Author response:
The following is the authors’ response to the original reviews
Reviewer #1 (Public review):
Summary:
In this manuscript, the authors discovered MYL3 of marine medaka (Oryzias melastigma) as a novel NNV entry receptor, elucidating its facilitation of RGNNV entry into host cells through macropinocytosis, mediated by the IGF1R-Rac1/Cdc42 pathway.
Strengths:
In this manuscript, the authors have performed in vitro and in vivo experiments to prove that MnMYL3 may serve as a receptor for NNV via macropinocytosis pathway. These experiments with different methods include Co-IP, RNAi, pulldown, SPR, flow cytometry, immunofluorescence assays, and so on. In general, the results are clearly presented in the manuscript.
Weaknesses:
For the writing in the introduction and discussion sections, the author Yao et al mainly focus on the viral pathogens and fish in Aquaculture, the meaning and novelty of results provided in this manuscript are limited, and not broad in biology. The authors should improve the likely impact of their work on the viral infection field, maybe also in the evolutionary field with the fish model.
(1) Myosin is a big family, why did authors choose MYL3 as a candidate receptor for NNV?
We appreciate your insightful question. We selected MYL3 as a candidate receptor based on a combination of proteomic screening and literature evidence, and functional validation. Increasing evidence indicated that myosins have been implicated in viral infections. For instance, myosin heavy chain 9 plays a role in multiple viral infections (Li et al., 2018), and non-muscle myosin heavy chain IIA has been identified as an entry receptor for herpes simplex virus-1 (Arii et al., 2010). Furthermore, myosin II light chain activation is essential for influenza A virus entry via macropinocytosis (Banerjee et al., 2014). Our previous studies hinted at a potential interaction between MYL3 and CP (Zhang et al., 2020). Huang et al also reported that Epinephelus coioides MYL3 might interact with native NNV CP by proteomic analysis of immunoprecipitation (IP) assay (Huang et al., 2020). Our Co-IP and SPR analyses confirmed a direct interaction between MYL3 and the RGNNV CP. Based on these studies, we selected MYL3 as a candidate receptor for NNV.
References
Huang PY, Hsiao HC, Wang SW, Lo SF, Lu MW, Chen LL. 2020. Screening for the Proteins That Can Interact with Grouper Nervous Necrosis Virus Capsid Protein. Viruses 12:1–20.
Li L, Xue B, Sun W, Gu G, Hou G, Zhang L, Wu C, Zhao Q, Zhang Y, Zhang G, Hiscox JA, Nan Y, Zhou EM. 2018. Recombinant MYH9 protein C-terminal domain blocks porcine reproductive and respiratory syndrome virus internalization by direct interaction with viral glycoprotein 5. Antiviral Res 156:10–20.
Arii J, Goto H, Suenaga T, Oyama M, Kozuka-Hata H, Imai T, Minowa A, Akashi H, Arase H, Kawaoka Y, Kawaguchi Y. 2010. Non-muscle myosin IIA is a functional entry receptor for herpes simplex virus-1.
Banerjee I, Miyake Y, Philip Nobs S, Schneider C, Horvath P, Kopf M, Matthias P, Helenius A, Yamauchi Y. 2014. Influenza A virus uses the aggresome processing machinery for host cell entry. Science (80- ) 346:473–477.
(2) What is the relationship between MmMYL3 and MmHSP90ab1 and other known NNV receptors? Why does NNV have so many receptors? Which one is supposed to serve as the key entry receptor?
We acknowledge the functional diversity of receptors for NNV. MmHSP90ab1 and MmHSC70 have been identified as receptors involved in NNV entry through clathrin-mediated endocytosis (CME), whereas MYL3 facilitates entry via macropinocytosis. These pathways serve as complementary mechanisms for the virus to enter host cells, potentially enhancing infection efficiency. While HSP90ab1 facilitates CME, MYL3 promotes macropinocytosis, both of which are critical for viral internalization, but through distinct endocytic mechanisms.
NNV likely utilizes multiple receptors to increase its host range and infection efficiency. The diversity of receptors ensures that the virus can infect a wide variety of host species. By employing HSP90ab1, HSC70, and MYL3, NNV can exploit different cellular pathways for entry, making it more adaptable to various host environments.
Regarding the identification of a key entry receptor, we agree this is a critical unresolved question. While HSP90ab1/HSC70 appear essential for CME-mediated entry, our data suggest MYL3 plays a distinct role in macropinocytic uptake. To systematically evaluate receptor hierarchy, we initially proposed comparative knockout studies targeting these candidate genes. However, we must acknowledge that current technical limitations in marine fish models – particularly the extended generation time for stable knockout cell lines and challenges in maintaining viable cell cultures post-editing – have delayed these experiments. Nevertheless, we are actively exploring strategies to overcome these obstacles and will continue to refine our approach to address these questions in future research.
(3) In vivo knockout of MYL3 using CRISPR-Cas9 should be conducted to verify whether the absence of MYL3 really inhibits NNV infection. Although it might be difficult to do it in marine medaka as stated by the authors, the introduction of zebrafish is highly recommended, since it has already been reported that zebrafish could serve as a vertebrate model to study NNV (doi: 10.3389/fimmu.2022.863096).
As noted in our manuscript from line 374 to 384, marine medaka is a relatively new model for studying viral infections and is not yet optimized for CRISPR-Cas9-mediated gene knockout. The technical challenges related to precise embryo microinjection and off-target effects using CRISPR-Cas9 in marine medaka complicate the establishment of knockout lines. These limitations, including the time required for multiple breeding generations and molecular screening, currently make this approach difficult to implement.
We fully agree with your suggestion to consider zebrafish as an alternative model. Zebrafish have been well-established as a vertebrate model for studying NNV, and their genetic tractability and well-developed CRISPR-Cas9 protocols provide a more accessible and efficient platform for generating knockout models. In our future studies, we plan to conduct CRISPR-Cas9-mediated knockout experiments targeting multiple NNV receptors in zebrafish. This will allow us to systematically evaluate the role of different receptors in NNV infection and elucidate their potential interactions. The findings from these studies will be included in a future publication, which will provide a more comprehensive understanding of the molecular mechanisms underlying NNV infection in vertebrate models.
(4) The results shown in Figure 6 are not enough to support the conclusion that "RGNNV triggers macropinocytosis mediated by MmMYL3". Additional electron microscopy of macropinosomes (sizes, morphological characteristics, etc.) will be more direct evidence.
Previous study has reported that dragon grouper nervous necrosis virus (DGNNV) enters SSN-1 cells primarily through micropinocytosis and macropinocytosis pathways. Electron microscopy observations revealed several kinds of membrane ruffling and large disproportionate macropinosomes were observed in DGNNV infected cells, indicating NNV infection could triggers micropinocytosis (Liu et al., 2005). In our study, the data from inhibitor treatments, co-localization of MmMYL3 with RGNNV CP, and dextran uptake assays also provide compelling evidence for the involvement of macropinocytosis in RGNNV entry via MmMYL3. These methods are well-established in the literature and have been used extensively to study viral entry pathways (Lingemann et al., 2019). Specifically, the dextran uptake assay has been widely utilized as a marker for macropinocytosis and has provided clear evidence of RGNNV internalization via this pathway. The use of macropinocytosis inhibitors, such as EIPA and Rottlerin, significantly reduced RGNNV entry, further supporting our conclusion. Nonetheless, we acknowledge the potential value of additional electron microscopy studies and will consider this approach in our future research.
References
Liu W, Hsu CH, Hong YR, Wu SC, Wang CH, Wu YM, Chao CB, Lin CS. 2005. Early endocytosis pathways in SSN-1 cells infected by dragon grouper nervous necrosis virus, J Gen Virol.
Lingemann M, McCarty T, Liu X, Buchholz UJ, Surman S, Martin SE, Collins PL, Munir S. 2019. The alpha-1 subunit of the Na+,K+-ATPase (ATP1A1) is required for macropinocytic entry of respiratory syncytial virus (RSV) in human respiratory epithelial cells, PLoS Pathogens.
(5) MYL3 is "predominantly found in muscle tissues, particularly the heart and skeletal muscles". However, NNV is a virus that mainly causes necrosis of nervous tissues (brain and retina). If MYL3 really acts as a receptor for NNV, how does it balance this difference so that nervous tissues, rather than muscle tissues, have the highest viral titers?
While MYL3 is highly expressed in cardiac and skeletal muscles, studies have shown that MYL3, like other myosin light chains, can also be present in non-muscle tissues. Additionally, proteins involved in viral entry do not always need to be the most highly expressed in the final target tissue, as long as they facilitate the initial infection process. For instance, rabies virus is a rhabdovirus which exhibits a marked neuronotropism in infected animals. Transferrin receptor protein 1 can serve as a receptor for rabies virus through CME pathway, but TfR1 expressed most abundantly in liver tissue not nervous system (Wang et al., 2023).
Viral tropism is often determined not only by the presence of an entry receptor but also by co-receptors, cellular factors, and post-entry mechanisms. While MYL3 may act as a receptor for NNV, other factors, such as cell-specific proteases, signaling molecules, and intracellular trafficking pathways, likely contribute to NNV’s preferential replication in the brain and retina.
Reference
Wang Xinxin, Wen Z, Cao H, Luo J, Shuai L, Wang C, Ge J, Wang Xijun, Bu Z, Wang J. 2023. Transferrin Receptor Protein 1 Is an Entry Factor for Rabies Virus. J Virol 97. doi:10.1128/jvi.01612-22
Reviewer #2 (Public review):
Summary:
The manuscript offers an important contribution to the field of virology, especially concerning NNV entry mechanisms. The major strength of the study lies in the identification of MmMYL3 as a functional receptor for RGNNV and its role in macropinocytosis, mediated by the IGF1R-Rac1/Cdc42 signaling axis. This represents a significant advance in understanding NNV entry mechanisms beyond previously known receptors such as HSP90ab1 and HSC70. The data, supported by comprehensive in vitro and in vivo experiments, strongly justify the authors' claims about MYL3's role in NNV infection in marine medaka.
Strengths:
(1) The identification of MmMYL3 as a functional receptor for RGNNV is a significant contribution to the field. The study fills a crucial gap in understanding the molecular mechanisms governing NNV entry into host cells.
(2) The work highlights the involvement of IGF1R in macropinocytosis-mediated NNV entry and downstream Rac1/Cdc42 activation, thus providing a thorough mechanistic understanding of NNV internalization process. This could pave the way for further exploration of antiviral targets.
Thanks for your review.
Reviewer #3 (Public review):
Summary:
The manuscript presents a detailed study on the role of MmMYL3 in the viral entry of NNV, focusing on its function as a receptor that mediates viral internalization through the macropinocytosis pathway. The use of both in vitro assays (e.g., Co-IP, SPR, and GST pull-down) and in vivo experiments (such as infection assays in marine medaka) adds robustness to the evidence for MmMYL3 as a novel receptor for RGNNV. The findings have important implications for understanding NNV infection mechanisms, which could pave the way for new antiviral strategies in aquaculture.
Strengths:
The authors show that MmMYL3 directly binds the viral capsid protein, facilitates NNV entry via the IGF1R-Rac1/Cdc42 pathway, and can render otherwise resistant cells susceptible to infection. This multifaceted approach effectively demonstrates the central role of MmMYL3 in NNV entry.
Thanks for your review.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) Line94: SPR analysis? The full name should be provided when it first shows.
We have defined SPR when it first appears at line 97 in the revised manuscript.
(2) Moreover, is it too many for a manuscript to have a total of nine figures in the main text? Some of them might be moved to the supplementary file.
We have merged the previous Fig 4 and Fig 5 and combined Fig 8 and Fig 9, reducing the number of figures to seven. For the specific details of the figure adjustments, please refer to the corresponding figure legends.
Reviewer #2 (Recommendations for the authors):
(1) Expand on the potential therapeutic implications of targeting MYL3 or the IGF1R pathway in aquaculture settings. Including a discussion of how inhibitors could be developed or tested in future research would give practical context to the findings.
Thanks for your valuable suggestion to expand on the therapeutic implications of targeting MYL3 and the IGF1R pathway in aquaculture. In response, we have discussed potential strategies for developing inhibitors, such as small molecules, peptides, or monoclonal antibodies targeting MYL3 to block its interaction with the viral capsid, and IGF1R inhibitors to prevent macropinocytosis-mediated viral entry. We propose using virtual screening platforms to identify these inhibitors, followed by in vivo testing in aquaculture models. Additionally, combining MYL3 and IGF1R inhibitors could provide a synergistic approach to enhance antiviral efficacy. The relevant discussions have been supplemented at lines 358 to 368 in the revised manuscript.
(2) It is recommended to include the data regarding the lack of interaction between the CMNV CP and MmMYL3 as a supplementary figure.
We have included supplementary data demonstrating that CMNV CP does not interact with MmMYL3, highlighting the specificity of MYL3 for RGNNV. For detailed information, please refer to Fig. S4.
Reviewer #3 (Recommendations for the authors):
Consider discussing the broader implications of these findings, particularly whether MYL3 might serve as a receptor for other viruses.
We appreciate this suggestion. It is important to note that viral receptors typically exhibit specificity for specific types of viruses. Receptor recognition is typically highly specific, and the binding interactions between viral proteins and host receptors often depend on the structural compatibility between the viral capsid/ viral envelope and the host receptor. Our study demonstrates that MYL3 serves as a receptor for NNV based on its direct interaction with the NNV capsid protein (CP). However, when we tested whether MYL3 interacts with CMNV (Covert Mortality Nodavirus), which is phylogenetically closer to NNV, we found that CMNV CP does not bind to MYL3. Given the lack of interaction between MYL3 and CMNV, it is unlikely that MYL3 serves as a receptor for more distantly related viruses. Since MYL3 does not interact with CMNV, a virus more closely related to NNV, it is less likely to function as a receptor for viruses that are more distantly related to NNV. The relevant discussions have been supplemented at lines 306 to 310 in the revised manuscript.
