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. 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 . Upon viConsidering 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 . Some moAlthough 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 . InflammViral infections also trigger different ER stresses due to the excessive synthesis and accumulation of viral proteins in the ER lumen or through signal hijacking . In respIn our previous unpublished experiments, we observed that RTN3 is dramatically upregulated upon RNA viral infection when using VSV as a stimulus.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.RTN3 mRNA upregulation (TNF mRNA levels that were used to indicate the effectiveness of VSV-eGFP or poly(I:C) treatment toward inflammatory induction in stimulated cells  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 , top or gulation , top acced cells , bottom.ed cells  and mRNAed cells . In addied cells . These rSince 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  and mRNATo 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 . In addiHA-RTN3 vs. THP-1HA-Ev)  , which eTo 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 , left. QIFNB1, IFIT2, IFIT1, TNF and the proinflammatory cytokines CCL20 and IL6 during VSV infection ], 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  expression. RT-PCR results showed that RTN3-overexpressing cells exhibited a deficiency in nfection . Similarnfection . Becausepression . These rRTN3 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  in human healthy donor PBMCs , in whicnfection . Furthernfection , while tnfection . CollectRIG1 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 -binding protein through mass spectrometry analysis , and TRInd RIG-I . Besidesnd RIG-I  as well nd RIG-I . Furthernd RIG-I , indicatnd RIG-I . CollectWe 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 , excludiSubsequently, we investigated the polyubiquitination of RIG-I upon VSV infection in the absence or presence of RTN3 overexpression. As shown in 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) . SimilarSubsequently, 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  and wereRtn3 in liver extracts were significantly higher in the VSV- and poly(I:C)-treated mice than in those of the PBS control mice  or VSV-eGFP separately via intravenous (I.V.) injection. Real-time PCR results showed that the mRNA levels of rol mice , and Rtnrol mice . These r+Gr1+ neutrophils  or the empty vector (HA-Ev) and then inoculated with VSV-eGFP via intraperitoneal (I.P.) injection to infect mice for another 12 hr . At 48 htrophils  dramatict change . Taken tBased 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 , and theRTN3 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 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 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 , neutropTherefore, we propose a working model that describes how RTN3 suppresses RIG-I-mediated antiviral innate immune responses . Upon RNIn 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.7 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.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  , 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.7 plaque-forming units (PFU)/g for 12 hr. For the in vitro study, cells were infected with VSV-eGFP  analyses) or SeV (MOI = 0.1 for immune response activation).VSV-eGFP and VSV  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 × 10RTN3 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  and a ClonExpressII One Step Cloning Kit  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.The plasmids used in the present study were constructed as follows. 6 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  was used for TRIM25-knockdown HEK293T cell selection, and immunoblot analysis was performed to determine the knockdown efficiency. For TRIM25 knockdown-HEK293T cells generation, the following TRIM25 shRNA sequences were used: (1), 5’- CCGGAACAGTTAGTGGATTTA-3’; (2), 5’-GAACTGAACCACAAGCTGATA-3’. Both sequences target the coding region exon1 of TRIM25 gene and cloned into pLKO.1 lentivirus expression vector. The same methods were performed for transient RTN3 knockdown with the following shRNA sequences: (1), 5’- CCCGATTGTCTATGAGAAGTA-3’; (2), 5’-CTTTGGCACCACGCTGATCAT-3’.HEK293T cells were seeded into six-well plates at a density of 0.5 × 10HEK293T 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  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.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 Total RNA was extracted from HEK293T or A549 cells dissolved in TRIzol reagent  and then reverse transcribed into cDNA using HiScript III RT SuperMix (Vazyme). A SYBR Green I Master Mix kit  was used for real-time PCR with a LightCycler 480 system (Roche). The following primers were used for the assay: Human For coimmunoprecipitation assays, cells transfected and stimulated with the appropriate ligands were lysed with IP buffer , 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  and further incubated with the indicated primary and secondary antibodies. The images were visualized using an Odyssey Sa system (LI-COR).@EZ slides  overnight and then transfected using Lipofectamine 2000. Twenty-four hours posttransfection, the cells were carefully washed with 1× PBS three times  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.WT HeLa cells were seeded in 8-well MillicellInfected mice were sacrificed at the indicated timepoints, and their livers and lungs were dissected and fixed in 4% PFA. After embedding, paraffin sections were stained with hematoxylin and eosin (H and E) or subjected to standard immunohistochemical staining (IHC) before being visualized by bright field microscopy.The data are presented as the means ± SD , 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. 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: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.Thank you for submitting your article "RTN3 inhibits RIGI-I-mediated antiviral responses by impairing TRIM25-mediated K63-linked polyubiquitination" for consideration by 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 : 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  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 ". : 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 .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-I6) 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. 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  and provided a new figure  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 , leading to the enhanced transcription of interferon and proinflammatory factors , along with the significantly compromised antiviral responses toward viral infection . Furthermore, we also provided results showing that RTN3 knockdown significantly upregulates K63-linked polyubiquitination of RIG-I under VSV infection .“RTN3 depletion augments antiviral immune responsesTo further elucidate the physiological function of RTN3 in antiviral responses, we depleted its expression by short hairpin RNAs (shRNAs) in human healthy donor PBMCs , in which RTN3 normally undergoes upregulation during viral infection . 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 . 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 . 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 , while the amplification of VSV was compromised . Collectively, depressed RTN3 expression facilitates antiviral innate immune and inflammatory responses upon viral infection.”And :“In contrast, RTN3 depletion enhanced the K63-linked polyubiquitination level of RIG-I .”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 . Furthermore, the enhanced K63 ubiquitination of RIG-I was also observed under RTN3 depletion . 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 .The following description has been added to the manuscript :“Immunoprecipitation using anti-RTN3 antibody in human PBMCs infected with VSV demonstrated endogenous interaction between RTN3 and TRIM25/RIG-I ”And :“In contrast, RTN3 depletion enhanced the K63-linked polyubiquitination level of RIG-I .”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 , 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 .4. Figure 4B : 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 , 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 . 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?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 .As described in our manuscript, the detection of the transcriptional level of The following description has been revised in the manuscript :“Interestingly, we observed that both TNF-α and IFN-β could upregulate RTN3 expression at both the protein  and mRNA levels ”.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 .3) Figure 3D, and S1F, immunofluorescence  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 . 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 . 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 :“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) .”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 ". 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 :“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 .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-ISince a new figure  was incorporated into the main figures, the panel orders have changed as follow:Original Figure 3 G → Figure 4HOriginal Figure 3 H → Figure 4—figure supplement 1HOriginal Figure S3 E → Figure 4—figure supplement 1INew panel → Figure 4—figure supplement 1EFor 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 , 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 , and promotes K63-linked and WT polyubiquitination of TRIM25 , 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  to detect RTN3-mediated endogenous K63-linked polyubiquitination of RIG-I under VSV infection. A similar result was obtained , 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 , which leading the higher expression levels of type I interferon and proinflammatory cytokines , eventually causing stronger resistance to viral infection . In addition, we also provided data showing that RTN3 knockdown significantly enhanced K63-linked polyubiquitination of RIG-I under VSV infection .The following description has been added to the manuscript :“RTN3 depletion augments antiviral immune responsesTo further elucidate the physiological function of RTN3 in antiviral responses, we depleted its expression by short hairpin RNAs (shRNAs) in human healthy donor PBMCs , in which RTN3 normally undergoes upregulation during viral infection . 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 . 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 . 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 , while the amplification of VSV was compromised . Collectively, depressed RTN3 expression facilitates antiviral innate immune and inflammatory responses upon viral infection.”And :“In contrast, RTN3 depletion enhanced the K63-linked polyubiquitination level of RIG-I .”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  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 RTN3Upon 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:eLife 6: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. Proc Natl Acad Sci U S A 111:5646-5651.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. J Mol Cell Biol 6:154-163.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.


Begomovirus, is a prolific monopartite begomovirus in the Indian sub-continent. CYVMV infects multiple crop plants to cause leaf curl disease. Plants have developed host RNA silencing mechanisms to defend the threat of viruses, including CYVMV. We characterized four RNA silencing suppressors, namely, V2, C2, and C4 encoded by CYVMV and betasatellite-encoded C1 protein (βC1) encoded by the cognate betasatellite, croton yellow vein betasatellite (CroYVMB). Their silencing suppressor functions were verified by the ability of restoring the β-glucuronidase (GUS) activity suppressed by RNA silencing. We showed here for the first time that V2 was capable of self-interacting, as well as interacting with the V1 protein, and could be translocalized to the plasmodesmata in the presence of CYVMV. The knockout of either V2 or V1 impaired the intercellular mobility of CYVMV, indicating their novel coordinated roles in the cell-to-cell movement of the virus. As pathogenicity determinants, each of V2, C2, and C4 could induce typical leaf curl symptoms in Nicotiana benthamiana plants even under transient expression. Interestingly, the transcripts and proteins of all four suppressors could be detected in the systemically infected leaves with no correlation to symptom induction. Overall, our work identifies four silencing suppressors encoded by CYVMV and its cognate betasatellite and reveals their subcellular localizations, interaction behavior, and roles in symptom induction and intercellular virus movement.Croton yellow vein mosaic virus (CYVMV), a species in the genus  Begomovirus of the family Geminiviridae cause severe diseases in several crops and reduce their potential yield (Bemisia tabaci)  that encode the coat protein  and precoat protein (AV2) in the viral sense strand, and the replication initiator protein , transcriptional activator protein , replication enhancer protein , and AC4 protein in the complementary strand. DNA-B contains two ORFs encoding nuclear shuttle protein  and movement protein  in the viral-sense and complementary strands, respectively  . The genectively . Both DNectively .Some monopartite begomoviruses are virulent alone , but theBegomovirus, is a prolific begomovirus in the Indian subcontinent. CYVMV was first reported from the weed host Croton sparsiflorus and later found on other crops such as Jatropha gossypifolia (Croton yellow vein mosaic virus (CYVMV), a species in the genus ypifolia , clusterypifolia , radish ypifolia , rapeseeypifolia , and Craypifolia . Under typifolia . Althougypifolia , it prodypifolia , which cypifolia . Dependiypifolia , which iBegomovirus infection triggers RNA silencing defense response in host plants . As a reIn this report, we demonstrated that similar to other begomoviruses, CYVMV and its cognate betasatellite CroYVMB encode four silencing suppressors  to counteract RNA silencing-mediated plant defense mechanisms. To decipher the mechanism of suppressor functions, we determined the sub-cellular localization of the suppressors both in the presence or absence of the viral infection. We further analyzed the interactions of CYVMV silencing suppressors as well as their roles in pathogenicity and cell-to-cell movement.Nicotiana benthamiana leaves and the Agrobacterium strain GV3101 were used for agroinfiltration assays as previously described  and virus-specific primers with attB adaptors. Each amplicon was first cloned into the pENTR/D-TOPO Gateway vector and then inserted into pEarleyGate103  via the LR reaction. For agroinfiltration, Agrobacterium pellet from 50 ml culture was resuspended in the infiltration buffer  and adjusted to an OD600 of 0.5. Four leaves from the same N. benthamiana plant were infiltrated with different combinations. For each combination, equal volumes of different cultures with same OD600 were mixed and used in agroinfiltration. Three biological replicates were taken for each experiment. All the primers used in this study are listed in The CYVMV infectious clone has been described previously . In the escribed . Moderatpromoter . The GUSpromoter . The kno4, pH 7.0, 5 mM dithiothreitol, 10 mM Na2EDTA, 0.1% sodium lauryl sarcosine, and 0.1% Triton-100) was added, and 100 μl extract was mixed with 40 μl 10 mM fluorescent substrate of 4-methylumbelliferyl β-D-glucoside (MUG) . After incubation at 37°C for 1 h, the reaction was stopped with 1 ml stop buffer (0.2 M sodium carbonate). After mixing well with the stop buffer, 100 μl of mixture was loaded onto a 96-well black microtiter plate . Two replicate wells were carried out for each sample. The methylumbelliferone (MU)  standard curve was included in each plate. Furthermore, 2.5 μM, 625 nM, 156 nM, and 39 nM MU gradients were used to generate the standard curve. The fluorescence intensity of MU was measured in a Molecular Devices SpectraMax M2 microplate reader .β-Glucuronidase staining was performed as described previously . For theV2, C2, C4, and βC1 genes were cloned into pEarleyGate103 with a C-terminal green fluorescent protein (GFP) fusion tag. These GFP fusion constructs were infiltrated individually or co-infiltrated with the CYVMV infectious clone into N. benthamiana leaves. After 3 days, infiltrated leaves were examined by a Leica confocal laser-scanning microscope to determine the subcellular localizations of the GFP fluorescent signal as described earlier ( earlier . Leaves Yeast two-hybrid (Y2H) assay was described previously . The codAgrobacterium tumefaciens GV3101 for N. benthamiana leaf infiltration. After a 48-h incubation, leaves were examined using a Leica TCS SP8 X confocal laser-scanning microscope, and the LAS X software  was used for image processing. The protein-protein interaction was indicated by the restoration of yellow fluorescent protein (YFP). YFP fluorescence was visualized with an excitation of 514 nm and emission collected from 530 to 600 nm. The bright-field signal to show the cell walls was collected with a PMT detector.The coding sequence of V2 was cloned into pSITE-cEYFP-N1 and pSITE-nEYFP-C1, respectively. Both constructs were then introduced into The pull-down assay was performed as described previously . V1 and HindIII and EcoRI to create a pGreenII-IR construct. Similarly, a V2-IR region was amplified using AR11 and AR15 primers and cloned into the pGreenII vector using HindIII and EcoRI to create a pGreenII-V2-IR construct. These two initial constructs served the partial dimeric portion of the knockout constructs. Furthermore, a V2 deleted portion of CYVMV was amplified using primer combination AR13 and AR14 and cloned into pGreenII-IR using SpeI and NotI to generate V2-knockout construct of CYVMV (CYVMV-KoV2). Similarly, a V1 deleted portion of the CYVMV was amplified using primers AR17 and AR19 and cloned into pGreenII-V2-IR using SpeI and NotI to generate V1-knockout construct of CYVMV (CYVMV-KoV1). Next, a portion of the CYVMV genome containing the IR and C1, C2, and C3 ORFs was amplified using AR17 and AR14 and cloned into pGreenII-IR using SpeI and NotI to generate a double-knockout construct of V1 and V2 ORFs of CYVMV (CYVMV-KoV1-V2). All three knockout constructs were designed to retain the multiple cloning sites of EcoRI, XmaI, SmaI, and SpeI from pGreenII-IR. CYVMV-KoV1-GFP and CYVMV-KoV2-GFP constructs were generated by amplifying the GFP gene using AR20 and AR21 primers and cloning it into EcoRI/SmaI sites of the corresponding knockout constructs. The GFP expression was driven by the viral promoter in the IR region. Primer details are given in To create infectious knockout constructs, it is essential to create partial tandem dimeric repeat constructs where the gene to knockout is absent. To develop such constructs, initially, the intergenic region (IR) of the CYVMV was amplified from the genome of CYVMV using primers AR11 and AR12 and cloned into pGreenII vector using N. benthamiana leaves. Inoculation of CYVMV agroinfectious clone served as a control. At 15 dpi, DNA was isolated from the agroinfiltrated tissue and systemically infected leaves. PCR was carried out using a set of abutting primers, i.e., AR40 and AR41, which were used to amplify the viral replicon. GFP epifluorescence was measured under UV light, and the intracellular GFP expression was analyzed using confocal microscopy. Maps of constructs and deletion mutants, episomal replicon released from the plasmid vector backbone, and locations of the abutting primers are shown in To understand the replication behavior of these constructs and the symptom phenotype produced by them, all the knockout constructs alone as well as with GFP were agroinfiltrated into N. benthamiana plants were agroinfiltrated individually for each of the suppressor protein constructs in pEarleyGate103 vector; their impact on symptom development, if any, was monitored regularly. To assess the transportation of the transcripts of the suppressor gene constructs in the systemically infected leaf tissues, RNA was isolated from the systemically infected leaves of the suppressor construct-inoculated plants, and a reverse-transcriptase PCR (RT-PCR) was performed for pulled samples of three systemically infected leaves of all the five replications corresponding to all of the four suppressor-inoculated plants. The expression of the suppressor proteins in the systemically infected leaves was analyzed through confocal microscopy as described earlier.A set of five Nicotiana benthamiana plants showed the leaf curl symptom 10 days after co-agroinfiltration of a partial tandem repeat construct of CYVMV and a dimeric construct of CroYVMB. V2 (357 bp), C2 (405 bp), and C4 (258 bp) genes from CYVMV and βC1 (357 bp) gene from CroYVMB were amplified (excluding their stop codon) using the DNA isolated from these agroinfiltrated plants. Amplicons were cloned and sequenced to investigate the putative RNA silencing suppressor function of their encoded products. Transient silencing suppression assays were conducted on four leaves of the same N. benthamiana plant. After infiltration with Agrobacterium carrying pGNGC-CBSm:GUS , the leaves showed a moderate level of GUS expression. In contrast, dramatically reduced GUS staining signals were observed in leaves co-infiltrated with Agrobacterium carrying pGNGC-CBSm:GUS and pRNAi-GG-GUS , indicating the suppression of the GUS expression by RNA silencing. When added with the third co-infiltrator of V2, C2, C4, or βC1 harbored by pEarleyGate103 , GUS signals were restored in all infiltrated leaves, demonstrating the silencing suppressor function of these four proteins  showed similar levels of GUS accumulation, which was significantly reduced by the co-infiltration of pRNAi-GG-GUS (GUS + GUS-hp). Consistent with our qualitative results, additional co-infiltrations of V2, C2, C4 or βC1 (GUS + GUS-hp + Suppressor) all suppressed RNA silencing and thereby restored GUS accumulation, with V2 being the most effective silencing suppressor (∼85% GUS restoration efficiency). The restoration values with C2, C4, and βC1 were around 50–60%. As a positive control, HC-Pro consistently restored the GUS signal -tagged V2 (V2-GFP) alone was found to aggregate as large clumps in various locations within the cytoplasm, mainly surrounding the nucleus . Co-infiWhether the CYVMV silencing suppressors are capable of physically interacting with one another and themselves are yet to be investigated. Virus might use such interactions for their own replication and spread. Thus, potential self- or mutual interactions among V2, C2, C4, and βC1 were tested by targeted Y2H assays . Only thin vivo self-interaction of V2 was tested using bimolecular fluorescence complementation. The full-length V2 gene was cloned to fuse with N- and C-terminal fragments of YFP and co-infiltrated onto the N. benthamiana leaves. Infiltration with V2-cYFP + nYFP-V2 constructs resulted in strong yellow fluorescent signals  constructs were created to infect d leaves  when usid leaves . Abuttind leaves . We usedither V1  or V2 . Since V1 and V2 ORFs were carried by two different constructs for complementation, the C2 protein might be incapable of activating the promoters of individual V1 and V2 ORFs to generate enough transcripts, which could lead to the complementation failure.via agroinfiltrating their respective overexpression constructs (fused with eGFP in pEarleyGate103) to N. benthamiana leaves. Five plants were infiltrated for each construct. Four, two, and three out of five infiltrated plants showed variable leaf curl symptoms with regard to V2-GFP, C2-GFP, and C4-GFP constructs, respectively   . Begomovs (TGMV) . We founnthetase . WhetherV2 is considered as the strongest PTGS suppressor encoded by begomoviruses . It may V2 can suppress both local and systemic RNA silencing , which ivia the direct interaction with V1. This model is consistent with our observation that the deletion of either V1 or V2 gene from CYVMV impairs virus mobility in planta. Since V1 and V2 gene transcription is controlled by a single viral promoter in the same direction  designed from C1 ORF. For the mini viral replicon assay, this is a commonly used method . Since V1 functions as a CP, V2 possibly mediates the CYVMV movement irection , their sd method . The prid method . Our worN. benthamiana plants  are involved in the development of the host symptom, which is consistent with the previously reported pathogenesis roles of their orthologs from other begomoviruses. For example, the C2 protein of bhendi yellow vein mosaic virus plays an important role in symptom determination and viral genome replication in a plants . A singla plants . The patase BAM1 . The symase BAM1  but not In conclusion, through this study, we characterized four silencing suppressor proteins encoded by CYVMV and its cognate betasatellite, showed their subcellular localizations, and established their roles in pathogenicity. In addition, we demonstrated the self-interaction of V2 protein and the interaction between V2 and the CP (product of V1 ORF). The absence of either protein inhibited the cell-to-cell movement of the virus. In future studies, the identification of host factors interacting with these suppressor proteins and/or repressing the expression of their coding genes would deepen our understanding of CYVMV pathogenicity mechanisms and provide new clues for the development of disease management strategies.The original contributions presented in the study are included in the article/HRP and AR conceived and designed the research. YZ, AR, HP, DM, and GK performed the experiments, collected the data, and analyzed the results. YZ, AR, HP, and HRP wrote the manuscript. BM and SM provided input to the experimental design and discussion. All the authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Biologic agents are increasingly used for many autoimmune and inflammatory conditions, as they are both steroid sparing and can potentially induce and maintain remission. Notably tumor necrosis factor (TNF) alpha antagonists are particularly useful in inflammatory bowel disease (IBD) such as Crohn’s disease (CD) and ulcerative colitis (UC).Infliximab is a chimeric monoclonal antibody that targets TNF alpha (cytokine involved in modulation of inflammatory responses) and neutralizes its effects. As infliximab is a generic TNF alpha inhibitor, it can cause non-specific immune mediated side effects in addition to its intended therapeutic effect on the target organ .We present a case of a gentleman developing a rare dermatological side effect of an acneiform reaction, after the use of infliximab for his CD. Monitoring anti-TNF alpha antibodies may help identify patients at a higher risk of developing adverse reactions. In addition, gut specific biologic agents (vedolizumab) may be the next preferable step in individuals with IBD who demonstrate reactions and/or intolerance to non-specific TNF alpha inhibitors. Increasingly, inflammatory bowel disease (IBD) is being treated with biologic agents, to try and achieve control of flares and in many cases they are able to achieve remission. These agents are often reserved for escalation of treatment after failure of previous first line treatments  or intolerance to these in addition to five aminosalicylates especially in ulcerative colitis (UC).There are different types of biologic agents used in IBD, including TNF-α inhibitors namely infliximab, adalimumab, golimumab, an IL-12/23 inhibitor (ustekinumab), and an α4β7 integrin inhibitor (vedolizumab). Of these, the most commonly used agents are those that have longer safety evidence notably infliximab and adalimumab [As these agents target the immune system and have variable levels of specificity to their target organ/lesions, their potential to cause a range of immune-mediated side effects is one of their main adverse risks. Listed for infliximab in particular, the most common side effects include infections, rashes, infusion reactions, hypersensitivity reactions, formation of autoantibodies, Lupus like syndrome, serum sickness, vasculitis, and exanthum . The cutWe present the case report of a 44-year-old gentleman with Crohn’s colitis, diagnosed in 2011, who was referred to Bedford Hospital by the IBD nurses due to a rash.He is a gentleman who had previously been documented as intolerant to five aminosalicylates, azathioprine, and six-mercaptopurine . His treatment was thus escalated to infliximab in September 2012. The dosing and interval were guided by British National Formulary (BNF) and NICE  guidance. He had a dose of 120 mg of infliximab at week 0, week 2, and week 6. This was followed by an eight-weekly regime of infliximab. The regular infusions were stopped in June 2015, on the patient’s own volition, as he felt well and asymptomatic from his IBD and thus felt he no longer needed it.He then developed symptoms consistent with a clinical flare-up of Crohn’s in March 2019 and a sigmoidoscopy showed moderate to severely active left colonic Crohn’s disease (CD), with histology suggestive of moderate to severe active chronic proctocolitis. A multidisciplinary team (MDT) meeting was held in May 2019 and a decision was made to restart infliximab infusions.The offending rash then developed after receiving the first two loading doses administered at week 0 and week 2. There was no difference in dosing of infliximab as compared to his initial regime. The rash was described as acneiform eruptions on the back and neck plus eczematous lesions on elbows Figures -2. The eHis past medical history included no previous significant dermatological problems. During review it was noted that his bowel frequency was up to 10 times per day with a stool consistency of type 6/7 mixed with blood as per the Bristol Stool Chart; he also had three deep mouth ulcers, which were limiting his oral intake.During his admission he had a dermatology review and they felt that this was an acneiform eruption, secondary to his anti-TNF alpha therapy. No biopsies were taken by the dermatology team. In literature the usual histopathological description given is of eosinophilic pustular folliculitis with eosinophils predominantly present within hair follicles .This is a rare reaction. From our literature review, it was noted that acneiform reactions in patients treated with anti-TNFα therapy have only been reported in 11 cases (to the best of our knowledge) (Table Recommended treatment for the patient was to stop infliximab and consider acitretinoid if needed. However, our patient continued to improve without the retinoids Figure .Gastroenterology opinion was to avoid TNF alpha inhibitors in the future and the patient was switched to more gut specific biologic, in this case vedolizumab as it offers a better safety profile in terms of systemic side effects .Although infliximab is a widely studied and used biologic agent in cases where patients do not respond to conventional treatment options for IBD, there are reported dermatological presentations associated with its use. Acneiform eruptions, however, are a rarely reported type of such reactions.Recommendations are to test antibody levels to the drug if there is suspicion that the drug might be the offending agent. Literature review suggests that the most common time in which such acneiform eruptions are found is roughly one to two months after starting the agent. This correlates with our patient’s time of presentation. In addition, the suggestions are that the drug be discontinued and potentially other TNF alpha inhibitors be avoided in the future. Newer more target organ specific biologic agents are required which have fewer systemic side effects. In our case vedolizumab proved a useful alternative.We felt obliged to write up this case report to add to the body of evidence with regard to association of this rather rare dermatological adverse effect with anti-TNF treatment to increase its awareness.Antibody levels in patients with prior therapy with infliximab should be done prior to restarting therapy to aid in predicting any risk of adverse reactions and hence considering therapy with alternate biologic agents. Vedolizumab has been shown to be more target organ specific and hence has lower risk of systemic side effects.