Porcine Enteric Coronavirus PEDV Induces the ROS-ATM and Caspase7-CAD-γH2AX Signaling Pathways to Foster Its Replication

Round 1

Reviewer 1 Report

In this manuscript authors investigated the interactions between PEDV infection and DNA damage response. How RNA viruses modulate DNA damage response is an understudied field. Authors should be commended by presenting a large body of high-quality mechanistic data in this manuscript. Having said that, I suggest the following points for authors to consider:

1.       Timepoints in the experiments: there are a large span of timepoints in the experiments throughout the manuscript, ranging from 0, 6, 12, 24, 25, 30, 36, to 48 hrs. It would help the audience to comprehend if a rationale is given.

2.       The effects of different treatments on PEDV: authors were using either the intracellular levels of viral N protein and/or infectious virus titres released in culture supernatants to demonstrate changes in PEDV replication. While granted, these two parameters reflect different stages in the virus life cycle. Interestingly, the reduction of infectious virus titres was much less pronounced at 12 hr in Figures 2 and 7. Whether these observations suggest different effects of the treatments on viral transcription/replication and assembly/release needs to be discussed.   

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Porcine enteric coronavirus PEDV induces the ROS-ATM and caspase7-CAD-gammaH2AX signalling pathways to foster its replication

By Xin Ming, Huan Chen et al (Corresponding author: Yingjuan Qian)

Submitted to Viruses (Editorial No. viruses-1864599)

 

General Comments

This is a comprehensive study on the interaction of porcine enteric diarrhea virus (PEDV) with cellular pathways such as the DNA damage response (DDR, involving ATM, ATR and DNA-PK compounds), reactive oxygen species (ROS) production and their correlation with PEDV replication. It is shown that stimulated ROS activates ATM-mediated breaks of dsDNA, and that the downstream substrate Chk2 is phosphorylated and fosters early viral replication. Furthermore, caspase 8, caspase 3 and 7, and caspase-activated DNAse are activated and lead to DNA breaks and fragmentation, followed by H2AX phosphorylation in the nucleus. PEDV replication can be blocked by H2AX, ATM and Chk2 inhibition.

The data were obtained by analysis of the viral replication cycle, plasmid usage, extensive Western blotting, and immunofluorescence, using comprehensive sets of commercially available reagents. The findings are carefully described and transparently explained in Fig. 8. The supply of original blots/gels has been much appreciated. The work is novel as another contribution to the interaction of RNA viruses with cellular DDR and ROS responses. This reviewer has only a few additional Specific Comments.

 

Specific Comments

Line

22        Alphacoronavirus … Coronaviridae… Please print in italics.

56        Consider phrasing: … 28 kb in size…

65        Chk2 … H2AX… others. Since many compounds were investigated which were (incompletely) identified by abbreviations, it is suggested to present a (alphabetical) list of the abbreviations used at the bottom of the title page.

83        … CV777, a vaccine strain, and HLJBY (…), a virulence-attenuated strain…

96        … 2 x SDS sample buffer… Please provide details.

105      Please provide a reference for the rabbit anti-PEDV N antibody.

129      This reagent should be listed under 2.2. Plasmids and reagents (line 86f).

154      See comment line 83.

205      Fig. 2. The effects of KU55933, siATM and siCh2 on viral infectivity titers at 6 h p.i. appear to be moderate (panels C, F, G) and have disappeared at 12 h p.i. Please comment.

222      Fig. 3. The legend of panel D should be improved.

253      … DSB… Please spell out at first mentioning. See comment line 65.

266      Fig. 4, panel B. The gammaH2AX staining pattern may be over-interpreted. If authors feel strongly about this point, the data should be complemented by showing analogous findings with ‘typical gammaH2AX foci’.

414      Consider omitting ‘for the first time’.

420      While the discussion comes over as very comprehensive, additional review of some of the following recent publications may be considered:

Xu X, Wang L, Liu Y, Shi X, Yan Y, Zhang S, Zhang Q. TRIM56 overexpression restricts porcine epidemic diarrhoea virus replication in Marc-145 cells by enhancing TLR3-TRAF3-mediated IFN-β antiviral response. J Gen Virol. 2022 May;103(5). doi: 10.1099/jgv.0.001748. PMID: 35503719.

 

Yan Q, Liu X, Sun Y, Zeng W, Li Y, Zhao F, Wu K, Fan S, Zhao M, Chen J, Yi L. Swine Enteric Coronavirus: Diverse Pathogen-Host Interactions. Int J Mol Sci. 2022 Apr 2;23(7):3953. doi: 10.3390/ijms23073953. PMID: 35409315; PMCID: PMC8999375.

 

Zhang K, Lin S, Li J, Deng S, Zhang J, Wang S. Modulation of Innate Antiviral Immune Response by Porcine Enteric Coronavirus. Front Microbiol. 2022 Feb 14;13:845137. doi: 10.3389/fmicb.2022.845137. PMID: 35237253; PMCID: PMC8882816.

 

Li S, Yang F, Ma C, Cao W, Yang J, Zhao Z, Tian H, Zhu Z, Zheng H. Porcine epidemic diarrhea virus nsp14 inhibits NF-κB pathway activation by targeting the IKK complex and p65. Anim Dis. 2021;1(1):24. doi: 10.1186/s44149-021-00025-5. Epub 2021 Oct 14. PMID: 34778885; PMCID: PMC8514322.

 

Li S, Zhu Z, Yang F, Cao W, Yang J, Ma C, Zhao Z, Tian H, Liu X, Ma J, Xiao S, Zheng H. Porcine Epidemic Diarrhea Virus Membrane Protein Interacted with IRF7 to Inhibit Type I IFN Production during Viral Infection. J Immunol. 2021 Jun 15;206(12):2909-2923. doi: 10.4049/jimmunol.2001186. Epub 2021 Jun 14. PMID: 34127522.

 

Lu Y, Cai H, Lu M, Ma Y, Li A, Gao Y, Zhou J, Gu H, Li J, Gu J. Porcine Epidemic Diarrhea Virus Deficient in RNA Cap Guanine-N-7 Methylation Is Attenuated and Induces Higher Type I and III Interferon Responses. J Virol. 2020 Jul 30;94(16):e00447-20. doi: 10.1128/JVI.00447-20. PMID: 32461321; PMCID: PMC7394890.

 

Possibly:

Li L, Fu F, Guo S, Wang H, He X, Xue M, Yin L, Feng L, Liu P. Porcine Intestinal Enteroids: a New Model for Studying Enteric Coronavirus Porcine Epidemic Diarrhea Virus Infection and the Host Innate Response. J Virol. 2019 Feb 19;93(5):e01682-18. doi: 10.1128/JVI.01682-18. PMID: 30541861; PMCID: PMC6384061.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf