Myosin light chain 3 serves as a receptor for nervous necrosis virus entry into host cells via the macropinocytosis pathway

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?

(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?

(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).

(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.

(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?

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.

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.