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RTN3 inhibits RIG-I-mediated antiviral responses by impairing TRIM25-mediated K63-linked polyubiquitination

  1. Ziwei Yang
  2. Jun Wang
  3. Bailin He
  4. Xiaolin Zhang
  5. Xiaojuan Li  Is a corresponding author
  6. Ersheng Kuang  Is a corresponding author
  1. Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, China
  2. Key Laboratory of Tropical Disease Control (Sun Yat-Sen University), Ministry of Education, China
  3. Zhongshan School of Medicine, Sun Yat-Sen University, China
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Cite this article as: eLife 2021;10:e68958 doi: 10.7554/eLife.68958

Abstract

Upon viral RNA recognition, the RIG-I signalosome continuously generates IFNs and cytokines, leading to neutrophil recruitment and inflammation. Thus, attenuation of excessive immune and inflammatory responses is crucial to restore immune homeostasis and prevent unwarranted damage, yet few resolving mediators have been identified. In the present study, we demonstrated that RTN3 is strongly upregulated during RNA viral infection and acts as an inflammation-resolving regulator. Increased RTN3 aggregates on the endoplasmic reticulum and interacts with both TRIM25 and RIG-I, subsequently impairing K63-linked polyubiquitination and resulting in both IRF3 and NF-κB inhibition. Rtn3 overexpression in mice causes an obvious inflammation resolving phenomenon when challenged with VSV, Rtn3-overexpressing mice display significantly decreased neutrophil numbers and inflammatory cell infiltration, which is accompanied by reduced tissue edema in the liver and thinner alveolar interstitium. Taken together, our findings identify RTN3 as a conserved negative regulator of immune and inflammatory responses and provide insights into the negative feedback that maintains immune and inflammatory homeostasis.

Introduction

The innate immune responses can be triggered by pathogen-associated molecular patterns (PAMPs) and then serve as the first line of defense against invading pathogens (Takeuchi and Akira, 2010). Upon viral infection, pattern recognition receptors (PRRs) detect viral RNA, DNA and other viral products and subsequently mediate the activation of downstream signaling pathways (Roers et al., 2016Loo and Gale, 2011). Both RIG-I and MDA5, two crucial members of the RIG-I-like receptor (RLR) family, sense cytoplasmic viral RNA and activate antiviral responses. Both RIG-I and MDA5 share the same domain architecture, comprising two N-terminal caspase activation recruitment domains (CARDs), a central DEAD box helicase/ATPase domain and a C-terminal regulatory domain (CTD) (Loo and Gale, 2011). MDA5 has been reported to recognize longer dsRNA molecules (>2 kb), while RIG-I prefers shorter (< 1–2 kb) or 5’ triphosphate-containing dsRNA (Peisley et al., 2012Peisley et al., 2011). Unlike MDA5, which is conformationally unaltered, upon recognition of dsRNA by the CTD domain, RIG-I unfolds and releases its CARDs from the inward folding state formed by its helicase domain, thereby transforming into an activated state (Jiang et al., 2011; Kowalinski et al., 2011). The CARD domain then recruits MAVS and activates the downstream signaling cascade to induce the expression of type I interferons, IFN stimulated genes (ISGs), inflammatory cytokines, and/or chemokines (Kowalinski et al., 2011).

Considering that RIG-I-mediated innate antiviral responses are important, drastic and rapid upon viral RNA recognition, multiple mechanisms have evolved to precisely regulate RIG-I-mediated antiviral signaling to maintain the balance between immunity and tolerance and prevent severe or even fatal unnecessary damage, which may be caused by excessive immune and inflammatory responses (Eisenächer and Krug, 2012). Some modifications of RIG-I reduce its stability. For example, LRRC25 targets RIG-I for ISG15-associated autophagy degradation (Du et al., 2018), whereas the E3 ubiquitin ligases RNF125, STUB1, c-Cbl, and CHIP induce the K48-linked polyubiquitination of RIG-I to promote its proteasome-dependent degradation (Arimoto et al., 2007Zhao et al., 2016). Alternatively, the posttranslational modification of RIG-I upon K63-linked polyubiquitination has been well shown to regulate RIG-I activation. TRIM25 and Riplet induce this modification in RIG-I (Gack et al., 2007Oshiumi et al., 2010), whereas the deubiquitination enzyme USP3 targets the RIG-I CARDs to cleave its K63-linked polyubiquitin (Cui et al., 2014). In addition, CKII and PKCα/β phosphorylate the RIG-I CTD and CARDs to negatively regulate its activation (Sun et al., 2011Maharaj et al., 2012).

Although the acute immune response and inflammation defend against viral infection and eliminate virus-induced damage, excessive and unrestricted responses can lead to excessive tissue injury and even organ failure. Several diseases have been shown to be related to chronic inflammation or autoimmunity, such as asthma, arthritis, periodontal disease, and neurodegenerative disorders (Chiang et al., 2017Chiang and Serhan, 2017Sugimoto et al., 2016Alessandri et al., 2013). Inflammation resolution has been shown to be an essential active process (Buckley et al., 2013), and pro-resolving mediators regulate inflammation resolution through multiple strategies, including the inhibition of signal transduction, the regulation of cytokines and chemokines, the cessation of leukocyte infiltration and the clearance of apoptotic cells (Sugimoto et al., 2016Ortega-Gómez et al., 2013).

Viral infections also trigger different ER stresses due to the excessive synthesis and accumulation of viral proteins in the ER lumen or through signal hijacking (Sano and Reed, 2013). In response to ER stress, two transcription factors, CHOP and ATF6, are activated, and the translational regulator eIF2α is phosphorylated and subsequently reprograms cellular transcription and translation (Oyadomari and Mori, 2004Walter et al., 2011). Although reticulon family members have shown to comprise a highly conserved housekeeping protein family in multiple species (Moreira et al., 1999), one reticulon member, RTN3, is transcriptionally upregulated under ER stress by CHOP or ATF6 (Wan et al., 2007).

In our previous unpublished experiments, we observed that RTN3 is dramatically upregulated upon RNA viral infection when using VSV as a stimulus. However, the mechanism and function of RTN3 upregulation during viral infection is unclear, raising the strong possibility that RTN3 is involved in antiviral and inflammatory responses. In the present study, we showed that RTN3 acts as a conserved negative regulator and suppresses RIG-I-mediated immune and inflammatory responses during RNA viral infections. We reveal that RTN3 exerts an inhibitory effect on RIG-I signalosome activation by impairing TRIM25-mediated RIG-I K63-linked polyubiquitination. Our study may contribute to a better understanding of a novel mechanism for inflammation resolution and the reconstitution of tissue homeostasis during viral infections.

Results

RTN3 is upregulated and self-aggregates upon RNA viral infection

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.

Figure 1 with 1 supplement see all
RTN3 is upregulated and self-aggregates upon RNA viral infection.

(A) Immunoblot analysis of HEK293T cells infected with VSV-eGFP (MOI = 1) or treated with poly(I:C) (5 μg/ml) at the indicated timepoints. (B–C) mRNA levels of RTN3 and TNF in the same samples shown in (A) were detected by real-time PCR. B. VSV-eGFP-infected group, C. poly(I:C)-treated group. (D) Immunoblot analysis of HEK293T cells treated with TNF-α (10 ng/ml) or IFN-β (20 ng/ml) at the indicated timepoints. (E) mRNA levels of RTN3 in the same samples shown in (D) were detected by RT-PCR. (F) Immunoblot analysis of PBMCs infected with VSV-eGFP (MOI = 2) at the indicated timepoints (top), and mRNA levels of RTN3 in the same samples were detected by RT-PCR (bottom). In (A, D, F), the data are representative of three independent experiments. In (B, C, E, F), the data are shown as the mean values ± SD (n = 3). *, p < 0.0332; **, p < 0.0021; ***, p < 0.0002; and ****, p < 0.0001 by Sidak’s multiple comparisons test.

Figure 1—source data 1

Uncropped blots with bands labeled AI file and raw unedited blots files for panels A, D, and F.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig1-data1-v2.zip
Figure 1—source data 2

Excel file of RT-PCR measurements and description of statistical tests for panel B.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig1-data2-v2.xlsx
Figure 1—source data 3

Excel file of RT-PCR measurements and description of statistical tests for panel C.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig1-data3-v2.xlsx
Figure 1—source data 4

Excel file of RT-PCR measurements and description of statistical tests for panel E.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig1-data4-v2.xlsx
Figure 1—source data 5

Excel file of RT-PCR measurements and description of statistical tests for panel F.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig1-data5-v2.xlsx

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.

RTN3 overexpression suppresses antiviral immune 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.

Figure 2 with 1 supplement see all
RTN3 overexpression suppresses antiviral immune responses.

(A) Luciferase assays of HEK293T cells transfected with an ISRE luciferase reporter (ISRE-Luc), an IFNB luciferase reporter (IFNβ-Luc) or an NF-κB luciferase reporter (NF-κB-Luc) together with an HA-tagged empty vector (HA-Ev, no wedge) or increasing amounts (wedge) of plasmid encoding RTN3 (HA-RTN3) followed by transfection with a plasmid encoding the RIG-I CARD domain [RIG-I (N)] to activate the pathway. Cell lysates were collected 24 hr posttransfection, the same as that described in the following experiments, if no additional annotation was performed. (B) Luciferase activity of HEK293T cells transfected with ISRE-Luc, together with HA-Ev or increasing amounts of plasmid HA-RTN3, followed by treatment with poly(I:C) (5 μg/ml) or SeV (MOI = 0.1) for 16 or 24 hr. (C) Fluorescence and phase contrast (PH) analyses of HEK293T cells transfected with HA-Ev or HA-RTN3, followed by infection with VSV-eGFP (MOI = 0.05) at the indicated timepoints (left). Scale bar, 100 μm. mRNA levels of VSV viral RNA in the same samples were analyzed by RT-PCR (right). (D) The percentage of eGFP-positive cells in the same samples shown in (C) was analyzed by flow cytometry. (E) Immunoblot analysis of HEK293T cells transfected with HA-Ev or HA-RTN3 followed by infection with VSV-eGFP (MOI = 1) at the indicated timepoints (left). Quantitative comparison of the indicated protein levels analyzed by gray intensity scanning of blots (right). (F) RT-PCR analysis of IFNB1, IFIT2, IFIT1, TNF, IL6, and CCL20 mRNA levels in HEK293T cells transfected with HA-Ev or HA-RTN3 followed by infection with VSV-eGFP (MOI = 1) at the indicated timepoints. In (E), the data are representative of three independent experiments. In (A, B, C, F), the data are shown as the mean values ± SD (n = 3). *, p < 0.0332; **, p < 0.0021; ***, p < 0.0002; and ****, p < 0.0001 by Sidak’s multiple comparisons test.

Figure 2—source data 1

Uncropped blots with bands labeled AI file and raw unedited blots files for panel E.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-data1-v2.zip
Figure 2—source data 2

Excel file of luciferase reporter assay measurements and description of statistical tests for panel A.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-data2-v2.xlsx
Figure 2—source data 3

Excel file of luciferase reporter assay measurements and description of statistical tests for panel B.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-data3-v2.xlsx
Figure 2—source data 4

Excel file of RT-PCR measurements and description of statistical tests for panel C.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-data4-v2.xlsx
Figure 2—source data 5

Excel file of blots quantification and description of statistical tests for panel E.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-data5-v2.xlsx
Figure 2—source data 6

Excel file of RT-PCR measurements and description of statistical tests for panel F.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-data6-v2.xlsx

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-1HA-RTN3 vs. THP-1HA-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-1HA-RTN3 cells compared to THP-1HA-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.

RTN3 depletion augments antiviral immune 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.

RTN3 knockdown enhances antiviral immune responses.

(A) RT-PCR analysis (top) and immunoblotting analysis (bottom) of RTN3 mRNA levels in PBMCs infected with Ctrl or RTN3 shRNA encoding lentivirus (shCtrl, shRNA #1, shRNA #2) for 12 hr and cultured for additional 24 hr (top). (B) Immunoblotting analys of PBMCs infected with Ctrl or RTN3 shRNA encoding lentivirus for 12 hr and cultured for another 24 hr, followed by infection with VSV-eGFP (MOI = 2) at the indicated timepoints (left). Quantitative comparison of the indicated protein levels was analyzed by gray intensity scanning of blots and is shown (right). (C–D) RT-PCR analysis of IFNB1, IFIT2, IFIT1, TNF, IL6, and CCL20 mRNA levels (C) and VSV viral genomic RNA mRNA levels (D) in PBMCs infected with Ctrl or RTN3 shRNA encoding lentivirus for 12 hr and cultured for another 24 hr, followed by infection with VSV-eGFP (MOI = 2) at the indicated timepoints. In (B), the data are representative of three independent experiments. In (A, C, D), the data are shown as the mean values ± SD (n = 3). *, p < 0.0332; **, p < 0.0021; ***, p < 0.0002; and ****, p < 0.0001 by Sidak’s multiple comparisons test.

Figure 3—source data 1

Uncropped blots with bands labeled AI file and raw unedited blots files for panels A and B.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig3-data1-v2.zip
Figure 3—source data 2

Excel file of RT-PCR measurements and description of statistical tests for panel A.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig3-data2-v2.xlsx
Figure 3—source data 3

Excel file of blots quantification and description of statistical tests for panel B.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig3-data3-v2.xlsx
Figure 3—source data 4

Excel file of RT-PCR measurements and description of statistical tests for panel C.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig3-data4-v2.xlsx
Figure 3—source data 5

Excel file of RT-PCR measurements and description of statistical tests for panel D.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig3-data5-v2.xlsx

RTN3 interacts with TRIM25 or RIG-I

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.

Figure 4 with 1 supplement see all
RTN3 separately interacts with TRIM25 and RIG-I and inhibits the K63-linked polyubiquitination of RIG-I.

(A) Coimmunoprecipitation (CoIP) and immunoblot analyses of HEK293T cells transfected with a plasmid encoding TRIM25 (Flag-TRIM25) or a Flag-tagged empty vector (Flag-Ev) together with GFP-tagged RTN3 (GFP-RTN3) or a GFP-tagged empty vector (GFP-Ev) in the indicated groups for 24 hr and followed by infection with VSV (MOI = 1) for 8 hr. (B) CoIP and immunoblot analyses of RIG1 KO HEK293T cells transfected with HA-Ev or HA-RTN3 together with GFP-tagged TRIM25 (GFP-TRIM25) or GFP-Ev for the indicated groups for 24 hr and followed by infection with VSV (MOI = 1) for 8 hr. (C) CoIP and immunoblot analyses of HEK293T cells transfected with a plasmid encoding RIG-I (Flag-RIG-I) or Flag-Ev together with GFP-RTN3 or GFP-Ev in the indicated groups for 24 hr and followed by infection with VSV (MOI = 1) for 8 hr. (D) Confocal microcopy analysis of HeLa cells transfected with GFP-RIG-I and mCherry-tagged RTN3 (mCherry-RTN3) or GFP-TRIM25 with mCherry-RTN3, and GFP-RIG-I, GFP-TRIM25, or mCherry-Ev, respectively. The dotted box (a, b) indicates the colocalization area. Scale bar, 5 μm. (E) Qualitative analysis of the fluorescence intensity of the selected area in (D––a, b). (F) Co-IP and immunoblot analyses of PBMCs infected with VSV-eGFP (MOI = 2) for 18 hr, cell lysates were immunoprecipitated with anti-RTN3 or IgG isotype. (G) IP and immunoblot analyses of HEK293T cells transfected with HA-Ev or increasing amounts of HA-RTN3 (+, ++) together with Flag-RIG-I and GFP-tagged TRIM25 (GFP-TRIM25) or GFP-Ev in the indicated groups for 24 hr and followed by infection with VSV (MOI = 1) for 8 hr. (H) CoIP and immunoblot analyses of HEK293T cells transfected with HA-Ub-K63 and Flag-Ev or increasing amounts of Flag-RTN3 (+, ++) together with GFP-RIG-I or GFP-Ev in the indicated groups for 24 hr and followed by infection with VSV (MOI = 1) for 8 hr. In (A–C) and (F–H), the data are representative of three independent experiments.

Figure 4—source data 1

Uncropped blots with bands labeled AI file and raw unedited blots files for panels A, B, C, F, G, and H.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig4-data1-v2.zip

RTN3 impairs the K63-linked polyubiquitination of RIG-I upon RNA viral infection

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.

RTN3 inhibition of antiviral responses is TRIM25-dependent

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.

Figure 5 with 1 supplement see all
RTN3-mediated inhibition of antiviral responses is TRIM25-dependent.

(A) Immunoblot analysis of control HEK293T cells (HEK293T-shCtrl) and TRIM25 knockdown HEK293T cells (HEK293T-shTRIM25) transfected with HA-Ev or HA-RTN3 and followed by treated with poly(I:C) (5 μg/ml) at the indicated timepoints. (left). Quantitative comparison of the indicated protein levels in (A) analyzed by gray intensity scanning of blots (right). (B) CoIP and immunoblot analysis of HEK293T-shCtrl and HEK293T-shTRIM25 cells transfected with HA-Ub-K63 and increasing amounts of Flag-RTN3 (+, ++) or Flag-Ev together with GFP-RIG-I or GFP-Ev in the indicated groups for 24 hr and followed by infection with VSV (MOI = 1) for 8 hr. (C) The structure of RTN3 and its truncated mutants. Noncytoplasmic domain (NC), transmembrane domain (TM), full length (FL), truncated (T) (top). Immunoblot analysis of HEK293T cells transfected with HA-Ev or HA-tagged truncated mutants for 24 hr (bottom). (D) Luciferase activity of HEK293T cells transfected with ISRE-Luc (top) and NF-κB-Luc (bottom) together with HA-Ev or increasing amounts (wedge) of HA-RTN3 and its truncated mutants and followed by transfection of RIG-I (N) as an activation of the pathway. In (A, B), the data are representative of three independent experiments. In (D), the data are shown as the mean values ± SD (n = 3). *, p < 0.0332; **, p < 0.0021; ***, p < 0.0002; and ****, p < 0.0001 by Sidak’s multiple comparisons test.

Figure 5—source data 1

Uncropped blots with bands labeled AI file and raw unedited blots files for panels A, B, and C.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig5-data1-v2.zip
Figure 5—source data 2

Excel file of blots quantification and description of statistical tests for panel A.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig5-data2-v2.xlsx
Figure 5—source data 3

Excel file of luciferase reporter assay measurements and description of statistical tests for panel D.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig5-data3-v2.xlsx

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.

RTN3 overexpression suppresses antiviral immune responses in mice

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.

Figure 6 with 1 supplement see all
RTN3 overexpression suppresses antiviral immune responses in mice.

(A) mRNA levels of Rtn3 were detected by RT-PCR in liver cells from mice treated with PBS, poly(I:C) (400 µg/mouse) or VSV-eGFP (1×107 PFU/g/mouse) for 12 hr. (B) Immunoblot analysis of liver cell lysates of the same samples shown in (A), with ‘# number’ indicating individual mice. (C) Timeline of in vivo experiments. For plasmid hydrodynamic injection, 20 µg/mouse; for viral infection (intraperitoneal injection), 1×107 PFU g−1/mouse VSV (VSV-eGFP) or 600 µl DMEM /mouse. (D) Immunoblot analysis of liver cell lysates from the same samples shown in (C). (E) Protein levels of Mcp-1, IL-12p70, Ifn-γ, and Tnf-α in serum samples from the mice shown in (C) were measured by flow cytometry using a BD CBA Mouse Inflammation kit. (F) The percentage of neutrophils in liver cell suspensions from the same mice shown in (C) was detected by flow cytometry. The density graph indicates the cell population groups for each mouse (left), and the percentage comparison for all mice is shown in the scatterplot (right). (G) The percentage of neutrophils among PBMCs from the same mice as in (C) was detected by flow cytometry. The density graph indicates the cell population grouped of each mouse (left), and the percentage comparison for all mice is shown in the scatterplot (right). In (A, E, F, G), the data are shown as the mean values ± SD (n = four in A, n = six in E-G). *, p < 0.0332; **, p < 0.0021; ***, p < 0.0002; and ****, p < 0.0001 by Sidak’s multiple comparisons test.

Figure 6—source data 1

Uncropped blots with bands labeled AI file and raw unedited blots files for panels B and D.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig6-data1-v2.zip
Figure 6—source data 2

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

https://cdn.elifesciences.org/articles/68958/elife-68958-fig6-data2-v2.xlsx

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+Ly6ChiF480+ macrophages, Ly6ChiF4/80lo 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.

RTN3 overexpression suppresses inflammation 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.

Figure 7 with 1 supplement see all
Rtn3 overexpression promotes inflammation resolution in mice.

(A) Microcopy analysis of liver and lung tissue sections from the same mice as in (Figure 5C) with hematoxylin and eosin (H and E) staining, and HA-Rtn3 exotic expression was visualized by immunohistochemical staining (IHC). (B) Inflammation scores of liver tissue sections (n=4, 4, 4, 4) from the same mice shown in (A). The data were obtained and analyzed using ImageJ and visualized with GraphPad Prism 8. (C) IHC score assessment of liver tissue sections as in (A, middle), data was obtained and calculated with Image J plugin ‘IHC Profiler’ (D) Inflammation scores of lung tissue sections (n=4, 4, 4, 4) from the same mice shown in (A). The data were obtained and analyzed using ImageJ and visualized with GraphPad Prism 8. In (B–D), data are shown as the mean values ± SD (n = 4). *, p < 0.0332; **, p < 0.0021; ***, p < 0.0002; ****, p < 0.0001; by Sidak’s multiple comparisons test.

Figure 7—source data 1

Excel file of inflammation scores and description of statistical tests for panel B.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig7-data1-v2.xlsx
Figure 7—source data 2

Excel file of IHC scores and description of statistical tests for panel C.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig7-data2-v2.xlsx
Figure 7—source data 3

Excel file of inflammation scores and description of statistical tests for panel D.

https://cdn.elifesciences.org/articles/68958/elife-68958-fig7-data3-v2.xlsx

Discussion

In the present study, with respect to the conserved and ubiquitous expression and ER subcellular localization of reticulon members, we demonstrate that RTN3 is involved in the negative regulation of antiviral responses upon RNA viral infection. Our results demonstrate that RTN3 is upregulated upon infection with VSV or poly(I:C) stimulation and then self-aggregates along the ER membrane. A potential mechanism for RTN3 induction may be ER stress upon acute viral infection. As a UPR target gene, the promoter region of the RTN3 gene harbors binding sites for the ER-stress-related transcription factors ATF6 and CHOP. As a consequence, RTN3 upregulation inhibited RIG-I-mediated immune responses, but had only slight and minimal effects on MDA5- and TLR3-induced activation. We further demonstrated that RTN3 overexpression significantly attenuates the RIG-I-mediated production of proinflammatory cytokines.

In assessments of whether RIG-I is the predominant target through which RTN3 inhibits antiviral responses, our results demonstrated that RTN3 separately interacts with RIG-I and TRIM25 and that their interaction is augmented upon VSV stimulation. Furthermore, we showed that RTN3 overexpression impairs the K63-linked polyubiquitination of RIG-I in a TRIM25-dependent manner but does not interfere with RIG-I stability or interaction. Notably, the K63-linked polyubiquitination of TRIM25 was promoted while that of RIG-I was inhibited by RTN3 overexpression that led to RTN3 self-aggregation and the formation of a scaffold-like structure, indicating that RTN3 may interfere with the oligomerization of TRIM25 and its E3 ubiquitin ligase activity to promote RIG-I polyubiquitination. Furthermore, mapping analysis of truncated RTN3 constructs showed that RTN3 transmembrane domain 1 (TM1) is crucial for impairing RIG-I K63-linked polyubiquitination and inhibiting immune responses, indicating that a proper topological structure of RTN3 on the ER membrane is essential for its activity. Thus, the underlying mechanism of how RTN3 interferes with this process is worth further investigation.

Consistent with the in vitro results, in vivo experiments in mice showed that RTN3 is upregulated upon VSV infection or poly(I:C) stimulation. Importantly, our results provide evidence that RTN3 overexpression decreases the production of inflammatory factors and the neutrophil population in the liver and blood during RNA viral infection. The native neutrophil population may be primarily affected by the downregulation of cytokines and chemokines, and the circulating neutrophil population is affected secondarily. Because neutrophils have the capacity to damage tissue (Kolaczkowska and Kubes, 2013Ley et al., 2007), neutrophil inhibition is crucial in the inflammatory response (Ortega-Gómez et al., 2013Soehnlein and Lindbom, 2010). The suppression of chemokine CXCL8 (IL-8; Figure 2—figure supplement 1H) levels by RTN3 indicates that it may be a potential mediator of inflammation resolution by indirectly restricting neutrophil recruitment. Although the decreased macrophage populations in Rtn3-ovrerexpressing mice were not significant, we did observe a decreasing trend for these populations (Figure 6—figure supplement 1C). The histological results demonstrated that RTN3 overexpression attenuated the infiltration of lymphocytes and greatly relieved tissue inflammation and edema. Taken together, these results provide evidence that RTN3 functions to maintain tissue homeostasis and initiate the resolution of inflammation by inhibiting RIG-I-mediated antiviral responses and even terminating acute inflammation.

Therefore, we propose a working model that describes how RTN3 suppresses RIG-I-mediated antiviral innate immune responses (Figure 8). Upon RNA viral infection, the RIG-I signalosome undergoes TRIM25-mediated activation and induces the production of IFNs, cytokines and chemokines, leading to acute immune responses and inflammation. Subsequently, RTN3 is upregulated in an ER stress- and/or inflammation-dependent manner, which is directly associated with acute viral infection. Aggregated RTN3 on the endoplasmic reticulum interacts with TRIM25 and RIG-I, inhibiting the K63-linked polyubiquitination of RIG-I via the E3 ligase activity TRIM25 to block the RIG-I signaling cascade and attenuate innate immune responses. As a result, the production of IFNs, cytokines and chemokines is attenuated, and the resolution of host inflammation is simultaneously initiated.

The working model of the inhibition of RIG-I mediated antiviral responses by RTN3 Upon RNA virus infection, the RIG-I signalosome is activated by TRIM25-mediated K63-linked polyubiquitination, and then triggers IRF3 and NF-κB activation and the expression of interferon, cytokines and inflammatory factors.

On the other hand, RTN3 is up-regulated by acute viral infection and interferon/inflammatory factors in an ER stress-dependent manner. RTN3 is subsequently aggregated on the endoplasmic reticulum, interacts with RIG-I and TRIM25, rapidly diminishes the K63-linked polyubiquitination of RIG-I and hence suppresses antiviral immune and inflammatory responses. The inflammatory response is consequently mitigated and the resolution of acute inflammation is initiated.

In summary, our present studies demonstrate that a conserved reticulon protein, RTN3, which is ubiquitously expressed in various tissues and organs, is upregulated upon RNA viral infection. In turn, upregulated RTN3 inhibits RIG-I signalosome activation by impairing the TRIM25-RIG-I axis. Our findings demonstrate a novel negative feedback mechanism for acute immune responses and inflammation resolution and the potential reconstitution of tissue homeostasis during viral infection.

Materials and methods

Mice and mouse experiments

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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.

Cell culture

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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-1HA-RTN3) and control THP-1 cells (THP-1HA-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.

Antibodies and reagents

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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.

Viral titer and infection

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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).

Plasmids and transfection

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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.

Generation of gene-modified cell lines

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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’.

Luciferase reporter assays

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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.

Real-time PCR

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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.

Coimmunoprecipitation and immunoblot analysis

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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).

Confocal microcopy

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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.

Histology

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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.

Statistical analyses

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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.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

References

    1. Sano R
    2. Reed JC
    (2013) ER stress-induced cell death mechanisms
    Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1833:3460–3470.
    https://doi.org/10.1016/j.bbamcr.2013.06.028

Decision letter

  1. Michaela Gack
    Reviewing Editor; Cleveland Clinic Lerner Research Institute, United States
  2. Sara L Sawyer
    Senior Editor; University of Colorado Boulder, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This study is of broad interest to readers in the field of antiviral innate immunity and those studying inflammation and the molecular mechanisms that resolve inflammation. Kuang and colleagues identify and characterize a new regulatory protein that helps to maintain immune homeostasis following activation of the cytoplasmic RNA sensor RIG-I. The authors present a combination of in vivo and in vitro data that highlight a delicate regulatory circuit in innate immunity to RNA virus infection.

Decision letter after peer review:

Thank you for submitting your article "RTN3 inhibits RIGI-I-mediated antiviral responses by impairing TRIM25-mediated K63-linked polyubiquitination" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Sara Sawyer as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential Revisions:

1) Functional analyses in RTN3 knockdown or knockout cells, ideally in primary cells.

2) Mechanistic and interaction studies with endogenous RTN3 to confirm the proposed molecular mechanism.

3) Addressing the specific issues about controls that have been pointed out by reviewers.

Reviewer #1:

In this manuscript "RTN3 inhibits RIG-I-mediated antiviral responses by impairing TRIM25-mediated K63-linked polyubiquitination", Yang et al. report that the reticulon member RTN3 is upregulated upon VSV infection or poly(I:C) stimulation, and that its interaction with the E3 ligase TRIM25 and the sensor RIG-I impairs innate immune signaling to maintain immune homeostasis and resolve inflammation. The authors show that this interaction inhibits the K63-linked ubiquitination of RIG-I mediated by TRIM25, which is a process necessary for RIG-I signaling. The upregulation of RTN3 expression following RNA virus infection was confirmed in vivo, and the authors further demonstrated that neutrophil numbers, lymphocyte infiltration, and inflammation including edema were reduced in mice in which RTN3 was overexpressed. Altogether, this study identifies RTN3 as a regulatory protein that controls RNA virus infection associated upregulation of the innate immune/inflammatory response in order to alleviate tissue inflammation.

The conclusions of this paper are overall supported by the data, and the experiments which represent both in vitro mechanistic studies and in vivo functional analysis, are well designed. The study demonstrates that enforced RTN3 expression inhibits RIG-I mediated innate immune responses and alleviates inflammation. However, additional extensive analyses detailing the effect of RTN3 gene targeting on antiviral innate immunity, as well as additional experiments with endogenously expressed RTN3, are needed to strengthen the physiological role of RTN3 in inflammation/immunity.

Major Strengths:

The authors performed a very thorough characterization of RTN3 and its mechanism. They convincingly show binding of RTN3 to both TRIM25 and RIG-I, and the inhibitory effect that this interaction mode has on K63-linked ubiquitination and antiviral signaling of RIG-I. It is clear from the in vitro and in vivo data that endogenous RTN3 is upregulated upon VSV infection or poly(I:C) stimulation, and that its enforced expression dampens the activation of downstream signaling mediators and the induction of proinflammatory genes. The authors performed detailed mapping studies to identify the domain responsible for RTN3's ability to block antiviral signaling, which suggested that proper localization of RTN3 on ER membranes is required for RTN3's immune dampening activity. The in vivo data convincingly show upregulation of endogenous Rtn3 upon VSV infection, and that the neutrophil population is decreased and inflammation is alleviated when Rtn3 expression is enforced.

The authors utilized a number of cell lines to support their findings, and numerous assays to validate their hypothesis and proposed model.

Weaknesses:

The design of the experiments solely relies on RTN3 overexpression. The study needs to be further strengthened by experiments that analyze: (a.) endogenous RTN3/TRIM25/RIG-I interaction or complex formation during virus infection or dsRNA stimulation; and (b.) the effect of RTN3 knockdown or knockout on RIG-I antiviral/proinflammatory signaling. RTN3 gene-targeting analysis in primary cells (human or mouse cells) would be preferred, if possible.

The overall writing and structure of the manuscript could be further improved for the reader's benefit.

1. Most of the experiments are done with RTN3 overexpression, which clearly showed an immune dampening effect. What is the effect of RTN3 knockdown or knockout on RIG-I signaling and proinflammatory gene induction in response to virus infection or poly(I:C) stimulation? Such gene-targeting data, especially if performed in primary cells (human or mouse cells) if possible, would greatly strengthen the physiological role of RTN3.

2. The authors performed extensive interaction and mechanistic studies with overexpressed RTN3, which convincingly showed that RTN3 binds to both TRIM25 and RIG-I and blocks the TRIM25-mediated K63-linked ubiquitination of RIG-I. Data assessing endogenous TRIM25-RTN3 binding/co-localization during infection or poly(I:C) stimulation, and the effect of RTN3 silencing on RIG-I signaling would further bolster the proposed mechanism.

3. Figure 3H: There is not an appreciable effect of HA-RTN3 on K63-ubiquitination of TRIM25. Overall, it is unclear how enhanced TRIM25 K63-ubiquitination/auto-ubiquitination by RTN3 would lead to suppressed TRIM25 catalytic activity and thereby dampened TRIM25-mediated RIG-I K63-ubiquitination. Overall, this part of the mechanism is not very clear. This reviewer suggests moving Figure 3H to the Supplementary Data, or the authors may further discuss this.

4. Figure 4B (IP, right 3 lanes): Although there was no effect observed for HA-RTN3 on RIG-I ubiquitination in the absence of TRIM25, it is unclear why the overall RIG-I ubiquitination signal in shTRIM25 cells is as high as that in shCtrl cells. One would expect overall reduced RIG-I K63-ubiquitination when TRIM25 is silenced, according to reduced downstream signaling in shTRIM25 cells as shown in Figure 4A. This reviewer is wondering whether the reason for the still-high RIG-I ubiquitination in shTRIM25 cells could be because the authors blotted the IP with a pan-Ub antibody, which would also recognize K48-linked ubiquitination of RIG-I, but not with HA antibody that would specifically recognize the overexpressed HA-tagged K63-Ub. The authors should repeat this experiment and blot the IP with HA antibody instead of Ub antibody (here the authors could use a differently-tagged RTN3)

Reviewer #2:

In this manuscript Yang et al., explore the potential role of RTN3, a member of the reticulon family, in inhibition of RIG-I and TRIM25 -mediated responses. The authors show that RTN3 expression is induced during Vesicular Stomatitis virus (VSV) infection or double stranded RNA (dsRNA) stimulation, as well as TNF stimulation. Overexpression of RTN3 reduces IFNbeta and NF-κB responses upon stimulation with RIG-I (N) or VSV infection. RTN3 interacts with RIG-I and TRIM25 and is proposed that RTN3 inhibits K63-linked polyubiquitination of RIG-I resulting in inhibition of inflammation. The authors performed mouse in vivo experiments with overexpression of RTN3, which show reduced neutrophil infiltration. The authors conclude that RTN3 is a "proresolving mediator' of immune and inflammatory responses.

The main strength of this manuscript is the effort in performing in vivo mouse experiments to elucidate in a closer physiological setting the potential role of RTN3 in immunity and inflammatory responses, although the overexpression approach reduces the significance of the findings.

The weaknesses of the manuscript include the very heavy reliance on overexpression assays and the complete lack of RTN3 knockdown or knockout experiments to confirm phenotypes. The overexpression experiments also have the weakness that may not represent the real levels of RTN3 and these levels may never be reached during infection. Additional weaknesses include lack of important controls, overstatements that are not supported by the data, lack of analysis of endogenous ubiquitination, and a model/conclusion that seems not very well supported by the data.

Overall, these studies could be of importance and significant to the field if experiments are designed to address more closely the endogenous role of RTN3. Due to the lack of endogenous analysis and reliance of overexpression assays, the study is likely to have only moderate impact in the field.

Specific issues:

1) Figure 1D – why do the authors focus on TNF as the potential cytokine that induces RTN3 expression? What about type-I IFNs?

2) Figure 2A, Luciferase reporter assays: these assays are not well designed to address the role of RTN3 upon overexpression of RIG-I (N). These experiments are biased towards inhibitory effects of the overexpressed protein. RIG-I (N) needs to be carefully titrated in the absence of RTN3.

3) Figure 3D, and S1F, immunofluorescence (colocalization) experiments: need controls with each protein overexpressed alone. Also, for S1F, the authors claim there is aggregation in the endoplasmic reticulum. There is no evidence or markers of localization in the ER. This must be based on the known localization of RTN3, however, the RIG-I and TRIM25 have been proposed to localized to other compartments, including the mitochondria and in some cases in the Golgi or in mitochondrial-associated endoplasmic reticulum membranes (MAMs). Without more evidence of localization it is not clear where RTN3 localizes with RIG-I/TRIM25.

4) Lines 184 -188: "Since TRIM25 directly targets RIG-I, the coimmunoprecipitation assays were performed in RIG-I knock out HEK293T cells and the result showed that TRIM25-RTN3 interaction did not required RIG-I, we thus concluded that RTN3 directly binds to TRIM25 and excluded the possibility that RTN3 may interacts with TRIM25 through the interaction between TRIM25 and RIG-I (Figure S3 C)". : This statement is not correct. The fact that TRIM25 and RTN3 interact in RIG-I knockout cells doesn't mean that the interaction between RTN3 and TRIM25 is "direct". The interactions is independent of RIG-I, but the authors need to do in vitro binding assays using purified proteins if they want to conclude that interactions are "direct". TRIM25 has other interacting factors besides RIG-I.

5) Figures 3G, 3H and S3E, ubiquitination assays: there are a few issues with these experiments.

A) there is extensive background of Ubiquitin in the asbcence of the immunoprecipitated protein (lane 1 in figures 3G and 3H, in the IP and the IB for Ub).

B) these experiments are performed with overexpressed HA-Ub-K63, but the immunoblot is performed with anti-Ub. The immunoblot is not specific for HA-Ub-K63. In addition, these experiments do not show the covalently modified form of ubiquitinated RIG-I (or TRIM25 in figure 3H).

C) All these experiments need to show the levels of overexpressed HA-Ub-K63 in the WCL to control for the transfection efficiencies.

D) these experiments should also be performed without overexpression of ubiquitin to determine the endogenous ubiquitination of RIG-I

6) The conclusion that RTN3 is a "proresolving mediator' of immune and inflammatory responses, cannot be made when using only overexpression assays. The levels of RTN3 that are present in overexpression assays are not physiological and most likely do not represent real conditions. 'proresolving' mediator of inflammation is also an odd definition of these observation s. RIG-I has multiple negative regulators, which keep the pathway from overactivation. There is really no evidence that RTN3 acts at the 'resolving' stage of inflammation.

7) The model shown in figure S7. This implies that there are two different cells, one that is undergoing 'inflammation' and another different cell that is 'resolving' inflammation. This also is not really supported by any data. The authors would need to do single-cell an analysis of infected vs non-infected cells, as well as more detailed analysis of which cytokines are controlling inflammation or dampening neutrophil numbers. The 'resolving' cell could have nothing to do with infection, and could be responding to cytokines. In addition, this conclusion would also need the use of knockout mice.

https://doi.org/10.7554/eLife.68958.sa1

Author response

1. Most of the experiments are done with RTN3 overexpression, which clearly showed an immune dampening effect. What is the effect of RTN3 knockdown or knockout on RIG-I signaling and proinflammatory gene induction in response to virus infection or poly(I:C) stimulation? Such gene-targeting data, especially if performed in primary cells (human or mouse cells) if possible, would greatly strengthen the physiological role of RTN3.

Thank you for your suggestion. Here, we performed RTN3 knockdown experiments in PBMCs (human normal primary cells) and provided a new figure (Figure 3) that shows that RTN3 knockdown positively manipulated antiviral immune and inflammatory responses. As shown in this figure, the phosphorylation levels of TBK1, p65 and IRF3 were augmented substantially during VSV infection (Figure 3B), leading to the enhanced transcription of interferon and proinflammatory factors (Figure 3C), along with the significantly compromised antiviral responses toward viral infection (Figure 3D). Furthermore, we also provided results showing that RTN3 knockdown significantly upregulates K63-linked polyubiquitination of RIG-I under VSV infection (Figure 4—figure supplement 1F,1G).

“RTN3 depletion augments antiviral immune 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 h, and the infected cells were then settled for the following 24 h 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.”

And (Page 7, Line 236-237):

“In contrast, RTN3 depletion enhanced the K63-linked polyubiquitination level of RIG-I (Figure 4—figure supplement 1F, 1G).”

2. The authors performed extensive interaction and mechanistic studies with overexpressed RTN3, which convincingly showed that RTN3 binds to both TRIM25 and RIG-I and blocks the TRIM25-mediated K63-linked ubiquitination of RIG-I. Data assessing endogenous TRIM25-RTN3 binding/co-localization during infection or poly(I:C) stimulation, and the effect of RTN3 silencing on RIG-I signaling would further bolster the proposed mechanism.

As the reviewer suggested, we performed IP analyses in human PBMCs using anti-RTN3 antibody and IgG isotype. Consistent endogenous interactions (RTN3 interacts with both TRIM25 and RIG-I) were observed in PBMCs, and the interaction was moderately strengthened upon VSV stimulation (Figure 4F). Furthermore, the enhanced K63 ubiquitination of RIG-I was also observed under RTN3 depletion (Figure 4—figure supplement 1F, 1G). In addition, with regard to the perturbation of RIG-I signaling when RTN3 was knocked down, we also proved that depletion of RTN3 enhanced the antiviral immune and inflammatory responses after virus infection (Figure 3B, 3C).

The following description has been added to the manuscript (Page 6-7, Lines 218-220):

“Immunoprecipitation using anti-RTN3 antibody in human PBMCs infected with VSV demonstrated endogenous interaction between RTN3 and TRIM25/RIG-I (Figure 4F)”

And (Page 7, Line 236-237):

“In contrast, RTN3 depletion enhanced the K63-linked polyubiquitination level of RIG-I (Figure 4—figure supplement 1F, 1G).”

3. Figure 3H: There is not an appreciable effect of HA-RTN3 on K63-ubiquitination of TRIM25. Overall, it is unclear how enhanced TRIM25 K63-ubiquitination/auto-ubiquitination by RTN3 would lead to suppressed TRIM25 catalytic activity and thereby dampened TRIM25-mediated RIG-I K63-ubiquitination. Overall, this part of the mechanism is not very clear. This reviewer suggests moving Figure 3H to the Supplementary Data, or the authors may further discuss this.

Thank you for your comments and suggestion. We repeated these experiments, including both K63-linked polyubiquitination and WT polyubiquitination of TRIM25 mediated by overexpression of RTN3, and we detected the ubiquitination level with an anti-HA antibody but not an anti-ubiquitin antibody. A clearer tendency in the promotion of TRIM25 K63-linked polyubiquitination can now be observed (Figure 4—figure supplement 1H, 1I), probably because RTN3 might damper the delivery of K63-ubiquitin form TRIM25 to RIG-I. The fine-tuned manner of how RTN3 manipulates TRIM25 on its catalytic activity in RIG-I ubiquitination and activation is critical and of great interest; however, it is another story, and we will further explore the mechanism in our new projects.

As the reviewer and editors suggested, we have moved these results to the supplementary data (Figure 4—figure supplement 1).

4. Figure 4B (IP, right 3 lanes): Although there was no effect observed for HA-RTN3 on RIG-I ubiquitination in the absence of TRIM25, it is unclear why the overall RIG-I ubiquitination signal in shTRIM25 cells is as high as that in shCtrl cells. One would expect overall reduced RIG-I K63-ubiquitination when TRIM25 is silenced, according to reduced downstream signaling in shTRIM25 cells as shown in Figure 4A. This reviewer is wondering whether the reason for the still-high RIG-I ubiquitination in shTRIM25 cells could be because the authors blotted the IP with a pan-Ub antibody, which would also recognize K48-linked ubiquitination of RIG-I, but not with HA antibody that would specifically recognize the overexpressed HA-tagged K63-Ub. The authors should repeat this experiment and blot the IP with HA antibody instead of Ub antibody (here the authors could use a differently-tagged RTN3).

According to the suggestion of the reviewer, we repeated this experiment with Flag-tagged RTN3 and detected the ubiquitination level with an anti-HA antibody. The new result shows that the overall ubiquitination level is inferior in shTRIM25 cells compared with shCtrl cells (Figure 5B), which is consistent with the weaker phosphorylation levels of TBK1, P65 and IRF3, as shown in Figure 5A.

Previous studies have reported that TRIM4 and MEX3C are two positive regulators of RIG-I-mediated antiviral immune responses, both of which could significantly promote the K63-linked polyubiquitination of RIG-I upon virus infection (Kuniyoshi et al., 2014; Yan et al., 2014). We speculate that the remaining K63-linked polyubiquitination of RIG-I in TRIM25 knockdown cells was possibly caused by the compensatory effect of other regulators, such as TRIM4 or MEX3C, upon VSV infection.

Reviewer #2:

In this manuscript Yang et al., explore the potential role of RTN3, a member of the reticulon family, in inhibition of RIG-I and TRIM25 -mediated responses. The authors show that RTN3 expression is induced during Vesicular Stomatitis virus (VSV) infection or double stranded RNA (dsRNA) stimulation, as well as TNF stimulation. Overexpression of RTN3 reduces IFNbeta and NF-κB responses upon stimulation with RIG-I (N) or VSV infection. RTN3 interacts with RIG-I and TRIM25 and is proposed that RTN3 inhibits K63-linked polyubiquitination of RIG-I resulting in inhibition of inflammation. The authors performed mouse in vivo experiments with overexpression of RTN3, which show reduced neutrophil infiltration. The authors conclude that RTN3 is a "proresolving mediator' of immune and inflammatory responses.

The main strength of this manuscript is the effort in performing in vivo mouse experiments to elucidate in a closer physiological setting the potential role of RTN3 in immunity and inflammatory responses, although the overexpression approach reduces the significance of the findings.

The weaknesses of the manuscript include the very heavy reliance on overexpression assays and the complete lack of RTN3 knockdown or knockout experiments to confirm phenotypes. The overexpression experiments also have the weakness that may not represent the real levels of RTN3 and these levels may never be reached during infection. Additional weaknesses include lack of important controls, overstatements that are not supported by the data, lack of analysis of endogenous ubiquitination, and a model/conclusion that seems not very well supported by the data.

Overall, these studies could be of importance and significant to the field if experiments are designed to address more closely the endogenous role of RTN3. Due to the lack of endogenous analysis and reliance of overexpression assays, the study is likely to have only moderate impact in the field.

Specific issues:

1) Figure 1D – why do the authors focus on TNF as the potential cytokine that induces RTN3 expression? What about type-I IFNs?

As described in our manuscript, the detection of the transcriptional level of TNF was used to monitor the effectiveness of VSV-eGFP infection or poly(I:C) treatment toward RTN3 expression and inflammation. We observed a similar upregulation tendency of TNF and RTN3 expression during these processes; thus, we detected the relationship between TNF-α and RTN3 expression and found that TNF-α can induce RTN3 expression. We also tested the effect of IFN-β on RTN3 induction and found that IFN-β exhibits a similar induction of RTN3 expression. We have added the data and shown that IFN-β induces RTN3 upregulation (Figure 1D bottom and Figure 1E bottom).

The following description has been revised in the manuscript (Page 4, lines 120-122):

“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)”.

2) Figure 2A, Luciferase reporter assays: these assays are not well designed to address the role of RTN3 upon overexpression of RIG-I (N). These experiments are biased towards inhibitory effects of the overexpressed protein. RIG-I (N) needs to be carefully titrated in the absence of RTN3.

Thank you for your suggestion. We repeated these experiments with optimized amounts of RIG-I(N) and RTN3. In our new data, we showed dose-dependent inhibition of ISRE-luc, IFNβ-luc and NF-κB-luc by RTN3 (Figure 2A).

3) Figure 3D, and S1F, immunofluorescence (colocalization) experiments: need controls with each protein overexpressed alone. Also, for S1F, the authors claim there is aggregation in the endoplasmic reticulum. There is no evidence or markers of localization in the ER. This must be based on the known localization of RTN3, however, the RIG-I and TRIM25 have been proposed to localized to other compartments, including the mitochondria and in some cases in the Golgi or in mitochondrial-associated endoplasmic reticulum membranes (MAMs). Without more evidence of localization it is not clear where RTN3 localizes with RIG-I/TRIM25.

According to the reviewer’s suggestion, we have added images of the ctrl groups, including GFP-TRIM25, GFP-RIG-I and mCherry-RTN3, separately to the main panel (the previous Figure 3D, now renamed Figure 4D). In revised Figure 1—figure supplement 1G, we also added images of mCherry-Ev with/without poly(I:C) stimulation.

To strengthen our conclusion that RTN3 aggregates in the endoplasmic reticulum, we supplemented the ER marker calnexin in confocal IF images, which clearly showed the colocalization of RTN3 and calnexin within the aggregates after poly(I:C) stimulation (Figure 1—figure supplement 1I). These images clearly confirmed the known localization of RTN3 in the ER, either non-aggregated or aggregated, as well as RTN3 colocalization with RIG-I/TRIM25 in the ER.

The following description has been added to the manuscript (Page 4-5, lines 136-138):

“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).”

4) Lines 184 -188: "Since TRIM25 directly targets RIG-I, the coimmunoprecipitation assays were performed in RIG-I knock out HEK293T cells and the result showed that TRIM25-RTN3 interaction did not required RIG-I, we thus concluded that RTN3 directly binds to TRIM25 and excluded the possibility that RTN3 may interacts with TRIM25 through the interaction between TRIM25 and RIG-I (Figure S3 C)". This statement is not correct. The fact that TRIM25 and RTN3 interact in RIG-I knockout cells doesn't mean that the interaction between RTN3 and TRIM25 is "direct". The interactions is independent of RIG-I, but the authors need to do in vitro binding assays using purified proteins if they want to conclude that interactions are "direct". TRIM25 has other interacting factors besides RIG-I.

Thank you for your comment and suggestion. We agree that this is an imprecise statement, and the description of “direct interaction” requires data from an in vitro pulldown assay. Considering that the specific transmembrane domains of RTN3 inserted in the endoplasmic reticulum membrane are required for the interaction with TRIM25/RIG-I and its inhibitory function, as elucidated in Figure 5 C, 5D and Figure 5—figure supplement 1, we speculated that the interaction between RTN3 and TRIM25 or RIG-I would need a certain condition. Thus, we have replaced our statement “…directly binds…” with “…independent on RIG-I”.

The following description has been adjusted in the manuscript (Page 6, lines 211-212):

“thus we concluded that RTN3 binds to TRIM25 independent of RIG-I…”

5) Figures 3G, 3H and S3E, ubiquitination assays: there are a few issues with these experiments.

A) There is extensive background of Ubiquitin in the asbcence of the immunoprecipitated protein (lane 1 in figures 3G and 3H, in the IP and the IB for Ub).

B) These experiments are performed with overexpressed HA-Ub-K63, but the immunoblot is performed with anti-Ub. The immunoblot is not specific for HA-Ub-K63. In addition, these experiments do not show the covalently modified form of ubiquitinated RIG-I (or TRIM25 in figure 3H).

C) All these experiments need to show the levels of overexpressed HA-Ub-K63 in the WCL to control for the transfection efficiencies.

D) These experiments should also be performed without overexpression of ubiquitin to determine the endogenous ubiquitination of RIG-I

Since a new figure (new Figure 3) was incorporated into the main figures, the panel orders have changed as follow:

Original Figure 3 G → Figure 4H

Original Figure 3 H → Figure 4—figure supplement 1H

Original Figure S3 E → Figure 4—figure supplement 1I

New panel → Figure 4—figure supplement 1E

For comment A:

We have optimized our protocols and improved these images of polyubiquitination assays, in order to decrease the Ubs background in the ctrl lane. As shown in (Figure 4H, Figure 4—figure supplement 1E), the improved images clearly show that RTN3 inhibit RIG-I K63- linked polyubiquitination, although there is still a weak background, which might be caused by nonspecific interaction of the beads or anti-GFP antibody.

For comment B, C:

As the reviewer suggested, we detected the K63-linked or WT polyubiquitination with anti-HA antibody, and HA-K63 Ub bands of WCL also supplemented. Consistent results were obtained which again demonstrated that overexpressed RTN3 inhibits K63-linked polyubiquitination of RIG-I (Figure 4H, Figure 5B), and promotes K63-linked and WT polyubiquitination of TRIM25 (Figure 4—figure supplement 1H, 1I), upon VSV infection.

For the detection of “covalently modified form of ubiquitinated RIG-I (or TRIM25)”, we have optimized our protocols and improved the images to greatly diminish the nonspecific or degraded bands of ubiquitinated RIG-I. In fact, the background and nonspecific bands of Ub-linked RIG-I are closely related to the intensity in the western blot analysis of immunoprecipitated sample, if the exposure becomes weak, these background and nonspecific bands could not even be seen.

For comment D:

We have performed an additional experiment using anti-K63-linkage Specific Polyubiquitin Rabbit mAb (Cell Signaling Technology, #5621) to detect RTN3-mediated endogenous K63-linked polyubiquitination of RIG-I under VSV infection. A similar result was obtained (Figure 4—figure supplement 1E), clearly elucidating the inhibitory function of RTN3 toward endogenous RIG-I K63-linked polyubiquitination.

6) The conclusion that RTN3 is a "proresolving mediator' of immune and inflammatory responses, cannot be made when using only overexpression assays. The levels of RTN3 that are present in overexpression assays are not physiological and most likely do not represent real conditions. 'proresolving' mediator of inflammation is also an odd definition of these observation s. RIG-I has multiple negative regulators, which keep the pathway from overactivation. There is really no evidence that RTN3 acts at the 'resolving' stage of inflammation.

Thank you for your comments and suggestion. We agree that “proresolving mediator” is not an appropriate definition and then it has changed to “negative regulator”.

To strengthen our speculation of the inhibitory role of RTN3 in modulating RIG-I-mediated antiviral immune responses, we depleted RTN3 expression using shRNA in human PBMCs and challenged these cells with VSV. Consist with overexpression experiments, RTN3-depleted cells exhibited the enhanced phosphorylation level of TBK1, p65 and IRF3 upon VSV infection (Figure 3B), which leading the higher expression levels of type I interferon and proinflammatory cytokines (Figure 3C), eventually causing stronger resistance to viral infection (Figure 3D). In addition, we also provided data showing that RTN3 knockdown significantly enhanced K63-linked polyubiquitination of RIG-I under VSV infection (Figure 4—figure supplement 1F, 1G).

The following description has been added to the manuscript (Page 6, Lines 183-195):

“RTN3 depletion augments antiviral immune 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 h, and the infected cells were then settled for the following 24 h 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.”

And (Page 7, Line 236-237):

“In contrast, RTN3 depletion enhanced the K63-linked polyubiquitination level of RIG-I (Figure 4—figure supplement 1F, 1G).”

7) The model shown in figure S7. This implies that there are two different cells, one that is undergoing 'inflammation' and another different cell that is 'resolving' inflammation. This also is not really supported by any data. The authors would need to do single-cell an analysis of infected vs non-infected cells, as well as more detailed analysis of which cytokines are controlling inflammation or dampening neutrophil numbers. The 'resolving' cell could have nothing to do with infection, and could be responding to cytokines. In addition, this conclusion would also need the use of knockout mice.

Thank you for your insightful comment and suggestion. We agree that our model that drawing two different cells to symbolize “inflammation” and “resolving inflammation” was imprecise, since the inflammation and resolution happen at the tissue level but not in a single cell. Our studies reveal that RTN3 negatively regulates RIG-I-mediated antiviral immune and inflammatory responses, thus the working model was reprogrammed and improved to diagram the inhibition and mechanism of RIG-I mediated antiviral responses by RTN3.

We moved it (the previous Figure S7) to main figures as Figure 8 and figure legend was added.

Finally, RTN3 knockout mice is not available currently, probably because RTN3 is an essential housekeeping gene for cell survival and RTN3 knockout causes cell death.

Following description had been added to the manuscript:

“Figure 8. The working model of the inhibition of RIG-I-mediated antiviral responses by RTN3

Upon RNA virus infection, the RIG-I signalosome is activated by TRIM25-mediated K63-linked polyubiquitination and then triggers IRF3 and NF-κB activation and the expression of interferons, cytokines and inflammatory factors. On the other hand, RTN3 is upregulated by acute viral infection and interferon/inflammatory factors in an ER stress-dependent manner. RTN3 is subsequently aggregated on the endoplasmic reticulum, interacts with RIG-I and TRIM25, rapidly diminishes the K63-linked polyubiquitination of RIG-I and hence suppresses antiviral and inflammatory responses. The inflammatory response is consequently mitigated, and the resolution of acute inflammation is initiated.”

Finally, we greatly appreciate all the reviewers and editors for the helpful comments and invaluable suggestions. We have improved our manuscript according to the comments and suggestions and addressed all of the points as described above.

References:

Grumati, P., G. Morozzi, S. Holper, M. Mari, M.I. Harwardt, R. Yan, S. Muller, F. Reggiori, M. Heilemann, and I. Dikic. 2017. Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy. eLife 6:

Kuniyoshi, K., O. Takeuchi, S. Pandey, T. Satoh, H. Iwasaki, S. Akira, and T. Kawai. 2014. Pivotal role of RNA-binding E3 ubiquitin ligase MEX3C in RIG-I-mediated antiviral innate immunity. Proc Natl Acad Sci U S A 111:5646-5651.

Yan, J., Q. Li, A.P. Mao, M.M. Hu, and H.B. Shu. 2014. TRIM4 modulates type I interferon induction and cellular antiviral response by targeting RIG-I for K63-linked ubiquitination. J Mol Cell Biol 6:154-163.

https://doi.org/10.7554/eLife.68958.sa2

Article and author information

Author details

  1. Ziwei Yang

    1. Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China
    2. Key Laboratory of Tropical Disease Control (Sun Yat-Sen University), Ministry of Education, Guangzhou, China
    Contribution
    Formal analysis, Investigation, Methodology, Writing - original draft
    Contributed equally with
    Jun Wang and Bailin He
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3362-0946
  2. Jun Wang

    Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China
    Contribution
    Formal analysis, Methodology
    Contributed equally with
    Ziwei Yang and Bailin He
    Competing interests
    No competing interests declared
  3. Bailin He

    Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China
    Contribution
    Formal analysis, Methodology
    Contributed equally with
    Ziwei Yang and Jun Wang
    Competing interests
    No competing interests declared
  4. Xiaolin Zhang

    Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  5. Xiaojuan Li

    1. Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China
    2. Key Laboratory of Tropical Disease Control (Sun Yat-Sen University), Ministry of Education, Guangzhou, China
    Contribution
    Conceptualization, Supervision, Funding acquisition
    For correspondence
    lixjuan3@mail.sysu.edu.cn
    Competing interests
    No competing interests declared
  6. Ersheng Kuang

    1. Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China
    2. Key Laboratory of Tropical Disease Control (Sun Yat-Sen University), Ministry of Education, Guangzhou, China
    Contribution
    Conceptualization, Supervision, Funding acquisition, Project administration, Writing - review and editing
    For correspondence
    kuangersh@mail.sysu.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4976-3311

Funding

National Natural Science Foundation of China (81871643)

  • Ersheng Kuang

National Natural Science Foundation of China (32061143008)

  • Ersheng Kuang

National Natural Science Foundation of China (81971928)

  • Xiaojuan Li

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

Acknowledgements

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.

Ethics

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.

Senior Editor

  1. Sara L Sawyer, University of Colorado Boulder, United States

Reviewing Editor

  1. Michaela Gack, Cleveland Clinic Lerner Research Institute, United States

Publication history

  1. Preprint posted: December 2, 2020 (view preprint)
  2. Received: March 30, 2021
  3. Accepted: July 12, 2021
  4. Version of Record published: July 27, 2021 (version 1)
  5. Version of Record updated: September 9, 2021 (version 2)

Copyright

© 2021, Yang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Further reading

    1. Cell Biology
    Lisa M Strong et al.
    Research Article Updated

    Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double-membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12–5–16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12–5–16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven-bladedß -propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207–230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 ß-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12–5–16 L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12–5–16 L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand and ATG8 lipidation on the other.