An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

Reviewer #1 (Public Review):

This study offers valuable insights into host-virus interactions, emphasizing the adaptability of the immune system. Readers should recognize the significance of MDA5 in potentially replacing RIG-I and the adversarial strategy employed by 5'ppp-RNA SCRV in degrading MDA5 mediated by m6A modification in different species, further indicating that m6A is a conservational process in the antiviral immune response.

However, caution is warranted in extrapolating these findings universally, given the dynamic nature of host-virus dynamics. The study provides a snapshot into the complexity of these interactions, but further research is needed to validate and extend these insights, considering potential variations across viral species and environmental contexts. Additionally, it is noted that the main claims put forth in the manuscript are only partially supported by the data presented.

Reviewer #2 (Public Review):

This manuscript by Geng et al. aims to demonstrate that MDA5 compensates for the loss of RIG-I in certain species, such as teleofish miiuy croacker. The authors use siniperca cheats rhabdovirus (SCRV) and poly(I:C) to demonstrate that these RNA ligands induce an IFN response in an MDA5-dependent manner in m.miiuy derived cells. Furthermore, they show that MDA5 requires its RD domain to directly bind to SCRV RNA and to induce an IFN response. They use in vitro synthesized RNA with a 5'triphosphate (or lacking a 5'triphosphate as a control) to demonstrate that MDA5 can directly bind to 5'-triphosphorylated RNA. The second part of the paper is devoted to m6A modification of MDA5 transcripts by SCRV as an immune evasion strategy. The authors demonstrate that the modification of MDA5 with m6A is increased upon infection and that this causes increased decay of MDA5 and consequently a decreased IFN response.

- One critical caveat in this study is that it does not address whether ppp-SCRV RNA induces IRF3-dimerization and type I IFN induction in an MDA5 dependent manner. The data demonstrate that mmiMDA5 can bind to triphosphorylated RNA (Fig. 4D). In addition, triphosphorylated RNA can dimerize IRF3 (4C). However, a key experiment that ties these two observations together is missing.
- Specifically, although Fig. 4C demonstrates that 5'ppp-SCRV RNA induces dimerization (unlike its dephosphorylated or capped derivatives), this does not proof that this happens in an MDA5-dependent manner. This experiment should have been done in WT and siMDA5 MKC cells side-by-side to demonstrate that the IRF3 dimerization that is observed here is mediated by MDA5 and not by another (unknown) protein. The same holds true for Fig. 4J.
- Fig 1C-D: these experiments are not sufficiently convincing, i.e. the difference in IRF3 dimerization between VSV-RNA and VSV-RNA+CIAP transfection is minimal.
- Fig. 2N and 2O: why did the authors decide to use overexpression of MDA5 to assess the impact of STING on MDA5-mediated IFN induction? This should have been done in cells transfected with SCRV or polyIC (as in 2D-G) or in infected cells (as in 2H-K). In addition, it is a pity that the authors did not include an siMAVS condition alongside siSTING, to investigate the relative contribution of MAVS versus STING to the MDA5-mediated IFN response. Panel O suggests that the IFN response is completely dependent on STING, which is hard to envision.
- Fig. 3F and 3G: where are the mock-transfected/infected conditions? Given that ectopic expression of hMDA5 is known to cause autoactivation of the IFN pathway, the baseline ISG levels should be shown (ie. In absence of a stimulus or infection). Normalization of the data does not reveal whether this is the case and is therefore misleading.
- Fig. 4F and 4G: can the authors please indicate in the figure which area of the gel is relevant here? The band that runs halfway the gel? If so, the effects described in the text are not supported by the data (i.e. the 5'OH-SCRV and 5'pppGG-SCRV appear to compete with Bio-5'ppp-SCRV as well as 5'ppp-SCRV).
- My concerns about Fig. 5 remain unaltered. The fact that MDA5 is an ISG explains its increased expression and increased methylation pattern. The authors should at the very least mention in their text that MDA5 is an ISG and that their observations may be partially explained by this fact.

Reviewer #3 (Public Review):

In this manuscript, the authors explored the interaction between the pattern recognition receptor MDA5 and 5'ppp-RNA in the Miiuy croaker. They found that MDA5 can serve as a substitute for RIG-I in detecting 5'ppp-RNA of Siniperca cheilinus rhabdovirus (SCRV) when RIG-I is absent in Miiuy croaker. Furthermore, they observed MDA5's recognition of 5'ppp-RNA in chickens (Gallus gallus), a species lacking RIG-I. Additionally, the authors documented that MDA5's functionality can be compromised by m6A-mediated methylation and degradation of MDA5 mRNA, orchestrated by the METTL3/14-YTHDF2/3 regulatory network in Miiuy croaker during SCRV infection. This impairment compromises the innate antiviral immunity of fish, facilitating SCRV's immune evasion. These findings offer valuable insights into the adaptation and functional diversity of innate antiviral mechanisms in vertebrates.

Author response:

The following is the authors’ response to the original reviews.

eLife assessment

The authors present evidence suggesting that MDA5 can substitute as a sensor for triphosphate RNA in a species that naturally lacks RIG-I. The key findings are potentially important for our understanding of the evolution of innate immune responses, but the evidence is incomplete, as additional biochemical and functional experiments are needed to unambiguously assign MDA5 as a bona fide sensor of triphosphate RNA in this model. This also leaves the title as overstating its case.

We would like to thank the editorial team for these positive comments on our manuscript and the constructive suggestions to improve our manuscript. According to the suggestions and valuable comments of the referees, we have added substantial amounts of new data and analysis to substantiate our claims, and the manuscript, including the title, has been carefully revised to better reflect our conclusions. We are now happy to send you our revised manuscript, we hope the modified manuscript addresses your and the reviewers’ concerns satisfactorily and is suitable for publication in eLife now.

Reviewer #1 (Public Review):

This study offers valuable insights into host-virus interactions, emphasizing the adaptability of the immune system. Readers should recognize the significance of MDA5 in potentially replacing RIG-I and the adversarial strategy employed by 5'ppp-RNA SCRV in degrading MDA5 mediated by m6A modification in different species, further indicating that m6A is a conservational process in the antiviral immune response.

However, caution is warranted in extrapolating these findings universally, given the dynamic nature of host-virus dynamics. The study provides a snapshot into the complexity of these interactions, but further research is needed to validate and extend these insights, considering potential variations across viral species and environmental contexts.

We concur with the viewpoint that virus-host coevolution complicates the derivation of universal conclusions. To address this challenge, incorporated additional experiments and data based on the suggestions of the reviewers. These experiments were carried out across diverse models, including two distinct vertebrate species (M. miiuy and G. gallus), two different viruses (SCRV and VSV), and the synthesis of corresponding 5’ppp-RNA probes. We believe that these supplementary data bolster the evidence supporting the immune replacement role of MDA5 in the recognition of 5'ppp-RNA in RIG-I deficient species (Figure 1C-1E, Figure 2O and 2P, Figure 4). Moreover, we have duly incorporated references in both the introduction and discussion sections to further support our conclusion that MDA5 in T. belangeri, a mammal lacking RIG-I, possesses the ability to detect RNA viruses posed as RIG-I agonists (doi: 10.1073/pnas.1604939113). Lastly, meticulous revisions have been undertaken in the manuscript, including adjustments to the title, to ensure harmonization with our research outcomes.

Reviewer#2 (Public Review):

This manuscript by Geng et al. aims to demonstrate that MDA5 compensates for the loss of RIG-I in certain species, such as teleost fish miiuy croaker. The authors use siniperca cheats rhabdovirus (SCRV) and poly(I:C) to demonstrate that these RNA ligands induce an IFN response in an MDA5-dependent manner in M. miiuy derived cells. Furthermore, they show that MDA5 requires its RD domain to directly bind to SCRV RNA and to induce an IFN response. They use in vitro synthesized RNA with a 5'triphosphate (or lacking a 5'triphosphate as a control) to demonstrate that MDA5 can directly bind to 5'-triphosphorylated RNA. The second part of the paper is devoted to m6A modification of MDA5 transcripts by SCRV as an immune evasion strategy. The authors demonstrate that the modification of MDA5 with m6A is increased upon infection and that this causes increased decay of MDA5 and consequently a decreased IFN response.

The key message of this paper, i.e. MDA5 can sense 5'-triphosphorylated RNA and thereby compensate for the loss of RIG-I, is novel and interesting, yet there is insufficient evidence provided to prove this hypothesis. Most importantly, it is crucial to test the capacity of in vitro synthesized 5'-triphosphorylated RNA to induce an IFN response in MDA5-sufficient and -deficient cells. In addition, a number of important controls are missing, as detailed below.

To further support the notion that MDA5 is capable of detecting 5'ppp-RNA in species lacking RIG-I, we conducted additional experiments. Initially, we isolated the RNA from SCRV and VSV viruses. Subsequently, we synthesized 5'ppp-RNA probes that corresponded to the genome termini of SCRV and VSV in vitro. Then, these RNAs were treated with Calf intestinal phosphatase (CIAP) to generate dephosphorylated derivatives. Next, we separately tested the activation ability of various RNAs on IRF3 dimer and IFN response in MKC (M. miiuy kidney cell line) and DF-1 (G. gallus fibroblast cell line) cells, and determined that the immune activation ability of SCRV/VSV viruses depends on their triphosphate structure (Figure 1C-1E, Figure 4C and 4J). In addition, the knockdown of MDA5 inhibited the immune response mediated by SCRV RNA (Figure 2P and 2Q). Finally, we incorporated essential experimental controls (Figure 4B and 4I). We think that the inclusion of these supplementary experimental data significantly enhances the credibility and further substantiates our hypothesis.

The authors describe an interaction between MDA5 and STING which, if true, is very interesting. However, the functional implications of this interaction are not further investigated in the manuscript. Is STING required to relay signaling downstream of MDA5?

To better explore the role of STING in MDA5 signal transduction, we constructed a STING expression plasmid and synthesized specific siRNA targeting STING. Next, we found that co-expression of STING and MDA5 significantly enhance MDA5-mediated IFN-1 response during SCRV virus infection (Figure 2N). Conversely, silencing of STING expression restored the MDA5-mediated IFN-1 response (Figure 2O). These findings provide important evidence for the critical involvement of STING in the immune signaling cascade mediated by MDA5 in response to 5'ppp-RNA viruses.

The second part of the paper is quite distinct from the first part. The fact that MDA5 is an interferon-stimulated gene is not mentioned and complicates the analyses (i.e. is there truly more m6A modification of MDA5 on a per molecule basis, or is there simply more total MDA5 and therefore more total m6A modification of MDA5).

For the experimental data analysis in Figure 5E and 5F, we first compared the m6A-IP group to the input group, and then normalized the control group (IgG group of 5E and Mock group of 5F) to a value of “1”. Given the observed variability in MDA5 expression levels within the input group of Mock and SCRV virus-infected cells, our analysis represents the actual m6A content of each MDA5 molecule. To enhance clarity, we have updated the label on the Y-axis in Figure 5E and 5F.

Finally, it should be pointed out that several figures require additional labels, markings, or information in the figure itself or in the accompanying legend to increase the overall clarity of the manuscript. There are frequently details missing from figures that make them difficult to interpret and not self-explanatory. These details are sometimes not even found in the legend, only in the materials and methods section. The manuscript also requires extensive language editing by the editorial team or the authors.

We acknowledge the valuable feedback from the reviewer and have made significant improvements to our manuscript based on the recommendations provided in the "Recommendation for the authors" section. Furthermore, we have conducted a thorough review of the entire article, resulting in substantial enhancements to the format, clarity, and overall readability of our manuscript.

Reviewer#3 (Public Review):

Summary: In this manuscript, the authors investigated the interaction between the pattern recognition receptor MDA5 and 5'ppp-RNA in a teleost fish called Miiuy croaker. They claimed that MDA5 can replace RIG-I in sensing 5'ppp-RNA of Siniperca cheats rhabdovirus (SCRV) in the absence of RIG-I in Miiuy croaker. The recognition of MDA5 to 5'ppp-RNA was also observed in the chicken (Gallus gallus), a bird species that lacks RIG-I. Additionally, they reported that the function of MDA5 can be impaired through m6A-mediated methylation and degradation of MDA5 mRNA by the METTL3/14-YTHDF2/3 regulatory network in Miiuy croaker under SCRV infection. This impairment weakens the innate antiviral immunity of fish and promotes the immune evasion of SCRV.

Strengths:
These findings provide insights into the adaptation and functional diversity of innate antiviral activity in vertebrates.

Weaknesses:
However, there are some major and minor concerns that need to be further addressed. Addressing these concerns will help the authors improve the quality of their manuscript.One significant issue with the manuscript is that the authors claim to be investigating the role of MDA5 as a substitute for RIG-I in recognizing 5'ppp-RNA, but their study extends beyond this specific scenario. Based on my understanding, it appears that sections 2.2, 2.3, 2.5, 2.6, and 2.7 do not strictly adhere to this particular scenario. Instead, these sections tend to investigate the functional involvement of Miiuy croaker MDA5 in the innate immune response to viral infection. Furthermore, the majority of the data is focused on Miiuy croaker MDA5, with only a limited and insufficient study on chicken MDA5. Consequently, the authors cannot make broad claims that their research represents events in all RIG-I deficient species, considering the limited scope of the species studied.

We agree with the reviewer's perspective that functional analysis of MDA5 in M. miiuy may not adequately represent all species lacking RIG-I. To address this concern, we have incorporated additional experimental data utilizing different model systems, including two different vertebrate species (M. miiuy and G. gallus), two distinct viruses (SCRV and VSV), and the synthesis of two corresponding 5’ppp-RNA probes. While the functional characterization of G. gallus MDA5 remains relatively limited compared to M. miiuy, our current experimental findings provide support for two key observations. Firstly, the triphosphate structure of the VSV virus is pivotal in activating the innate immune response in G. gallus against the virus (Figure 1D and 4J). Secondly, G. gallus MDA5 can recognize 5’ppp-RNA (Figure 4I, 4K and 4L). Consequently, although we cannot definitively establish the immune surrogate function of MDA5 in all RIG-I-deficient species, our research data further substantiates this hypothesis. Moreover, we have adopted a more cautious attitude in summarizing our experimental conclusions, thereby enhancing the rigor of our manuscript language.

The current title of the article does not align well with its actual content. It is recommended that the focus of the research be redirected to the recognition function and molecular mechanism of MDA5 in the absence of RIG-I concerning 5'ppp-RNA. This can be achieved through bolstering experimental analysis in the fields of biochemistry and molecular biology, as well as enhancing theoretical research on the molecular evolution of MDA5. It is advisable to decrease or eliminate content related to m6A modification.

Following the reviewer's recommendations, we have revised the title to emphasize that our main research focus is a teleost fish devoid of RIG-I. Furthermore, we have conducted additional molecular experiments to further elucidate the 5'ppp-RNA recognition function of MDA5 in RIG-I-deficient species. In an attempt to analyze the potential molecular evolution of MDA5 resulting from RIG-I deficiency, we collected MDA5 coding sequences from diverse vertebrates. However, due to multiple independent loss events of RIG-I in fish, fish with or without RIG-I genes in the phylogenetic tree cannot be effectively clustered separately, making it extremely difficult to perform this aspect of analysis. Consequently, we have regrettably opted to forgo the molecular evolution analysis of MDA5.

Our article topic is to reveal an antagonistic phenomenon between fish receptor and RNA viruses. The MDA5 of RIG-I-lost fish has evolved the ability to recognize 5’ppp-RNA virus and mediate IFN response to resist SCRV infection. Conversely, the m6A methylation mechanism endows the SCRV virus with a means to weaken the immune capacity of MDA5. Therefore, we believe that the latter part is an important part of the arms race between the virus and its host, and should be retained.

Additionally, the main body of the writing contains several aspects that lack rigor and tend to exaggerate, necessitating significant improvement.

We appreciate the reviewer’s comment and have improved the manuscript addressing the points raised in the “Recommendation for the authors”. We have added corresponding experiments to strengthen the verification of the conclusions, and in addition, we are more cautious in summarizing the language of the conclusions.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) The evidential foundation within the Result 1 section appears somewhat tenuous.

Firstly, the author derives conclusions regarding the phenomenon of RIG-I loss in lower vertebrates by referencing external literature and conducting bioinformatics analyses. It is pertinent to inquire whether the author considered fortifying these findings through additional WB/PCR experiments, particularly for evaluating RIG-I expression levels across diverse vertebrates, encompassing both lower and higher orders.

Firstly, the species we analyzed are mostly model species with excellent genomic sequence information in the database. Secondly, the RIG-I protein sequences (at least some domain sequences) are relatively conserved in vertebrates. Therefore, the credibility of evaluating the existence of RIG-I in these species through homology comparison is high. Therefore, we do not intend to conduct additional PCR/WB experiments to confirm this.

Additionally, following the identification of RIG-I loss, the author postulates MDA5 as a substitute of RIG-I, grounding this speculation in the analysis of MDA5 and LGP2 protein structures. It is imperative to address whether the author could enhance the manuscript by supplying expression data for MDA5 and LGP2 across different vertebrates and elucidating further why MDA5 is posited as the compensatory mechanism for RIGI loss.

Like MDA5, LGP2 is also an interferon-stimulating gene, so they both likely exhibit high sensitivity to viral infections. Therefore, we think that comparing the expression data of these two genes is difficult to evaluate their function. In mammals, the regulatory mechanisms of LGP2 to RIG-I and MDA5 were complicated and ambiguous. To evaluate the potential function of LGP2 in M. miiuy, we further constructed LGP2 plasmid and synthesized siRNA targeting LGP2. Then, our results indicate that mmiLGP2 can enhance the antiviral immune response mediated by mmiMDA5 (Figure 1H and 1I), further indicating the regulatory role of mmiLGP2 in RLR signaling, rather than acting as a compensatory receptor for RIG-I.

Also, is it conceivable that other receptors contribute to this compensatory effect in lower vertebrates?

5’ triphosphate short blunt-end double-strand RNA is the ligand of RIG-I as contained in the panhandle of negative-strand viral genomes. We mainly focus on the immune recognition and compensatory effects of other receptors on RIG-I loss, and MDA5, as the protein with the most similar structure, first attracted our attention. In addition, IFIT proteins have been reported to recognize triphosphate single-stranded RNA (doi: 10.1038/nature11783). However, we used SCRV and VSV RNA as viral models, both of which have negative stranded genomes and meet the ligand standards of RIG-I, rather than IFIT. Therefore, we excluded the IFIT protein from our research scope.

(2) The article exclusively employs a singular type of 5'PPP-RNA virus and one specific lower vertebrate species, thereby potentially compromising the robustness of the assertion that this phenomenon is prevalent in lower vertebrates. To bolster this claim, could the author consider incorporating data from an alternative 5'PPP-RNA virus and a different lower vertebrate species?

To address this concern, we have incorporated additional experimental data utilizing different model systems, including two different vertebrate species (M. miiuy and G. gallus) and two distinct viruses (SCRV and VSV). While the functional characterization of G. gallus MDA5 remains relatively limited compared to M. miiuy, our current experimental findings provide support for two key observations. Firstly, the triphosphate structure of the VSV virus is pivotal in activating the innate immune response in G. gallus against the virus (Figure 1D and 4J). Secondly, G. gallus MDA5 can recognize 5’ppp-RNA (Figure 4I, 4K and 4L). Consequently, these experimental results further confirmed the conservatism of this immune compensation mechanism.

(3) A nuanced consideration of the statement in Result 5 is warranted. Examination of the results under SCRV infection conditions suggests dynamic fluctuations in MDA5 expression levels, challenging the veracity of the statement implying "increased expression", which contradicts the proposed working model of this article.

Because MDA5 acts as a receptor and plays a recognition immune role in the early stages of virus infection, the expression of MDA5 in the early stage of SCRV infection rapidly increases. In the later stage of infection, the expression of MDA5 may gradually decrease again due to the negative feedback mechanism in the host body to prevent excessive inflammation. However, compared to the uninfected group, the expression of MDA5 was significantly increased in the SCRV-infected group, so we believe that the term "increased expression" is not a problem. In addition, the m6A mechanism can weaken the function of MDA5, but it still cannot prevent the overall increase of MDA5 expression, which is not contradictory to the working model in this article.

Additionally, the alterations in m6A levels in miiuy croaker under SCRV infection conditions warrant clarification. Could the author employ m6A dot blotting to supplement the findings related to total m6A levels?

Our previous studies (doi: 10.4049/jimmunol.2200618) have suggested that the total m6A level is increased after SCRV infection in miiuy croaker. We cited this conclusion in the discussion of our manuscript.

(4) It would be beneficial if the editors could assist the author in enhancing the language of the manuscript.

We have carefully checked the full article and modified it with Grammarly tools, and we believe that the grammar, format, and readability of our articles have been greatly improved.

Reviewer #2 (Recommendations For The Authors):

Figure 1

(1) Figure 1B - some clarification needs to be added about this figure in the text. It is unclear what the main point is that the authors would like to convey.

What we want to emphasize is that some species with RIG-I, such as zebrafish, have also experienced RIG-I loss events, but have undergone whole genome replication events before the loss, thus preserving a copy of RIG-I. This indicates that loss events of RIG-I are very common in vertebrates and do not occur randomly. We have elaborated on this point in the results and discussion.

(2) Figure 1C - is not very informative other than showing Mm MDA5 and LGP2 side-by-side. It would be more useful to show a comparison of human RIG-I/MDA5 alongside Mm and Gg MDA5. Are there any conserved/shared key residues between hRIG-I/hMDA5 versus mmMDA5?

Homologous proteins are often known to adopt the same or similar structure and function. We have added human RIG-I domain information to this figure (Figure 1F). By comparing the domain information of human RIG-I with M. miiuy MDA5 and LGP2, M. miiuy MDA5 has a similar structure to human RIG-I, making it most likely to compensate for the missing RIG-I. While M. miiuy LGP2 lacks the CARD domain, which is crucial for signal transduction, so we will shift our focus to M. miiuy MDA5. In addition, we collected protein sequences of MDA5 and RIG-I from various vertebrates to identify key residues evolved in recognizing 5'ppp-RNA by M. miiuy MDA5. However, unfortunately, no potential residues were found during the comparison process.

Figure 2

(1) Figure 2B - It would be important to demonstrate MDA5-Flag expression by immunoblot and compare MDA5-Flag overexpression to endogenous MDA5 expression using the anti-MDA5 antibody from panel 2A. If IF is used, more cells need to be visible in the field.

After transfecting the MDA5 plasmid into MKC, endogenous MDA5 expression was detected using MDA5 antibodies. The results showed a significant increase in MDA5 protein levels, indicating that MDA5 antibodies can specifically recognize MDA5 protein. In addition, we retained the original immunofluorescence images to better demonstrate the subcellular localization of MDA5.

(2) Figure 2C - The 1:1 stoichiometry of MDA5:MAVS (in the absence of any stimulus) is quite surprising. How does the interaction between MDA5 and MAVS change upon stimulation with an RNA ligand (SCRV, poly(I:C))?

We do not believe that the actual stoichiometry between MDA5 and MAVS is what you described as 1:1. In fact, the proportion of proteins in the complex depends on many factors in the experimental results with Co-IP. Firstly, the MDA5 plasmid in this study has a 3 × Flag tag, while the MAVS only has a 1x Myc tag, which makes the antibody more sensitive for detecting MDA5-Flag. In addition, the Co-IP results are also affected by multiple factors such as the type of antibody and the number of recoveries, making it difficult to estimate the actual ratio of MDA5 to MAVS. Based on the above reasons and the fact that the detection of the interaction strength between MDA5 and MAVS after infection seems to be off-topic, we did not continue to explore this point.

(3) Figure 2D - The interaction between MDA5 and STING is a very interesting finding but is not elaborated on in the paper (even though the interaction between MDA5 and STING is mentioned in the abstract). The manuscript would be strengthened if the interaction between MDA5 and STING is further investigated. For example, does the IFN response that is reported in panels 2E to 2H require the presence of STING? Does mmMDA5 signal via STING in response to a DNA ligand?

We appreciate the referee's suggestion to study the mutual influence between MDA5 and STING. We found that co-expression of STING and MDA5 can enhance MDA5-mediated IFN-1 response during SCRV virus infection, while knocking down STING can restore MDA5-mediated IFN-1 (Figure 2N and 2O). This indicates that STING plays an important signaling role in the immune response of MDA5 to RNA viruses. We understand the importance of cGAS/STING pathways in identifying exogenous DNA, so exploring the MDA5 pathway for DNA ligand recognition is an interesting and meaningful perspective. But this seems to be detached from the theme of our article, so we didn't continue to explore this point.

(4) Figures 2F and 2H - the authors demonstrate that SCRV induces a type I IFN response in an MDA5-dependent manner. While SCRV is a single-stranded negative-sense RNA virus that contains 5'ppp-RNA, it cannot be excluded that MDA5 is activated here in response to a double-stranded RNA intermediate of viral origin or even a host-derived RNA whose expression or modification is altered during infection. To demonstrate in an unambiguous manner that MDA5 senses 5'ppp-RNA, it is crucial to use the in vitro synthesized 5'ppp-RNA (and its dephosphorylated derivative as a control) from Fig. 4 in these experiments.

We transfected 5 'ppp SCRV and 5' ppp VSV (and their dephosphorylated derivatives) synthesized in vitro into MKC cells and DF-1 cells, respectively. The results showed that 5’ppp-RNAs significantly promoted the formation of IRF3 dimers, while their dephosphorylated derivatives did not (Figure 4C and 4J). In addition, we extracted virus RNA from the SCRV and VSV viruses and dephosphorylated them with Calf intestinal phosphatase (CIAP). These RNAs were transfected into MKC and DF-1 cells and found that the immune response mediated by virus RNAs was much higher than the dephosphorylated form (Figure 1C-1E). The above results indicate that the immune response activated by SCRV and VSV is indeed dependent on their triphosphate structure. Finally, the IRF3 dimer and IFN induction activated by SCRV RNA can be inhibited by si-MDA5 (Figure 2P and 2Q), further demonstrating the involvement of MDA5 in the immune response mediated by 5’ppp-RNA ligands.

(5) In mice and humans, MDA5 is known to collaborate with LGP2 to jointly induce an IFN response. Does M.miiuy express LGP2? If so, it would be informative to include a siRNA targeting LGP2 in the experiments in panel F. In mammals, LGP2 potentiates the response via MDA5 while it may inhibit RIG-I activation.

M.miiuy express LGP2. We constructed an LGP2 plasmid and synthesized si-LGP2 to investigate the impact of LGP2 on MDA5-mediated immune processes (Figure 1G-1I). The results showed that LGP2 can enhance the IFN response mediated by MDA5 during SCRV virus infection, similar to that in mammals.

(6) Minor comment - Is the poly(I:C) used in this figure high or low molecular weight poly(I:C)? HMW poly(I:C) preferentially stimulates MDA5, while LMW poly(I:C) preferentially stimulates RIG-I.

We used poly(I:C)-HMW as a positive control for activating MDA5. We have modified the relevant information in Figure 2 and its legend.

Figure 3

(1) Figure 3F/G - The normalization in this Figure is difficult to interpret. It would be better to split Figure 3G into 4 separate graphs and include the mock-infected cells alongside the infected samples (as done in Figure 2).

To better demonstrate the function of the RD domain of MDA5 in M. miiuy, we have changed the experimental plan, as shown in figure 3F. We detected the induction of antiviral factors by overexpression of MDA5 and MDA5-△RD under poly (I:C)-HMW stimulation. This can indicate that the RD domain of MDA5 has a conserved function in the recognition of poly(I:C)-HMW in M. miiuy, and can serve as a positive control for the recognition of SCRV virus by the RD domain.

Figure 4

(1) Figure 4B - A number of important controls are missing. Was the immunoprecipitation of RNA successful? This could be shown by running a fraction of the immunoprecipitated material on an RNA gel and/or by showing that the input RNA was depleted after IP. In addition, a control IP (Streptavidin beads without biotinylated RNA) is missing to ensure that MDA5 does not stick non-specifically to the Streptavidin resin.

We appreciate the referee's suggestions. We rerun this experiment and added a non-biomarker RNA IP control group, and the results showed that MDA5 did not adsorb non-specific onto the beads (Figure 4B). In addition, based on the referee's suggestion, we tested the consumption of RNA before and after immunoprecipitation, and the results showed that biotin-labeled RNA, rather than non-biotin-labeled RNA, could be adsorbed by beads, indicating the success of RNA precipitation. However, we think that this is not necessary for the final presentation of the experimental results, so we did not show this in the figure.

(2) Figure 4B - It is unclear why there is such a large molecular weight difference between endogenous MDA5 and MDA5-Flag (110 kDa versus 130/140 kDa). Why is there less MDA5-Flag retrieved than endogenous MDA5?

After careful analysis, we believe that the significant difference in molecular weight between endogenous MDA5 and MDA5 Flag may be due to three reasons. Firstly, MDA5 flag has a 3× Flag tag. Secondly, as shown in the primer table, we constructed MDA5 between the NotI and XbaI cleavage sites in the pcDNA3.1 vector, which are located at the posterior position in the vector. This means that the Flag tag has a certain distance from the starting codon of MDA5, and these sequences on the vector can also be translated and increase the molecular weight of the exogenous MDA5 protein. Finally, in order to facilitate the amplification of the primers, the F-terminal primers of MDA5 contain a small portion of the 3'UTR sequence (excluding the stop codon). These above reasons may have led to significant differences in molecular weight. In addition, in order to supplement important experimental controls, we have conducted a new RNA pull-down experiment as shown in Figure 4B.

(3) Minor point: Figure 4B - please clarify in the figure whether RNA or protein is immunoprecipitated and via which tags.

We have conducted a new RNA pull-down experiment as shown in Fig 4B, and we have clearly labeled the relevant information in the figure.

(4) Figure 4E - the fraction of MDA5 that binds 5'ppp-RNA seems incredibly minor. And why is this experiment done using 5'OH-RNA as a competitor, rather than simply incubating MDA5 and 5'OH-RNA together and demonstrating that these do not form a complex?

The proportion of MDA5 combined with 5’ppp-RNA is influenced by many conditions, including the concentration and purity of the probe and purified protein. In addition, the dosage ratio between the RNA probe and MDA5 protein in the EMSA experiment can also have a significant impact on the results. Therefore, it is not possible to accurately determine the actual binding force between MDA5 and RNA. In the EMSA experimental program, both cold probes (5’ppp-RNA) and mutated cold probes (5’OH-RNA and 5’pppGG-RNA) are crucial for demonstrating the specific binding between MDA5 and 5’ppp-RNA, as they can exclude false positive errors caused by factors such as the presence of biotin in the purified MDA5 protein itself.

(5) Figure 4B/4C/4F - These experiments would be strengthened by including an MDA5 mutant that cannot bind to RNA. These mutants are well-described in mammals. If these residues are conserved, it is straightforward to generate this mutant.

As shown in Figure 3, the MDA5 of M. miiuy has an RD domain that can recognize the SCRV virus. We constructed MDA5-△RD mutant plasmids with 6x His-tags and purified them for EMSA experiments (Figure 4E). The experimental results further indicate that MDA5, rather than MDA5-△RD, can bind to 5’ppp-SCRV (Figure 4G). This further confirms the crucial role of the RD domain in recognizing the 5'ppp-RNA virus.

(6) Minor point: Figure 4E: please clarify in which lanes MDA5 has been added.

Thank you for the referee's suggestion. We have synthesized new 5'ppp-RNA probes (5’ppp-SCRV and their dephosphate derivatives) and rerun this experiment, and relevant information has been added in the Figure (Figure 4F).

Figure 5

(1) Figure 5C - As MDA5 is an interferon-stimulated gene (as shown in panel G/H/I)) the increased MDA5 expression could simply explain the increase in the amount of m6A-MDA5 that is immunoprecipitated after infection. Could this figure be improved by doing a fold change between input vs m6A-IP OR uninfected vs SCRV-infected conditions? This would reveal whether the modification of MDA5 with m6A is really increased after infection.

As shown in Figure 5F below, our data indicates that the proportion of m6A-modified MDA5 does indeed increase after SCRV infection, rather than solely due to the increased expression of MDA5 itself.

(2) Figure. 5E/F - The y-axis is unclear: relative MDA5 m6A levels. Relative to what? Input? Mock infected?

For experiments in Figure 5E/F, we first compared the m6A-IP group with the input group, and then normalized the control group (IgG group of 5E and Mock group of 5F) to “1”. We have replaced the Y-axis name with a clearer one (Figure 5E and 5F).

(3) General comment - It is not mentioned in the text that MDA5 is an interferon-stimulated gene. This would account for the increase in expression (qPCR) after viral infection or poly(I:C) transfection, hence there is no novelty in this finding. In addition, the authors suggest that MDA5 increases at the protein level (by immunoblot) but the increase on these blots is not convincing (figure 5H/5I).

We understand that the increase in expression of MDA5 as an interferon-stimulated gene after viral infection is a common phenomenon. We present this to further validate the m6A sequencing transcriptome data, and to demonstrate that although m6A modification interferes with MDA5 expression during viral infection, it cannot prevent the increase of mRNA level of MDA5. In addition, we rerun the experiment and the results showed that the expression of MDA5 protein can indeed be specifically activated by the SCRV virus and poly(I:C)-HMW.

Figure 6

(1) Figure 6E - What was the MOI of the virus used in this experiment? It is not mentioned in the figure legend.

MOI=5, we have added this point in the figure legend.

Figure 7

(1) Figure 7J - This graphic is somewhat misleading and should be altered to better reflect the conclusions that are drawn in the manuscript. The graphic suggests that MAVS and STING interact, but this is not demonstrated in the paper. In addition, the paper does not demonstrate whether MAVS or STING (or both) are needed downstream of MDA5 to relay signalling. Finally, please draw an arrow from type I IFNs to increased expression of MDA5 to illustrate that MDA5 is an ISG.

Thank you for the referee's suggestion. We have revised the images to more accurately match the conclusions of the manuscript (Figure 7J). Firstly, we have separated the STING protein from the MAVS protein. Secondly, arrows have been used to indicate that MDA5 is an IFN-stimulated gene. Finally, as we have added relevant experiments to demonstrate the importance of MITA protein in the signaling process of MDA5-activated IFN response. In addition, the function of MAVS binding to MDA5 protein and promoting its signal transduction is very conserved, and there is a good research background even in fish with RIG-I deficiency (10.1016/j.dci.2021.104235). Therefore, in Figure 7J, we still chose to bind MAVS to MDA5 protein and use it as a downstream signal transducer of MDA5.

Discussion
(1) There is very little discussion about METTL and YTHDF proteins in the discussion despite the fact that the last 2 figures are entirely devoted to these proteins.

Based on the referee's suggestion, we have added relevant content about METTL and YTHDF proteins in the discussion. In addition, the basic mechanism and function of METTL and YTHDF proteins were briefly described in the introduction.

Reviewer #3 (Recommendations For The Authors):

Please refer to the specific suggestions and recommendations. They include proposals for experimental additions, improved methodologies, and suggestions to resolve writing-related concerns.

Major concerns

(1) I suggest changing the article title to "Functional Replacement of RIG-I with MDA5 in Fish Miiuy Croaker", or a similar title, to make it more focused and closely aligned with the content of the article.

Following the reviewer's recommendations, we have revised the title to emphasize our primary research subject is a teleost fish that lacks RIG-I. In addition, we have changed “5’ppp-RNA” to “5’ppp-RNA virus” to emphasize the interaction between the virus and the receptor. We believe that the revised title is more in line with the content of the article.

(2) Due to the inherent limitations in genome sequencing, assembly, and annotation for the Miiuy croaker, comprehensive annotation of immune-related genes remains incomplete. To address this critical gap, it is recommended that authors establish experimental protocols, such as Fluorescence In Situ Hybridization (FISH), to confirm the absence of RIG-I in the Miiuy croaker. They should simultaneously employ MDA5 probes as a positive control for validation purposes.

The miiuy croaker has good genomic information at the chromosomal level (doi: 10.1016/j.aaf.2021.06.001). In addition, studies have shown that RIG-I is absent in the orders of Perciformes (doi: 10.1016/j.fsirep.2021.100012), while miiuy croaker belongs to the order Perciformes, so it does indeed lose the RIG-I gene. Therefore, we do not intend to use FISH technology to prove this.

(3) Similarly, it is recommended that the authors first provide evidence of the presence of 5'ppp at the 5' terminus of the genome RNA of SCRV, as demonstrated in the study by Goubau et al. (doi: 10.1038/nature13590, Supplementary figure 1). This evidence is crucial before drawing conclusions about the compensatory role of MDA5 in recognizing 5'ppp RNA viruses, using SCRV as the viral model.

As suggested by the referee, we extracted SCRV RNA from SCRV virus particles and assessed the 5’-phosphate-dependence of stimulation by SCRV RNA. Calf intestinal phosphatase (CIAP) treatment substantially reduced the stimulatory activity of SCRV RNA in MKC cells of M. miiuy (Figure 1C and 1E). In addition, similar results were obtained by transfecting VSV-RNA isolated from VSV virus into DF-1 cells of G. gallus (Figure 1D). The above evidences confirm the presence of triphosphate molecular features between SCRV and VSV viruses, and indicating that birds and fish lacking RIG-I have other receptors that can recognize 5’ppp-RNA.

(4) The 62-nucleotide (nt) 5'ppp-RNA utilized in this study was obtained from Vesicular Stomatitis Virus (VSV). In order to provide direct evidence, it is necessary to include a 62-nt 5'ppp-RNA that is directly derived from SCRV itself.

We adopted this suggestion and synthesized a 67-nucleotide 5’ppp-SCRV RNA probe. We found that 5’ppp-SCRV activates dimerization of IRF3 and binds to MDA5 of M. miiuy in a 5’-triphosphate-dependent manner (Figure 4A-4F).

(5) Given that RNAs with uncapped diphosphate (PP) groups at the 5′ end also activate RIG-I, similar to RNAs with 5′-PPP moieties, and the 5′-terminal nucleotide must remain unmethylated at its 2′-O position to allow RNA recognition by RIG-I, it is necessary for the authors to conduct additional experiments to supplement and validate these two distinguishing features of RIG-I in RNA recognition. This will provide more reliable evidence for the replacement of RIG-I by MDA5 in RNA recognition.

Thank you for the reviewer's professional suggestions. We understand that exploring the combination of 5’pp-RNA and 2′-O-methylated RNA with MDA5 can further demonstrate the alternative function of MDA5. But we think that the use of 5’ppp-RNA and their dephosphorylation derivatives can fully demonstrate that the MDA5 of M. miiuy and G. gallus have evolved to recognize 5’triphosphate structure like human RIG-I. Therefore, we do not intend to conduct any additional experiments

(6) In section 2.3, the authors assert that Miiuy croaker recognizes SCRV through its RD domain. This claim is supported by their data showing that cells overexpressed with the MDA5 ΔRD mutant lost the ability to inhibit SCRV replication. As a result, the authors draw the conclusion that "these findings provide evidence that MDA5 may recognize 5'-triphosphate-dependent RNA (5'ppp-RNA) through its RD domain." However, to strengthen their argument, the authors should first demonstrate that during SCRV infection, MDA5-mediated antiviral immune response is indeed initiated by recognizing the 5'ppp part of the SCRV RNA, rather than the double-strand part (which can exist in ssRNA virus) of the viral RNA, as this is naturally a ligand for MDA5. Additionally, the authors should treat the isolated SCRV RNA with CIP to remove the phosphate group and examine the binding of MDA5 with SCRV RNA before and after treatment. They should also transfect CIP-treated or untreated SCRV RNA into MDA5 knockdown and wild-type MKC cells to investigate the induction of antiviral signaling and levels of viral replication. Finally, the authors should verify the binding ability of the mutants with isolated SCRV RNA, with or without CIP treatment, to determine which domain of MDA5 is responsible for SCRV 5'ppp-RNA recognition.

We understand the reviewer's concern that MDA5 may be identified by binding to dsRNA in the SCRV virus. Based on the reviewer's suggestion, we extracted SCRV RNA and obtained its dephosphorylated RNA using Calf intestinal phosphatase (CIAP). Next, we transfected them into MDA5-knockdown and wild-type MKC cells, and detected the dimerization of IRF3 and IFN reaction. The results indicate that SCRV RNA does indeed activate immunity in a triphosphate-dependent manner, and knockdown of MDA5 prevents immune activation of SCRV RNA (Figure 1C and 1E, Figure 2P and 2Q). Finally, we synthesized a 5'ppp-SCRV RNA probe and demonstrated that MDA5 binds to 5'ppp-SCRV through the RD domain (Figure 4E-4G). We believe that these results can better demonstrate that MDA5 recognizes 5’ppp-RNA through its RD domain and addresses the concerns of the reviewers.

(7) Similarly, merely presenting Co-IP data demonstrating the interaction between Miiuy croaker MDA5 and STING in overexpressed EPC cells does not justify the claim that "in vertebrates lacking RIG-I, MDA5 can utilize STING to facilitate signal transduction in the antiviral response". This is because interactions observed through overexpression may not accurately reflect the events occurring during viral infection or their actual antiviral functions. To provide more robust evidence, it is essential to conduct functional experiments after STING knockout (or at least knockdown). Furthermore, it is important to note that Miiuy Croaker alone cannot adequately represent all "vertebrates lacking RIG-I".

We found that co-expression of STING and MDA5 can enhance MDA5-mediated IFN-1 response during SCRV virus infection, while knocking down STING can restore MDA5-mediated IFN-1 response (Figure 2N and 2O). This indicates that STING plays an important signaling role in the immune response of MDA5 to RNA viruses. In addition, loss of RIG-I is a common phenomenon in vertebrates, and STING of birds such as chickens (doi: 10.4049/jimmunol.1500638) and mammalian tree shrews (doi: 10.1073/pnas.1604939113) can also bind to MDA5, indicating that STING can indeed play a crucial role in MDA5 signaling in species with RIG-I deficiency. We have added this section to our discussion and elaborated on our observations in more cautious language.

(8) In the manuscript, a series of experiments were conducted using an antibody (Beyotime Cat# AF7164) against endogenous MDA5. The corresponding immunogen for this MDA5 antibody is a recombinant fusion protein containing amino acids 1-205 of human IFIH1/MDA5 (NP_071451.2). However, the amino acid sequences of IFIH1/MDA5 differ substantially between humans and Miiuy croaker, which could introduce errors in the results. Therefore, it is essential to employ antibodies specifically designed for targeting Miiuy croaker's own MDA5 in the experiments.

As shown in Figure 2B, endogenous MDA5 antibodies can detect the MDA5 portion that is forcibly overexpressed by plasmids, suggesting that the MDA5 antibody can indeed specifically recognize the MDA5 protein of M. miiuy.

(9) It is recommended to investigate the phosphorylation of IRF3 in order to confirm the downstream signaling pathway during viral infection when MDA5 is knocked down or overexpressed.

Due to the lack of available phosphorylation antibodies for fish IRF3, we used IRF3 dimer experiments to detect downstream signaling (Figure 1C and 1D, Figure 2P, Figure 4C and 4J).

(10) The use of poly I:C as a mimic for dsRNA to investigate MDA5's recognition of 5'ppp-RNA in hosts lacking RIG-I, as well as the examination of the regulatory role of MDA5 m6A methylation upon activation by 5'ppp-RNA, may be inappropriate. Poly I:C does not possess 5'ppp, and while it has been identified as a ligand for MDA5 in various studies, MDA5 cannot serve as a substitute for RIG-I in recognizing poly (I:C). Therefore, the authors should utilize 5'ppp-dsRNA as the mimic and include the corresponding 5'ppp-dsRNA control without a 5'triphosphate as the negative control (both available from InvivoGen). This approach will specifically elucidate the mechanisms involved when MDA5 functions similarly to RIG-I in the recognition of 5'ppp-RNA.

In our study, we used poly(I:C)-HMW, a known dsRNA mimetic that can be preferentially recognized by MDA5 rather than RIG-I, as a positive control for activating MDA5. What we want to demonstrate is that, like poly(I:C)-HMW (positive control), SCRV can also promote MDA5-mediated IFN immunity, further indicating the important role of MDA5 in 5’ppp-RNA virus invasion. We have clearly labeled the type of poly(I:C) in the figures and legends to avoid misunderstandings for readers.

(11) In Figure 2, Figure 3, and Figure 6, the appearance of virus plaques is not readily apparent, and it is necessary to replace these images with clearer photographs. It appears that MKC or MPC cells are not appropriate for conducting plaque assays. To accurately assess viral proliferation, the authors should measure key indicators throughout the process, such as the production of positive-strand RNAs (+RNAs), replication intermediates (RF), and transcription of subgenomic RNAs. This approach is preferable to solely measuring the M and G protein genes from the virus genome as positive results can still be observed in contaminated cells.

As pointed out by the reviewer, we also think that the virus plaque images in Figure 2K and Figure 3D are not clear enough, so we have replaced them with new clear images (Figure 2J and Figure 3D). But we think that other images can clearly display the proliferation of the SCRV virus, so we did not replace them. In addition, the primers we currently use do measure +RNA, so the replication level of the SCRV virus can be accurately evaluated without being affected by virus contamination. Because the regions where the two pairs of primers are located belong to the SCRV-M and SCRV-G protein genes, we label them as SCRV-M and SCRV-G to distinguish between the two pairs of genes. To avoid reader misunderstanding, we have modified the Y-axis label in the figures (Figure 2I and 2K, Figure 3E, Figure 6E and 6O).

(12) There is a substantial disparity in the molecular size of M. miiuy MDA5 between endogenous and exogenously expressed proteins, as shown in Figure 2A and 2C-D. Please provide clarification.

Please refer to the response to Reviewer 2's question regarding Figure 4B above.

(13) The manuscript incorporates the evolutionary perspective, but lacks specific evolutionary analysis. Thus, it is essential to include relevant analysis to comprehend the evolutionary dynamics and positive selection on MDA5 and LGP2 in the absence of RIG-I in Miiuy croaker. This can be achieved through theoretical calculations using appropriate algorithms, such as the branch models and branch-site models based on the maximum-likelihood method implemented in the phylogenetic analysis by maximum likelihood (PAML) package.

In fact, we have analyzed the molecular evolution of MDA5 and LGP2. Unfortunately, even when analyzing only the MDA5/LGP2 CDS sequences in fish, we found that the topologies of gene trees of MDA5/LGP2 were largely consistent with the species tree. Thus, species with or without RIG-I in the gene trees cannot effectively separate clusters, making it extremely difficult to analyze the molecular evolution of MDA5/LGP2 caused by RIG-I deficiency. Consequently, we gave up this aspect of analysis.

(14) If the narrative regarding m6A methylation goes beyond the activation of MDA5 through recognition of 5'ppp-RNA and represents a regulatory mechanism for all MDA5 activation events, it is not relevant to the theme of "An arms race under RIG-I loss: 5'ppp-RNA and its alternative recognition receptor MDA5." Therefore, all investigations in this paper should focus solely on events when MDA5 recognizes 5'ppp-RNA. Any data associated with the broader regulatory mechanisms and m6A methylation of MDA5 should be excluded from this manuscript and instead be included in a separate study dedicated to exploring this specific topic.

Our theme aims to showcase RNA viruses, rather than an interaction between 5'ppp-RNA and host virus receptors, which our current topic cannot accurately express. Therefore, we made two main changes: firstly, we limited the study species to M. miiuy, although some studies on the functional substitution of MDA5 for RIG-I involved birds. Secondly, change “5’ppp-RNA” to “5’ppp-RNA virus”. We believe that the revised title is more in line with our current research contents.

(15) The running title appears to be hastily done.

We modified it to “MDA5 recognizes 5’ppp-RNA virus in species lacking RIG-I”.

(16) There are many descriptions that are not strongly related to the main theme of the article in the introduction section, making it lengthy and fragmented. Please focus on the research background of RIG-I and MDA5, including their structures, functions, and regulatory mechanisms, as well as the research progress on the compensatory effect of MDA5 in the absence of RIG-I and its evolutionary adaptation mechanism in other species.

Based on the suggestions of the reviewers, we have removed some of the less relevant content in the introduction and added research progress on the compensatory effect of MDA5 in the evolutionary adaptation mechanism of tree shrews in the absence of RIG-I.

(17) Lines 149-156 in the "Results" section include content that resembles an "Introduction" It is important to avoid duplicating information in the results section. Therefore, the authors are encouraged to revise this paragraph to ensure conciseness in the article.

We have streamlined this section to enhance the article's conciseness and clarity.

(18) In the "Results" section, at line 177, the authors assert, "As depicted in Figure 1F-1H," which should be corrected to Figure 2F-2H. Furthermore, the y-axis of the two figures on the right-hand side of Figure 2H represents the ISG15 genes. At line 182, "as demonstrated in Figure 1I-1L," should be revised as "as illustrated in Figure 2I-2L". The authors demonstrated a lack of attention to detail.

Thank you to the reviewer for pointing out our errors, and we have made the necessary corrections.

(19) In lines 197-198, the authors stated that "MDA5-ΔRD showed an inability to interact with SCRV." However, Figure 3D did not reveal any significant difference, thus it is advisable to repeat this experiment at least once.

We have replaced this virus spot image with a new one (Figure 3D).

(20) In lines 200-201 of the "2.3 RD domain is required for MDA5 to recognize SCRV" section, the authors report that the expression of antiviral genes was induced by the overexpression of both MDA5 and MDA5-ΔRD, even in the absence of infection (Figure 3F). Why does the expression of antiviral genes increase in the absence of viral RNA stimulation? Please provide a reasonable explanation.

In the absence of viral infection, overexpression of viral receptor proteins may still transmit erroneous signaling, affecting the body's immunity. We speculate that due to the preservation of the CARD domain by MDA5 and MDA5-ΔRD, they can still induce the expression of antiviral factors without ligands, although this induction effect is much smaller than that of viral infection. However, in order to better demonstrate the function of the RD domain of MDA5 in M. miiuy, we have changed the experimental plan, as shown in the figure 3F. We detected the induction of antiviral factors by overexpression of MDA5 and MDA5-△RD under poly (I:C)-HMW stimulation. This can indicate that the RD domain has a conserved function in the recognition of poly(I:C)-HMW in M. miiuy, and can serve as a positive control for the recognition of SCRV virus invasion by the RD domain of MDA5.

(21) Please provide the GeneBank accession number of M. miiuy MDA5.

The GeneBank accession number of M. miiuy MDA5 was added in the section 4.5 plasmids construction.

(22) The content of lines 228-233 in the "Results" section bears resemblance to that of the "Introduction." To ensure the avoidance of information duplication, it is recommended to remove this paragraph from the results section.

This section has been streamlined.

(23) The bands of mmiMDA5 in the 5'ppp-RNA and dsRNA lanes in Figure 4B are weak and almost unobservable. Please replace them with clear images.

We have rerun this experiment and replaced the images (Figure 4B).

(24) In Figure 5G and at line 253, there are only results presented for the SCRV infection group, while no results are shown for the control group. This raises the question of why the control group results are missing. It is necessary to provide a reasonable explanation or correction for this issue.

The "0 h" infection time point of the SCRV virus is the control group, and we have replaced it with a more intuitive image (Figure 5G).

(25) In Figure 7C, it would be necessary to include the western blot result of YTHDF protein expression in order to verify the efficiency of YTHDF siRNA.

In fact, we have attempted to detect the endogenous expression of YTHDF protein using available commercial antibodies. Unfortunately, only the YTHDF2 antibody can specifically recognize the endogenous protein expression of YTHDF2 in M. miiuy. In addition, the knockdown effect of si-YTHDF2 has been validated by YTHDF2 antibody (doi: 10.4049/jimmunol.2200618).

(26) In line 422 of the "4.3 Cell culture and treatment" section, the paragraph raises a question regarding the nature of Miiuy croaker kidney cells (MKCs) and spleen cells (MPCs) - whether they are cell lines or freshly isolated cells (or primary cultures) derived from kidney and spleen tissues. If these cells are indeed cell lines, it is requested to provide detailed information about the sources and properties of the cells (such as whether they are epithelial cells or other mixed cell types) and the generations of propagation. Alternatively, if the cells were freshly isolated or primary cultures obtained from fish, the method for cell isolation should be provided. The source and stability of cells are extremely important for ensuring the repeatability and reliability of experimental outcomes.

M. miiuy kidney cells (MKCs) and spleen cells (MPCs) are cell lines derived from the kidney and spleen tissues of M. miiuy, with passages ranging from 20 to 40 times. These details have been incorporated into section 4.3.

(27) There are many inaccurate descriptions in the text, which employ concepts that are too broad. These descriptions need to be narrowed down to specific species or objects. Here are a few examples, along with the necessary revisions. Other similar instances should also be revised accordingly. For instance, in line 119, "fish MDA5" should be changed to "Miiuy croaker MDA5." Similarly, in line 166, "fish MDA5-mediated signaling pathway" should be changed to "Miiuy croaker MDA5-mediated signaling pathway." In line 174, "fish MDA5" should be revised to "Miiuy croaker MDA5." Additionally, in line 185, "antiviral responses of teleost" should be changed to "antiviral responses of Miiuy croaker." In line 197, "interact with SCRV" should be revised to "interact with 5'ppp-RNA of SCRV." In line 337, "loss of RIG-I in the vertebrate" should be modified to "loss of RIG-I in Miiuy croaker and chicken." Similarly, in line 338, "MDA5 of fish" should be changed to "MDA5 of Miiuy croaker." Lastly, in line 348, "RIG-I deficient vertebrates" should be revised to "RIG-I deficient Miichthys miiuy and Gallus gallus."

Thank you for the reviewer's suggestions. We have made revisions to these inaccurate descriptions and reviewed the entire manuscript to address similar statements with broad concepts.

(28) Finally, it should be noted that a similar discovery has already been reported in tree shrews (Ling Xu, et al., Proc Natl Acad Sci., 2016, 113(39):10950-10955). This article shares similarities with that research report, therefore it is necessary to discuss in detail the relationship between the two in the discussion and compare and analyze the evolutionary patterns of MDA5 from it.

Based on the reviewer's suggestions, we have compared the similarities and differences between these two reports during the discussion and analyzed the evolutionary dynamics of MDA5 in these vertebrates lacking RIG-I.

Minor concerns:

Thank you to the reviewer for their meticulous examination to our manuscript, we have made revisions to the following suggestions.

(1) At line 120, the sentence "SCRV(one 5'ppp-RNA virus)" should have a space between "SCRV" and "(one 5'ppp-RNA virus)". Please make this correction.

Corrected.

(2) At lines 147-148, the sentence "However, the downstream gene of TOPORSa is missing a RIG-I" is not accurate and needs modification.

We have modified this sentence.

(3) At line 184, "findings indicate" should be corrected to "findings indicated".

Corrected.

(4) At line 189, "a 5'ppp-RNA virus" should be deleted and the text seems redundant.

Deleted.

(5) At line 198, "replication. (Figure 3C-3E)", please remove the punctuation between "replication" and "(Figure 3C-3E)".

Corrected.

(6) At line 416 in "Materials and methods" section, "4.2 Sample and challenge" should be corrected to "4.2 Fish and challenge".

Corrected.

(7) At line 419, the authors state that "The experimental procedure for SCRV infection was performed as described", please briefly describe the SCRV infection method and the infectious dose.

Based on the reviewer's suggestions, we have added relevant descriptions of SCRV infection in section 4.2.

(8) There are several formatting issues in the "Materials and Methods" section. For instance, in line 424, there is no space between the number and letter in "100 μg/ml" and "26 ℃" should be corrected to "26℃". Additionally, in line 430, "Cells" should be corrected to "cells".

Corrected.

(9) At line 446, "50 ng/ul" and "100 mU/ul" should be corrected to "50 ng/μl" and "100 mU/μl".

Corrected.

(10) At line 459, "primers 1)" should be corrected to "primers".

Corrected.

(11) At lines 461-464, the description "For protein purification, MDA5 plasmids with 6× His tag was constructed based on pcDNA3" seems to be no direct logical connection between protein purification and the plasmid construction. Please make the necessary corrections.

Corrected.

(12) At line 548, "cytoplasmic" should be corrected to "Cytoplasmic".

Corrected.

(13) At line 549, "5× 107" should be corrected to "5 × 107".

Corrected.

(14) At line 557, "MgCl2" should be corrected to "MgCl2".

Corrected.

(15) At line 558, "6 %" should be corrected to "6%".

Corrected.

(16) At line 565, "50μg" should be corrected to "50 μg".

Corrected.

(17) At line 571, "300{plus minus}50 bp." should be corrected to "300 {plus minus} 50 bp."

Corrected.

(18) At lines 592-593, the sentence "After several incubations, the m6A level was quantified colorimetrically at a wavelength of 450 nm" does not read smoothly, please improve it.

Revised.

(19) At line 786, "MDA5 recognize" should be corrected to "MDA5 recognized".

Corrected.

(20) At lines 788 and 798, "Pulldown" should be corrected to "Pull-down".

Corrected.

(21) At lines 790 and 796, "bluestaining" should be corrected to "blue staining".

Deleted.

(22) At line 825, "SCRV and infection" should be corrected to "SCRV infection".

Corrected.

(23) At lines 826-827, "SCRV (H) and poly(I:C) (I) infection" should be corrected to "SCRV infection (H) and poly(I:C) stimulation (I)".

Corrected.