and genes were downregulated during RTN3 overexpression (Figure 2—figure supplement 1H). These results suggest that RTN3 overexpression preferentially suppresses RIG-I-mediated antiviral immune and inflammatory responses.

To further elucidate the physiological function of RTN3 in antiviral responses, we depleted its expression by short hairpin RNAs (shRNAs) in human healthy donor PBMCs (Figure 3A), in which RTN3 normally undergoes upregulation during viral infection (Figure 1F). Within our experience, complete or vast obliteration of the expression of RTN3 by knockout or knockdown leads to cell death; hence, RTN3 expression were moderately depleted with transient shRNA transduction (Figure 3A). PBMCs were infected with control or RTN3 shRNA-encoding lentivirus for 12 hr, and the infected cells were then settled for the following 24 hr and subsequently infected with VSV-eGFP. Consistent with the decreased phosphorylation levels of TBK1, p65 and IRF3 in RTN3 overexpressing cells, RTN3 depletion significantly augmented the phosphorylation and activation of these critical kinases and transcription factors upon RNA viral infection (Figure 3B). Further RT-PCR analyses determined that the levels of IFNB1, IFIT2, IFIT1, TNF and the proinflammatory cytokines CCL20 and IL6 were enhanced by RTN3 knockdown during VSV infection (Figure 3C), while the amplification of VSV was compromised (Figure 3D). Collectively, depressed RTN3 expression facilitates antiviral innate immune and inflammatory responses upon viral infection.

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Next, we investigated the potential mechanism for the RTN3-mediated inhibition of antiviral responses. We noted that RTN3 may function as a tripartite motif-containing protein 25 (TRIM25)-binding protein through mass spectrometry analysis (Choudhury et al., 2017), and TRIM25 is a crucial E3 ligase for the K63-linked polyubiquitination modification of RIG-I to promote its activation upon RNA viral infection (Gack et al., 2007). To elucidate the relationship between RTN3 and TRIM25 or RIG-I, HEK293T cells were transfected with GFP-tagged RTN3 and Flag-tagged TRIM25 followed by stimulation with or without VSV and then subjected to coimmunoprecipitation and immunoblot analysis. The results showed that RTN3 interacted with TRIM25 under basal conditions and was slightly strengthened upon viral infection (Figure 4A, Figure 4—figure supplement 1A). Considering that the RTN3 overexpression leads to its aggregation in the ER and may cause nonspecific interactions (such as structural enfolding), we used GFP-TRIM25 to pull down endogenous RTN3 and confirmed its interaction with TRIM25 and that this interaction was strengthened by VSV infection (Figure 4—figure supplement 1B). Since TRIM25 directly targets RIG-I, the coimmunoprecipitation assays were performed in RIG1 knockout HEK293T cells and the results showed that the TRIM25-RTN3 interaction did not require RIG-I, thus we concluded that RTN3 binds to TRIM25 independent of RIG-I and excluded the possibility that RTN3 may interact with TRIM25 through the interaction between TRIM25 and RIG-I (Figure 4B). Besides, we speculated that RTN3 may also interact with RIG-I. Therefore, we performed coimmunoprecipitation assays and observed an interaction between TRIM25 and RIG-I (Figure 4C) as well as a similar VSV-promoting pattern as that detected for RTN3 interacting with TRIM25 (Figure 4—figure supplement 1C). Furthermore, confocal microscopy results showed the colocalization of RTN3 with RIG-I or TRIM25 on the extended ER framework (Figure 4D and E), indicating that RTN3 aggregation may provide a scaffold for TRIM25 oligomerization or the RIG-I signalosome complex. Immunoprecipitation using anti-RTN3 antibody in human PBMCs infected with VSV demonstrated endogenous interaction between RTN3 and TRIM25/RIG-I (Figure 4F). Collectively, these results suggested that RTN3 interacts with both RIG-I and TRIM25 and that this interaction could be enhanced by RNA viral infection.

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We next assessed how RTN3 suppresses RIG-I-mediated antiviral responses through its interaction with TRIM25. Interestingly, the protein levels of RIG-I and TRIM25 were not decreased by RTN3 overexpression in HEK293T cells (Figure 4—figure supplement 1D), excluding the possibility that RTN3 may target RIG-I or TRIM25 for their degradation. We also assessed whether the interaction between RIG-I and TRIM25 was inhibited or promoted by RTN3 overexpression. To this end, HEK293T cells were cotransfected with Flag-RIG-I- and GFP-TRIM25-expressing constructs or GFP-tagged empty vector (GFP-Ev) together with increasing amounts of RTN3-expressing plasmid, which was followed by VSV infection. Unfortunately, the interaction between RIG-I and TRIM25 was completely unchanged (Figure 4G), indicating that RTN3 negatively regulates RIG-I-mediated antiviral responses via other mechanisms.

Subsequently, we investigated the polyubiquitination of RIG-I upon VSV infection in the absence or presence of RTN3 overexpression. As shown in Figure 4H and Figure 4—figure supplement 1E, the K63-linked ubiquitination level of RIG-I was greatly decreased by RTN3 overexpression, potentially suppressing RIG-I signalosome formation and downstream signal transduction. In contrast, RTN3 depletion enhanced the K63-linked polyubiquitination level of RIG-I (Figure 4—figure supplement 1F and G). We further investigated the ubiquitination of TRIM25 in the presence of RTN3, as the autoubiquitination (K63-linked) of TRIM25 is essential for its catalytic activity (Gupta et al., 2018). Following the increasing expression of RTN3 in HEK293T cells, the K63-linked polyubiquitination of TRIM25 was significantly promoted (Figure 4—figure supplement 1H), consistent with the observed RTN3-mediated increase in the WT polyubiquitination of TRIM25 (Figure 4—figure supplement 1I). Taken together, these data indicate that RTN3 suppresses RIG-I-mediated antiviral innate immune responses by inhibiting K63-linked polyubiquitination upon viral RNA recognition, simultaneously suppressing the K63-linked ubiquitination of RIG-I and promoting the K63-linked polyubiquitination of TRIM25.

To elucidate whether TRIM25 promotes the ability of RTN3 to inhibit RIG-I-mediated immune responses, we further investigated whether TRIM25 deficiency abolishes the inhibition of RIG-I-mediated antiviral immune responses. To this end, endogenous TRIM25 expression was depleted in HEK293T cells by shRNA (HEK293T-shTRIM25), after which the phosphorylation of TBK1, p65, and IRF3 was detected in both HEK293T-shTRIM25 and HEK293T-shCtrl cells. Compared to the HEK293T-shCtrl cells, TRIM25 depletion counteracted the inhibition of TBK1, p65, and IRF3 phosphorylation induced by RTN3 overexpression when challenged with poly(I:C) (Figure 5A). Similarly, RTN3 overexpression in HEK293T-shTRIM25 cells only slightly impaired RIG-I K63-linked polyubiquitination upon VSV infection (Figure 5B). Taken together, these data suggest that TRIM25 has a crucial role in the RTN3-mediated suppression exerted of RIG-I activation.

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Subsequently, we evaluated which domain of RTN3 is responsible for its inhibitory activity toward RIG-I-mediated antiviral responses. RTN3 localizes to the ER membrane and comprises a noncytoplasmic domain (NC) that inserts into the ER lumen, three transmembrane domains (TM) and two cytoplasmic domains. A series of RTN3 truncations were constructed from the C terminus based on its domain structure (Figure 5C) and were then assessed for their ability to suppress RIG-I-mediated immune activation through ISRE-luc and NF-κB-luc reporter assays. The TM1-3-truncated construct (T4 mutant) was unable to inhibit RIG-I (N)-induced ISRE-luc activity, while other truncated mutants maintained similar inhibitory activities as full-length RTN3 (Figure 5D, top). The same phenomenon was also observed in NF-κB-luc reporter assays (Figure 5D, bottom). These results suggest that the first TM domain is required for the inhibitory activity of RTN3. Furthermore, ubiquitination analysis demonstrated that K63-linked polyubiquitination was impaired by the RTN3 mutants T1, T2 and T3 at levels similar to wild-type RTN3, whereas T4 mutants lacked this activity (Figure 5—figure supplement 1A). To further determinate the inhibitory function of the truncated mutants, we performed the coimmunoprecipitation assays and founded that T4 mutants no longer interacts with TRIM25 or RIG-I (Figure 5—figure supplement 1B and C). The confocal microscopy results showed that T4 mutants localized in the nuclear while other mutants colocalizing with RIG-I and TRIM25 in the cytoplasm as full length RTN3 did (Figure 5—figure supplement 1D). Luciferase assays using ISRE-luc and NF-κB-Luc reporters also showed that RTN3’s inhibitory function is insusceptible to the deletion of its noncytoplasmic domain or any cytoplasmic domain (Figure 5D). Collectively, our data suggest that RTN3 inhibits RIG-I-mediated innate immune antiviral responses in a TRIM25-dependent manner and that transmembrane domain 1 (aa 61–92) of RTN3 is indispensable for this activity, so long as RTN3 localizes on the endoplasmic reticulum membrane its inhibitory function is retained.

We next assessed whether RTN3 is upregulated and inhibits RIG-I-mediated innate immune antiviral responses in vivo during viral infection by treating C57BL/6 mice with PBS, poly(I:C) or VSV-eGFP separately via intravenous (I.V.) injection. Real-time PCR results showed that the mRNA levels of Rtn3 in liver extracts were significantly higher in the VSV- and poly(I:C)-treated mice than in those of the PBS control mice (Figure 6A), and Rtn3 protein levels were also markedly upregulated (Figure 6B). These results confirm that RTN3 expression is significantly induced by RNA viral infection in mice, at least in the liver.

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Excel file of CBA Mouse Inflammation Kit measurements and description of statistical tests for panel E.

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To further investigate whether RTN3 upregulation inhibits antiviral responses in vivo, mice were hydrodynamically injected with a plasmid encoding Rtn3 (HA-Rtn3) or the empty vector (HA-Ev) and then inoculated with VSV-eGFP via intraperitoneal (I.P.) injection to infect mice for another 12 hr (Figure 6C). At 48 hr, as expected, appropriate levels of plasmid-mediated RTN3 overexpression were detected in the livers of mice (Figure 6D). After the mice were sacrificed and then bled, the serum samples were incubated with precoated beads, and flow cytometry results showed that the levels of key inflammatory factors, including Mcp-1, IL-12p70, IFN-γ, and TNF-α were consistently decreased in the Rtn3-overexpressing mice upon viral infection, especially IFN-γ and TNF-α, both of which were markedly downregulated (Figure 6E). We next analyzed the immune cell population within the liver and PBMCs through flow cytometry analysis. Cells were grouped as indicated (Figure 6—figure supplement 1A and B), and upon VSV-eGFP infection, the population of CD11b⁺Gr1⁺ neutrophils (Figure 6F and G) dramatically decreased in the Rtn3-overexpressing mice, while other immune cell populations, including CD11⁺Ly6C^hiF480⁺ macrophages, Ly6C^hiF4/80^lo monocytes, CD45⁺CD3⁺ T cells and CD45⁺CD3^-CD11b⁺CD11c⁺ dendritic cells, showed no significant change (Figure 6—figure supplement 1C). Taken together, these results indicate that upon RNA viral infection, RTN3 is upregulated and subsequently suppresses antiviral immune and inflammatory responses, decreasing the population of neutrophils in mice.

Based on the observed inhibitory activity of RTN3 in mouse experiments, we speculated that RTN3 may promote the resolution of inflammation in vivo. To assess this possibility, the liver and lung tissue sections of mice were stained with hematoxylin and eosin (H and E), and microcopy analysis showed that upon VSV infection, RTN3 overexpression (indicated by RTN3 IHC) alleviated inflammatory cell infiltration and tissue edema in the liver (Figure 7A top and middle, 7B, 7C), which was accompanied by a thinning of the alveolar interstitium in the lung (Figure 7A bottom, 7D). These results indicated that RTN3 upregulation is induced by RNA viral infection to attenuate inflammation and protect against tissue injury caused by excessive inflammatory responses. Further experiments evaluating VSV-eGFP infection in the liver revealed that the VSV-infected cell population was increased in RTN3-overexpressing mice (Figure 7—figure supplement 1A and B), and the MFI of the GFP signal indicated that VSV-eGFP infection also increased to a certain extent. Collectively, these results indicate that RTN3 upregulation under RNA viral infection attenuates tissue inflammation and likely facilitates the resolution of inflammation.

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All animal experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University, China. Wild-type (WT) C57BL/6 mice were purchased from VITAL RIVER, Beijing, China and used for the animal experiments. For Rtn3-induction models, 8-week-old female mice received an intravenous (I.V.) injected of poly(I:C) (400 μg/mouse) or an equal volume of phosphate-buffered saline (PBS) as controls or VSV-eGFP [1 × 10⁷ plaque-forming units (PFU)/g/mouse] for 12 hr. For RTN3-overexpressing mouse models, 8-week-old female mice were hydrodynamically injected with a plasmid encoding Rtn3 (HA-Rtn3) or the empty vector (HA-Ev). Twenty micrograms of DNA diluted in 2 ml of 1× PBS was injected through the tail vein using a syringe with a 30-gauge needle. The injection was completed within 8–12 s, and 48 hr post DNA injection, the mice received an intraperitoneal (I.P.) injection of VSV-eGFP [1 × 10⁷ PFU/g/mouse] or an equal volume of DMEM for 12 hr.

HEK293T, RIG1 KO HEK293T (kindly provided by Dr. Yang Du [Zhongshan School of Medicine, Sun Yat-Sen University]), and HeLa cells were cultured in DMEM (Corning, 10–013-CV) supplemented with 10% fetal bovine serum (FBS) (Gibco, 10270–106), while A549 and THP-1 cells were cultured in RPMI 1640 medium (Corning, 10–040-CV) supplemented with 10% FBS. THP-1 cells stably overexpressing HA-RTN3 (THP-1^HA-RTN3) and control THP-1 cells (THP-1^HA-Ev) were cultured in RPMI 1640 medium supplemented with 10% FBS, whereas TRIM25 knockdown HEK293T cells (shTRIM25-HEK293T) were cultured in DMEM supplemented with 10% FBS. All cells were cultured in an incubator at 37°C under an atmosphere with 5% CO₂. HEK293T, A549, THP-1, and HeLa cells were obtained from ATCC (American Type Culture Collection). Identities of the cell lines used in this study were authenticated by STR profiling. Mycoplasma contamination status was tested as negative with Myco-Lumi Luminescent Mycoplasma Detection Kit (Beyotime, C0298M).

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy donors that was purchased from Guangzhou Blood Bank, using Lymphoprep (StemCell Technologies, Vancouver, BC) according to the manufacturer's instructions. The PBMCs were cultured in RPMI 1640 containing 10% FBS at a final concentration of 1×10⁶ cells/mL. All human blood works were approved by the medical ethics committee at Sun Yat-sen University.

The following antibodies were used in this study: anti-RTN3 (BA3533), purchased from BOSTER; anti-HA (M132-3), purchased from MBL; anti-Flag (AE005), anti-GFP (AE0122), were purchased from ABclonal; anti-β-actin (HC201-01), purchased from TransGen; anti-TRIM25 (67314–1-Ig), purchased from Proteintech; anti-GST (#2622), anti-Phospho- IKKα/β (#2697), anti-Phospho-TBK1 (#5483P), anti-TBK1 (#3504), anti-Phospho-IRF3 (#4947), anti-IRF3 (#4302), anti-Phospho-NF-κB p65 (#3033), anti-NF-κB p65 (#8242), anti-K63-linkage Specific Polyubiquitin (#5621), and anti-Ubiquitin (#43124) were purchased from Cell Signaling Technology; goat anti-mouse IgG IRDye680RD (C90710-09) and goat anti-rabbit IgG IRDye800CW (C80925-05) were purchased from Li-COR. Following reagents were used in this study: Polyinosinic–polycytidylic acid potassium salt [poly (I:C)] (P9852, average MW: 200,000–500,000), purchased from Sigma-Aldrich; Human recombinant TNF-alpha protein (10602-H01H), purchased from SinoBiological; Lipofectamine 2000, purchased from Invitrogen; anti-GFP beads (KTSM1301), purchased from Alpa-life; Glutathione Sepharose 4B (17-0756-01), purchased from GE Healthcare; The BD CBA Mouse Inflammation Kit (552364), purchased from BD Bioscience.

VSV-eGFP and VSV (kindly provided by Dr. Meng Lin, School of Life Science) were propagated and amplified in HEK293T cells and titrated on Vero cells with a standard plaque assay. SeV was a gift from Dr. Yang Du. For the in vivo study, VSV-eGFP was injected into mice at a titer of 1 × 10⁷ plaque-forming units (PFU)/g for 12 hr. For the in vitro study, cells were infected with VSV-eGFP (MOI = one for RTN3 induction and immune response activation; MOI = 0.05 for fluorescence and phase contrast (PH) analyses) or SeV (MOI = 0.1 for immune response activation).

The plasmids used in the present study were constructed as follows. RTN3 and TRIM25 were obtained from the HEK293T cDNA library and cloned into the eukaryotic expression vector pcDNA3.1 in-frame with an HA or Flag tag as well as the eukaryotic expression vectors pEGFP-C2 and pEBG. The HA-tagged RTN3 fragment was also subcloned into the pMSCV-PURO retrovirus expression vector, and the RTN3 fragment was truncated based on its structure and subcloned into pcDNA3.1 to generate the HA-tagged mutants T1, T2, T3, and T4. The Flag-RIG-I, Flag-RIG-I CARDs [RIG-I (N)], Flag-TLR3, Flag-TBK1, Flag-P65, Flag-MDA5, Flag-IRF3, HA-tagged Ub, and K63-Ub (K63 only) were kindly provided by Dr. Yang Du. The RIG-I fragment was also subcloned into pEGFP-C2. PrimeSTAR^@ MAX DNA Polymerase (Takara, RO45A) and a ClonExpressII One Step Cloning Kit (Vazyme, C11-02) were used to generate all the constructs. For transfection, HEK293T cells were seeded into 24- or 6-well plates overnight and then transfected using Lipofectamine 2000 according to the manufacturer’s protocol. Poly(I:C) was transfected into HEK293T cells using Lipofectamine 2000.

HEK293T cells were seeded into six-well plates at a density of 0.5 × 10⁶ cells/ml and incubated overnight. Then, 2.5 ml/well of a TRIM25 shRNA-encoding lentivirus was added to the plate, which was then placed in an incubator. Twenty-four hours postinoculation, the cells were infected with a second batch of virus for another 24 hr. The infected cells were then reseeded into six-well plates with fresh RPMI 1640 medium supplemented with 10% FBS and cultivated for 36 hr. Puromycin (2–4 μg/ml, Thermo Fisher, A113803) was used for TRIM25-knockdown HEK293T cell selection, and immunoblot analysis was performed to determine the knockdown efficiency. For TRIM25 knockdown-HEK293T cells generation, the following TRIM25 shRNA sequences were used: (1), 5’- CCGGAACAGTTAGTGGATTTA-3’; (2), 5’-GAACTGAACCACAAGCTGATA-3’. Both sequences target the coding region exon1 of TRIM25 gene and cloned into pLKO.1 lentivirus expression vector. The same methods were performed for transient RTN3 knockdown with the following shRNA sequences: (1), 5’- CCCGATTGTCTATGAGAAGTA-3’; (2), 5’-CTTTGGCACCACGCTGATCAT-3’.

HEK293T cells were seeded in 24-well plates overnight and then transfected using Lipofectamine 2000 with 100 ng of an ISRE-luciferase reporter (firefly luciferase), 100 ng of an IFNB luciferase reporter (firefly luciferase), or 100 ng of an NF-κB luciferase reporter (firefly luciferase) together with 20 ng pRL-TK plasmid (Renilla luciferase), 150 ng Flag-RIG-I (N), Flag-MDA5, or Flag-IRF3-5D mutant and increasing amounts of HA-RTN3. Twenty-four hours post transfection, the cells were collected, and luciferase activity was measured using a Dual-Luciferase Assay kit (Promega, E1960) with a Synergy2 Reader (Bio-Tek) according to the manufacturer’s protocol. The relative level of gene expression was determined by normalizing firefly luciferase activity to Renilla luciferase activity. Poly(I:C) (5 μg/ml) or SeV (MOI = 0.1) was also used as for activation in the assay without RIG-I (N) construct transfection.

Total RNA was extracted from HEK293T or A549 cells dissolved in TRIzol reagent (Invitrogen, 15596026) and then reverse transcribed into cDNA using HiScript III RT SuperMix (Vazyme). A SYBR Green I Master Mix kit (Roche, 04887352001) was used for real-time PCR with a LightCycler 480 system (Roche). The following primers were used for the assay: Human RTN3 forward, 5’-AGCTGGCTGTCTTCATGTGG-3’; reverse, 5’-CTCGGGCGATGCCAACATAG-3’. Human ACTB forward, 5’-CACCATTGGCAATGAGCGGTTC-3’; reverse, 5’-AGGTCTTTGCGGATGTCCACGT-3’. Mouse Rtn3 forward, 5’-AGCTGGCTGTCTTCATGTGG-3’; reverse, 5’-TCCCGGGCAATCCCAACA-3’. Mouse Gapdh forward, 5’-AGGCCGGTGCTGAGTATGTC-3’; reverse, 5’-TGCCTGCTTCACCACCTTCT-3’. Human IFNB1, forward, 5'-CCTACAAAGAAGCAGCAA-3'; reverse 5'-TCCTCAGGGATGTCAAAG-3'. Human IFIT1 forward, 5'-GCCTTGCTGAAGTGTGGAGGAA-3'; reverse, 5'-ATCCAGGCGATAGGCAGAGATC-3'. Human IFIT2 forward, 5'-GGAGCAGATTCTGAGGCTTTGC-3'; reverse, 5'-GGATGAGGCTTCCAGACTCCAA-3'. Human TNF forward, 5'-CTCTTCTGCCTGCTGCACTTTG-3'; reverse, 5'-ATGGGCTACAGGCTTGTCACTC-3'. Human IL6 forward, 5'-AGACAGCCACTCACCTCTTCAG-3'; reverse, 5'-TTCTGCCAGTGCCTCTTTGCTG-3'. Human CCL20 forward, 5'-AAGTTGTCTGTGTGCGCAAATCC-3'; reverse, 5'-CCATTCCAGAAAAGCCACAGTTTT-3'. Other primers used in the array were listed in Figure 2—figure supplement 1—source data 9.

For coimmunoprecipitation assays, cells transfected and stimulated with the appropriate ligands were lysed with IP buffer (Beyotime, P0013), after which whole cell extracts were collected and incubated with anti-GFP beads or Glutathione Sepharose 4B at 4°C for 1 hr or overnight. Then, the beads were washed 4–5 times with IP buffer, and the immunoprecipitants were eluted with 2× SDS loading buffer and then resolved by SDS-PAGE. Subsequently, the proteins were transferred to PVDF membranes (Millipore, ISEQ00010) and further incubated with the indicated primary and secondary antibodies. The images were visualized using an Odyssey Sa system (LI-COR).

WT HeLa cells were seeded in 8-well Millicell^@EZ slides (Millipore Ltd, PEZGS0816) overnight and then transfected using Lipofectamine 2000. Twenty-four hours posttransfection, the cells were carefully washed with 1× PBS three times (2 ml/well, 5 min/time) and then fixed with 4% PFA for 15 min at 4°C. After the cells were penetrated with 0.2% Triton X-100 at room temperature for 5 min, they were washed and blocked with 3% BSA in 1× PBS for 1 hr at 4°C and then assayed using the indicated primary and secondary antibodies. Microcopy was performed with a ZESS LSM800 system, and images were captured and processed using ZESS ZEN Imaging Software.

Infected mice were sacrificed at the indicated timepoints, and their livers and lungs were dissected and fixed in 4% PFA. After embedding, paraffin sections were stained with hematoxylin and eosin (H and E) or subjected to standard immunohistochemical staining (IHC) before being visualized by bright field microscopy.

The data are presented as the means ± SD (for in vivo experiments, the values are presented as the means ± SD of n mice), and Sidak’s multiple comparisons test was used for all statistical analyses performed with GraphPad Prism 8. Differences between two groups were considered significant at p value < 0.0332.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We thank all the members of our laboratory for their critical assistance and helpful discussions. This work is supported by grants from the Natural Science Foundation of China (81871643 and 32061143008) to EK and the Natural Science Foundation of China (81971928) to XL.

Human subjects: All human samples (PBMCs) were anonymously coded in accordance with the local ethical guidelines. The Ethics Review Board of Zhongshan School of Medicine, Sun Yat-Sen University approved this study. Approval No: ZhongshanSchool of Medicine Ethics [2021] NO.016.

Animal experimentation: All animal experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University, China. Approval No: SYSU-IACUC-2021-B0025.

© 2021, Yang et al.

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