Identification of novel antiviral host factors by functional gene expression analysis using in vitro HBV infection assay systems

Takuto Nosaka; Tatsushi Naito; Yu Akazawa; Kazuto Takahashi; Hidetaka Matsuda; Masahiro Ohtani; Tsutomu Nishizawa; Hiroaki Okamoto; Yasunari Nakamoto

doi:10.1371/journal.pone.0314581

Peer Review History

Original SubmissionJuly 29, 2024
29 Jul 2024 Author Response https://doi.org/10.1371/journal.pone.0314581.r001
23 Aug 2024 Decision Letter - Youkyung Choi, Editor PONE-D-24-31113Identification of novel antiviral host factors by functional gene expression analysis using in vitro HBV infection assay systemsPLOS ONE Dear Dr. Nakamoto, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Oct 07 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Additional Editor Comments: We invite you to submit a revised manuscript that addresses the points below. Specifically, different results from the infection and transfection experiments using PXB cells and HepG2-D11 cells need to be addressed. In addition, a rationale for HepG2 cells instead of HepG2-NTCP cells needs to be provided. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? 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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The study by Nosaka et al. describes fumarylacetoacetate hydrolase (FAH) as a novel antiviral host factor against HBV. The authors found that siRNA-mediated knockdown of STAT1 unexpectedly reduced HBV replication in primary human hepatocytes (PXB cells). They then performed RNA microarray and identified 43 host genes that upregulated in the absence of STAT1 in PXB cells. Individual knockdown of these 43 genes revealed FAH as an antiviral host factor in PXB cells as well as in HepG2 cells. Furthermore, overexpression of FAH or addition of dimethyl fumarate (DMF), an FAH metabolite, reduced HBV replication in a STAT1-independent manner. The study is important since HBV remains uncurable and identifying new host factors may inform new antiviral strategies. However, the study contains several issues that need to be addressed. 1. The authors performed infection-based experiments in PXB cells and transfection-based experiments in HepG2 cells. This is not a fair comparison. The authors should use HepG2 cells stably expressing the HBV receptor NTCP and perform infection experiments in these cells so that the results from the two different cell culture systems can be compared. 2. Fig. 1, siRNA was transfected 4 days before infection, but the experiment lasted 13 days. Since siRNA-mediated gene knockdown is transient, the knockdown efficiency during infection day 4-13 is problematic which can be an issue in data interpretation. 3. Fig. 1D, the arrow for the day 1 label should be extended to the text above it. It’s confusing otherwise. 4. Figure 2B, I understand that the genes are chosen focused on PXB but indicating the number of genes that differ with treatment on the HepG2.D11 graph could be informative. Or point out the genes of interest for the other cell type on this graph too. 5. Figure 2C, asterisk is not explained. 6. Fig. 3, qPCR validation of the knockdowns is needed. Axes that say “relative expressions” should be “relative expression” singular. Lines labeled “folds” should say “fold” or “fold change” singular. Also, whether the red color represents significance is not explained. 7. Clarification that any comparisons not shown on graphs are non-significant would be useful. 8. In some graphs the authors indicate n=3 and show the mean. It would be better to show individual data points. If n= 3 means total data points (not 3 experimental replicates) the data pool is a little small. 9. Line 266, should “transfection” be “replication”? 10. Line 270, 1.2-fold is a rather marginal effect. Is this difference biologically meaningful? 11. Line 275, should “extracellular” be “intracellular”? 12. The sentence on lines 278-280 is confusing, I suggest it’s changed to "these results suggest that FAH has anti-HBV effects, as determined by siRNA screening of genes altered by STAT1 knockdown in PXB cells.” 13. Fig. 5F is quite zoomed out, I wonder if a slightly closer view would show cell morphology better. 14. Line 367, the sentence reads as if this is a conclusion, but the data do not really establish that DMF exerts its antiviral effect by inducing autophagy and anti-HBV related genes. 15. Line 371, what does “human biotransformation experiment” mean? Reviewer #2: Authors of this manuscript investigated that fumarylacetoacetate hydrolase (FAH) is an antiviral host factor and methyl ester of FAH metabolite, dimethyl fumarate (DMF) has the antiviral effect in STAT1-independent pathway. First of all, authors explained that knockdown of STAT1 increased viral products in HBV-transfected HepG2 cells therefore STAT1 is antiviral. And then decreased HBV replication and products in STAT1-KD, HBV-infected PXB cells is that HBV replication is reduced by STAT1-KD. The explanation by authors was not clear and the opposite results from the infection and transfection experiment should be thoroughly investigated. To claim that STAT1 is antiviral, STAT1 overexpression should be conducted in HepG2-D11 cells with Western blotting with STAT1 and HBc and Northern and Southern blotting for HBV RNA and HBV DNA. If possible, also in PXB cells, too. Since working with PXB cell may have some limitations, authors did not conduct Northern and Southern blotting for HBV RNA and DNA with PXB cells. Then, Northern and Southern blotting should be conducted in si-STAT-RNA transfected cells HepG2-D11 cell for clarification. Again, Fig. 2 was a continuation of the opposite results between PXB and HepG2-D11 cells and the explanation was not clear. In the Fig. 3A, FAH KD increased HBsAg in PXB cells and in the Fig 3B, FAH and STAT1 double KD increased HBsAg and intracellular DNA with decreased HBV DNA and HBsAg in STAT1 single KD. In should be included with FAH single KD in Fig 3B (right graph) to compare intracellular DNA level. In Fig. 3D, intracellular DNA level was 25 times higher in FAH KD HepG2-D11 cells than control KD cells then why RNA level was only 2 times higher and extracellular DNA level was only increased little compared to intracellular DNA? Southern blotting for intracellular DNA and extracellular DNA should be conducted. Northern blotting for RNA should be conducted to compare with pgRNA, S and X mRNAs. At the same time, RT-qPCR for pgRNA only and for total RNA should be conducted. Again, in Fig 4D in FAH overexpression, compare to HBV RNA, intracellular DNA level was decreased so much and extracellular DNA level was not decreased significantly. Southern blotting to compare RC DNA and DL DNA levels and Northern blotting for pgRNA, S and X mRNAs are needed. Since DMF increased NRF2 expression (Fig 5G) and NRF2 regulate HMOX-1, then why HMOX-1 is not increased? It needs to be explained and/or speculated why so. Minor points, In lane 64, cccDNA drug resistance should be corrected to cccDNA persistence or else. In lanes 141, ‘the selected target genes’ and 96 primers were used. How these gene were selected? I cannot find in the manuscript. And primer information was hard to find (Table 2). In lanes 189, radioimmunoprecipitation assay (RIPA) lysis buffer was used for Western blotting. It is better to show the composition of the buffer, since there are many versions. Lanes 407-409, it was not clear. Lanes 421-423, this conclusion is not clear, which I already mentioned above. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy . Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0314581.r002
Revision 1
9 Oct 2024 Author Response Reviewer #1: 1. The authors performed infection-based experiments in PXB cells and transfection-based experiments in HepG2 cells. This is not a fair comparison. The authors should use HepG2 cells stably expressing the HBV receptor NTCP and perform infection experiments in these cells so that the results from the two different cell culture systems can be compared. We appreciate your important suggestion. As you pointed out, it is not possible to observe HBV entry in HepG2 cells. In this study, we were focused on the influence of host factors on HBV transcription and amplification in hepatocytes. Transfection experiments in HepG2 cells were performed to examine intracellular viral transcription and amplification. We consider that there is no difference between HepG2 and HepG2-NTCP cells with respect to intracellular viral transcription and amplification. Using PXB cells, it is possible to observe viral infection. We performed experiments in which the order of HBV infection and siRNA knockdown was switched in the molecules we have examined, including STAT1. Gene knockdown did not affect the process of HBV entry into hepatocytes (S2 Fig). HBV infection experiments using HepG2-NTCP cells, which are not commercially available, are being considered as the next project. Accordingly, we added the description to the Discussion section (page 31-32, lines 459-468) and Figure and legend of S2 Fig. 2. Fig. 1, siRNA was transfected 4 days before infection, but the experiment lasted 13 days. Since siRNA-mediated gene knockdown is transient, the knockdown efficiency during infection day 4-13 is problematic which can be an issue in data interpretation. We appreciate your comments. Compared to HepG2 cells, which are a conventional cell line, PXB cells are the primary hepatocytes which do not proliferate, and we are considering the possibility of long-term siRNA knockdown effect. Though the course of the knockdown effect is not known, however, we have confirmed that the knockdown effect is still sufficient at day 13 (Fig 1B). 3. Fig. 1D, the arrow for the day 1 label should be extended to the text above it. It’s confusing otherwise. We appreciate your suggestion. The arrow for the day 1 label was extended to the text above it (Fig 1D). 4. Figure 2B, I understand that the genes are chosen focused on PXB but indicating the number of genes that differ with treatment on the HepG2.D11 graph could be informative. Or point out the genes of interest for the other cell type on this graph too. We appreciate your important suggestion. We indicated the number of genes that were altered in si-STAT1 in HepG2.D11 cells. In HepG2.D11 cells, STAT knockdown increased expression of 340 genes [si-STAT1/si-Non-Target (Log2 ratio) > 1] and decreased expression of 266 genes [si-STAT1/si-Non-Target (Log2 ratio) < -1] (Fig 2D). Accordingly, we added the description to the Results section (page 22, lines 276-278) and modified Fig 2D. 5. Figure 2C, asterisk is not explained. We appreciate your comments. The asterisk in Figure 2E (before 2C) was a typographical error and removed. 6. Fig. 3, qPCR validation of the knockdowns is needed. Axes that say “relative expressions” should be “relative expression” singular. Lines labeled “folds” should say “fold” or “fold change” singular. Also, whether the red color represents significance is not explained. We appreciate your important suggestion. Knockdown experiments were performed in PXB cells using siRNA. The results for two genes, FAH and NNMT, in which knockdown efficiency was confirmed by qRT-PCR, are shown in Fig. 3A and B. The expression of the target molecule was significantly reduced by knockdown experiment. Results for other siRNAs were provided in Supplemental Figure 1. Axes that say “relative expressions” were modified to “relative expression” singular in Figure 3, 4, and 5. Lines labeled “folds” were modified to “fold change” singular in Supplemental Figure 1. The description that red color does not represent significance is described in legend of Supplemental Figure 1. 7. Clarification that any comparisons not shown on graphs are non-significant would be useful. We appreciate your comments. We clarified that any comparisons not shown on graphs are non-significant in Materials and Methods section (page 19, lines 211-212). 8. In some graphs the authors indicate n=3 and show the mean. It would be better to show individual data points. If n= 3 means total data points (not 3 experimental replicates) the data pool is a little small. We appreciate your important suggestion. The number of times the experiment was replicated is described in the figure legends. 9. Line 266, should “transfection” be “replication”? We appreciate your comments. We changed “transfection” to “replication” (page 24, line 306). 10. Line 270, 1.2-fold is a rather marginal effect. Is this difference biologically meaningful? We appreciate your comments. A 1.2-fold increase is a rather marginal effect, and this difference is not considered to have biologically meaningful. Unfortunately, it was not so easy to show large increases in the amount of HBV, especially in HBV-transfected cells. Considering the characteristics of HBV stable transfection cells, we set the fold change at 1.2 in order to select the genes that have an anti-HBV effect from among the 43 candidate genes. 11. Line 275, should “extracellular” be “intracellular”? We appreciate your comments. We changed “extracellular” to “intracellular” (page 24, line 315). 12. The sentence on lines 278-280 is confusing, I suggest it’s changed to "these results suggest that FAH has anti-HBV effects, as determined by siRNA screening of genes altered by STAT1 knockdown in PXB cells.” We appreciate your suggestion. The sentence was changed to "These results suggest that FAH has anti-HBV effects, as determined by siRNA screening of genes altered by STAT1 knockdown in PXB cells.” (page 25, lines 321-322) 13. Fig. 5F is quite zoomed out, I wonder if a slightly closer view would show cell morphology better. We appreciate your suggestion. The image of Fig 5F was enlarged to improve the visibility of the cell morphology. 14. Line 367, the sentence reads as if this is a conclusion, but the data do not really establish that DMF exerts its antiviral effect by inducing autophagy and anti-HBV related genes. We appreciate your suggestion. As you pointed out, the data do not really establish that DMF exerts its antiviral effect by inducing autophagy and anti-HBV related genes. The sentence was changed to “DMF, the methyl ester of the FAH metabolite, showed antiviral effects and the expression of genes related to autophagy and anti-HBV effects were altered.” (page 29, lines 406-408) 15. Line 371, what does “human biotransformation experiment” mean? We appreciate your suggestion. The words “human biotransformation experiment” were removed. (page 29, line 411) Reviewer #2: First of all, authors explained that knockdown of STAT1 increased viral products in HBV-transfected HepG2 cells therefore STAT1 is antiviral. And then decreased HBV replication and products in STAT1-KD, HBV-infected PXB cells is that HBV replication is reduced by STAT1-KD. The explanation by authors was not clear and the opposite results from the infection and transfection experiment should be thoroughly investigated. We appreciate your important suggestion. As you pointed out, in HBV-transfected HepG2 cells, knockdown of STAT1 increased viral products. And in HBV-infected PXB cells, knockdown of STAT1 decreased HBV replication. To investigate the mechanism by which STAT1 knockdown reduces HBV levels in primary human hepatocytes, RNA microarray analysis was performed and to comprehensively examine the molecular changes in PXB cells. In HBV-infected PXB cells, STAT1 knockdown enriched 24 gene sets (p < 0.05, FDR q-value < 0.25) (Fig 2A). Of the 24 gene sets, 21 were associated with metabolism and protein synthesis. However, STAT1 knockdown in HepG2.D11 cells and IRF2 knockdown in PXB cells did not result in similar molecular changes (Fig 2B). Accordingly, we added the description to the Results section (page 22, lines 266-272), Figures and legends of Fig 2A and B, and the Materials and Methods section (page 15, lines 162-168). Moreover, the following text is described in the Discussion section as a mechanism for the changes in the signal pathways related to metabolism and protein synthesis in the PXB cell infection system, but not in the HepG2 cell transfection system. Wilkening et al. performed a comparison of primary hepatocytes and hepatoma cell line HepG2 in the presence of different classes of promutagens [22]. The three promutagens caused DNA damage in primary human hepatocytes, but not in HepG2 cells. The most abundant isozyme of all P450s in the human liver, CYP3A4, is the most important isoform in drug metabolism in primary hepatocytes; however, CYP3A4 mRNA was not detected in HepG2 cells. In addition, the researchers detected mRNA P450 in primary hepatocytes, similar to that previously reported for human liver samples [23, 24]. Similarly, in this study, host gene expression in primary hepatocytes of the in vitro HBV infection assay system was markedly different from that in HepG2 cells with downregulation of STAT1. A comprehensive functional screen of genes altered by STAT1 knockdown in PXB cells identified FAH as the gene exhibiting anti-HBV activity. FAH is the last enzyme in the tyrosine catabolic pathway [25] and catalyzes the breakdown of fumarylacetoacetate into fumarate and acetoacetate [26]. (page 29-30, lines 411-424). To claim that STAT1 is antiviral, STAT1 overexpression should be conducted in HepG2-D11 cells with Western blotting with STAT1 and HBc and Northern and Southern blotting for HBV RNA and HBV DNA. If possible, also in PXB cells, too. Since working with PXB cell may have some limitations, authors did not conduct Northern and Southern blotting for HBV RNA and DNA with PXB cells. Then, Northern and Southern blotting should be conducted in si-STAT-RNA transfected cells HepG2-D11 cell for clarification. We appreciate your suggestion. STAT1 overexpression was conducted in HepG2.D11 cells. To overexpress STAT1 in HepG2.D11 cells, STAT1 vector was transfected (Fig 1G). Increased STAT1 expression was confirmed by quantitative real-time polymerase chain reaction (PCR) (Fig 1H) and western blot (Fig 1I). Overexpression of STAT1 decreased extracellular HBsAg and HBV DNA and intracellular HBV DNA and RNA levels (Fig 1J). Accordingly, we added the description to the Results section (page 20, lines 231-235), Figures and legends of Fig 1G-J, and the Materials and Methods section (page 18, lines 181-186 and page 19, lines 199-206). In addition, we conducted transfection experiments using STAT1 and FAH vectors in primary human hepatocytes, PXB cells, however, functional overexpression of the target molecule was not possible. Unlike conventional cell lines, PXB cells do not proliferate, so the amount of RNA and DNA recovered is small. Thus, we did not conduct Northern blotting or Southern blotting. As you suggested, Northern and Southern blotting should be conducted in si-STAT-RNA transfected cells HepG2.D11 cell for clarification. We plan to perform Northern and Southern blotting for HBV RNA and DNA in si-STAT-RNA transfected HepG2-D11 cells in three months, and the results will be published in an upcoming paper summarized below. < Summary of the upcoming paper; UNPUBLISHED and CONFIDENTIAL > Title: Functional Analysis of Fumarylacetoacetate Hydrolase (FAH) as a Hepatocyte Host Factor Gene Regulating Hepatitis B Virus Replication and Hepatocarcinogenesis Background: Recent molecular biological analyses have highlighted the importance of host factors in anti-viral and/or anti-tumor effects. We established a hepatitis B virus (HBV) infection assay system using primary human hepatocytes and extracted fumarylacetoacetate hydrolase (FAH) as a host factor gene that regulates viral life cycle by RNA microarray analysis (in submission). In this study, we investigated the anti-HBV and anti-tumor effects of FAH using resected hepatocellular carcinoma (HCC) tissue and HBV stable transfected HCC cell lines. Methods: In 60 resected HCC specimens, the intensity of immunohistological staining of FAH in tumor and non-tumor areas was determined by H-score using inform (akoya), and the correlation with prognosis was analyzed. The FAH index was calculated by H-score of tumor/non-tumor area. The 1.3 mer HBV genome of genotype C2 with basic core promoter and/or pre-core mutation (wt/wt; Low transcription, mu/mu; High t.) was transfected into HepG2 cells and HepG2.1-E10 (Low t.) and HepG2.D11 (High t.) were established. FAH vector and siRNA were transfected and MTT assay, apoptosis assay, and sphere formation assay were performed. The HBsAg was standardized by cell count (HBsAg index). RNA expression was analyzed for 205 anti-tumor and anti-viral related genes using TaqMan custom array plates. Results: The H-score of FAH in resected HCC specimens was significantly lower in tumor areas compared to non-tumor areas (tumor/non-tumor areas: 143/165). Recurrence and survival rates were worse in the　patient group with lower FAH index (p < 0.05). In HepG2.D11 and HepG2.1-E10, the viability index and HBsAg index were increased in the FAH knockdown and decreased in the FAH vector transduction experiments compared to controls. Furthermore, FAH overexpression in HepG2.D11 and HepG2.1-E10 induced apoptosis and decreased the number of spheres. In FAH overexpression, the expression of CCKN2B and CHEK1 (cell cycle) were elevated, NANOG and SALL4 (stem cell function) were decreased, and anti-HBV related genes, APOBEC3B and IRF7, were increased. Conclusions: Evaluation of FAH expression in HCC tissues and the functional analysis in HBV-transfected HepG2 cells suggest that the FAH gene may contribute to HBV reduction, inhibition of cell proliferation, and induction of cell apoptosis. Hepatocyte host factor FAH is shown to have the potential to exhibit both anti-tumor and anti-HBV effects, and may be a candidate for a new therapeutic strategy of viral carcinogenesis. Again, Fig. 2 was a continuation of the opposite results between PXB and HepG2-D11 cells and the explanation was not clear. We appreciate your suggestion. The opposite results between PXB and HepG2-D11 cells were observed. Thus, to investigate the mechanism by which STAT1 knockdown reduces HBV levels in primary human hepatocytes, RNA microarray analysis was performed and to comprehensively examine the molecular changes in PXB cells. In HBV-infected PXB cells, STAT1 knockdown enriched 24 gene sets (p < 0.05, FDR q-value < 0.25) (Fig 2A). Of the 24 gene sets, 21 were associated with metabolism and protein synthesis. However, STAT1 knockdown in HepG2.D11 cells and IRF2 knockdown in PXB cells did not result in similar molecular changes (Fig 2B). Accordingly, we added the description to the Results section (page 22, lines 266-272), Figures and legends of Fig 2A and B, and the Materials and Methods section (page 15, lines 162-168). In the Fig. 3A, FAH KD increased HBsAg in PXB cells and in the Fig 3B, FAH and STAT1 double KD increased HBsAg and intracellular DNA with decreased HBV DNA and HBsAg in STAT1 single KD. In should be included with FAH single KD in Fig 3B (right graph) to compare intracellular DNA level. We appreciate your suggestion. Intracellular HBV DNA level in FAH single KD was shown in Fig 3B. Knockdown of FAH and NNMT significantly elevated extracellular HBsAg and intracellular HBV DNA levels (Fig 3B). Accordingly, we added the description to the Results section (page 24, lines 316-318), Figure and legend of Fig 3B. In Fig. 3D, intracellular DNA level was 25 times higher in FAH KD HepG2-D11 cells than control KD cells then why RNA level was only 2 times higher and extracellular DNA level was only increased little compared to intracellular DNA? Southern blotting for intracellular DNA and extracellular DNA should be conducted. Northern blotting for RNA should be conducted to compare with pgRNA, S and X mRNAs. At the same time, RT-qPCR for pgRNA only and for total RNA should be conducted. Again, in Fig 4D in FAH over https://doi.org/10.1371/journal.pone.0314581.r003
7 Nov 2024 Decision Letter - Youkyung Choi, Editor <div>PONE-D-24-31113R1Identification of novel antiviral host factors by functional gene expression analysis using in vitro HBV infection assay systemsPLOS ONE Dear Dr. Nakamoto, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. The revised manuscript improved to make better understanding. However, a few things need to be corrected. Figures 1, 3, and S1 still contain plural descriptions in graph description and line labeling. A reference in line 164 needs to be formatted as other ones. Please submit your revised manuscript by Dec 22 2024 11:59PM. 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy . Reviewer #1: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0314581.r004
Revision 2
9 Nov 2024 Author Response Reviewer #1: The revised manuscript improved to make better understanding. However, a few things need to be corrected. Figures 1, 3, and S1 still contain plural descriptions in graph description and line labeling. A reference in line 164 needs to be formatted as other ones. We appreciate your important suggestion. We revised plural descriptions in graph description and line labeling in Figures 1, 3, and S1. We formatted a reference in line 164 as other ones. Attachments Attachment Submitted filename: Response to Reviewers; PONE-D-24-31113R1.docx https://doi.org/10.1371/journal.pone.0314581.r005
13 Nov 2024 Decision Letter - Youkyung Choi, Editor Identification of novel antiviral host factors by functional gene expression analysis using in vitro HBV infection assay systems PONE-D-24-31113R2 Dear Dr. Nakamoto, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Youkyung H. Choi, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0314581.r006
Formally Accepted
Acceptance Letter - Youkyung Choi, Editor PONE-D-24-31113R2 PLOS ONE Dear Dr. Nakamoto, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Youkyung H. Choi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0314581.r007

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