Review Reports

Response to Guest editors' comments

Thank you very much for your nice and constructive criticism on our manuscript. We really appreciate your suggestions and comments which significantly increased the quality of our manuscript.

We are very sorry for not clearly understanding some points raised by reviewers that have not been addressed well.

The information/corrections/revisions based on your following comments have been mentioned in the manuscript with purple color.

Point 1: The language appears to be unchanged on a sentence level (many have just been added to). If this has been altered from the original, please use the "compare documents" command in Word to highlight the areas where you have improved the language. Both a compared document and a clean document would be appreciated for the second round of review in this instance.

Response 1: We prepared three compared documents for clearly understanding of current/latest revision.

Compared file-1: Current/latest revised version vs first revision one (viruses-682900)- for checking latest revision with previous resubmitted version.

Compared file-2: Current/latest revised version vs original version (viruses-627423)- for checking latest revision with originally (First time) submitted one.

As we reconstructed and rearrange the whole manuscript; maybe, that is why it is hard to find the English corrections. Therefore, we prepared compared file-3 for English checking.

Compared file-3: After English proof of revised version vs before English proof- for checking the English corrections which have been done by professional native English proofing company.

Point 2: We still have concerns about balance and strength of evidence on how likely these in vitro studies reflect biologically-relevant systems. It is not balanced to simply suggest that these overexpression studies need to be confirmed in infection and liver tissue studies; there are studies

already showing the distribution of viral proteins in infection and primary liver tissues and these

should be discussed. Also, studies that do not support these aspects (e.g. HBV does not alter the

innate immune response, doi: 10.1053/j.gastro.2018.01.034) should be added for context in the

field.

Response 2: In accordance with your comment, we have added the descriptions about the correlations between “in vitro studies”, “in vivo studies (HBx-transgenic mice)”, and “the clinical studies (clinical specimens from HBV-mediated HCC)”. Please see the compared file-1.

The contradictory studies for localization and/or the effects of viral proteins have also been discussed.

Point 3: Moreover, the changes added to describe the systems remain insufficient (e.g. HBV-replicating HepG2): these should include description of constructs (1.3 mer, 1mer, sub-genomic), promoter type (e.g. CMV vs. native), how these are brought into the cell (transfection vs. AAV vs. electroporation). It is important to describe also the cell line used, which may already have altered mitochondrial dynamics. Indeed, the limitations of these studies justify a separate dedicated section.

Response 3: We have added the description about systems in detail throughout the manuscript. Please check the revised/latest version of the manuscript. However, altered mitochondrial dynamics have been always compared with the control experiments according to previous reports.

Point 4: Related to the biological context, the effect sizes of the phenotypes mentioned in the review are not described at all. To what extent these pathways (e.g. innate immunity, cell death, cell survival, etc.) activated by HBV proteins? Are these phenotypes based on functional assays or presumption based on gene upregulation? To what extent are these valid interpretations?

Response 4: We have added this information in detail throughout the manuscript based on the works/results conducted by the different research groups. Please check the compared file-1 and sections of HBx, Pol, HBsAg, and HBc, and figure 2 as well.

Cell survival: HBx-induced mROS activates transcription factors such as Foxo-4 in Chang cells stably expressing HBx (Chang-HBx) and in primary hepatic tissues from HBx-transgenic mice, and STAT-3 and NF- κB in HBx-transfected HepG2 cells. HBx associates with various factors such as MARCH5, COX-2, COX-III, AIM2, c-Jun, AP-1, Raf-1, etc, and involves in cell survival. HBsAg interacts with JTB which involved p65/ NF- κB to develop HCC (Please see HBx and HBsAg sections)

Cell death/Apoptosis: HBV viral proteins directly interact and/or influence the expression of Bax, hVDAC, HSP60, p53, Bad, Bid, Bim etc (Please see HBx and HBsAg sections).

Antiviral/Innate immunity: Involvement of MAVS, Parkin, IFN-β, LUBAC, RIG1, STING, IRF-3 etc. has been discussed in the section of HBx and Polymerase (Lines: 209-226; 248-260).

HBV replications: We have discussed about the direct HBV-mediated involvement of Par14/15, GRP78/75, hTid1 etc. (Please see the section of HBx, Polymerase and HBsAg; Lines: 195-208; 271-288).

Mitochondrial damage fission and mitophagy: Direct/indirect involvement of HBx with several factors such as PINK1, parkin, Mfn2, Drp1, LC3B etc have been discussed (Please see lines: 167-176)

Point 5: In summary, there is still limited clarity, critical interpretation and contextualisation of the data discussed in the review. We thank you for understanding that the high editorial quality of Viruses should be maintained and we look forward reviewing your revised manuscript.

Response 5: We revised the whole manuscript according to your suggestions and we hope now you will accept our corrections. Thank you again for giving the chance for revising the manuscript and these corrections significantly improved the standard of the manuscript.

Round 1

Reviewer 1 Report

The authors did revise the manuscript significantly by expanding scientific content and improving English. Not the text is quite clear and readable.

In particular, they tried to report which studies were obtained in which system. But this is still not clear for localization of HBV proteins. But since they can have different localization upon overexpression and during infection, this requires further clarification.

A passage on lines 190-210: Grp75 is not just ER- on mitochondria-residing protein but a protein that links both organelles in MAMs. So, the P protein could affect MAM integrity and thus have an impact on mitochondrial functions. This could be worth mentioning here

Line 84: Please indicate what is meant by the phrase „recent papers”

Please revise the phrase on lines 170-171.

Author Response

Thank you very much for reviewing our manuscript. We have revised the manuscript according to your suggestions and comments.

Point 1: In particular, they tried to report which studies were obtained in which system. But this is still not clear for localization of HBV proteins. But since they can have different localization upon overexpression and during infection, this requires further clarification.

Response 1: We have added the description of systems in detail throughout the current version of the manuscript. Please check the purple-colored text in the revised/latest version of the manuscript. Furthermore, we also correlated the points by discussing the data from clinically infected patients with their limitations as well.

Point 2: A passage on lines 190-210: Grp75 is not just ER- on mitochondria-residing protein but a protein that links both organelles in MAMs. So, the P protein could affect MAM integrity and thus have an impact on mitochondrial functions. This could be worth mentioning here.

Response 2: This is a very important point. Polymerase protein could affect MAM integrity and thus have an impact on mitochondrial functions which have been discussed in the Polymerase section (3.2). We have not found any published report on interaction/relation between grp75 and pol so far we searched. Therefore, we omitted the discussion regarding grp75 and pol.

Point 3: Line 84: Please indicate what is meant by the phrase „recent papers”.

Response 3: We have rewritten the sentence (Lines 85-86).

Point 4: Please revise the phrase on lines 170-171.

Response 4: We corrected the phrase including the paragraph of the manuscript (Lines 229-243).

Reviewer 2 Report

The review can be accepted in the present form

Author Response

Thank you very much for accepting the revisions.

Reviewer 3 Report

The authors have made substantial efforts towards editing the manuscript and addressing the concerns of the reviewers. The manuscript has significantly improved, making for a much more comprehensive and organized review article. The updated manuscript is suitable for publication in Viruses.

Author Response

Thank you very much for your appreciation and for accepting the revisions.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The Review article entitled “Hepatitis B viral proteins and mitochondrial dynamics” written by Md. Golzar Hossain covers the literature surrounding how Mitochondria or mitochondrial proteins are alerted by Hepatitis B virus infection. The effects of virus infection on mitochondrial dynamics and activity is an important topic as mitochondria are central in regulating many cellular pathways. Moreover, this topic has not been reviewed giving an article written on this topic the potential to be high relevance to the field. However, the current version of this manuscript requires an extensive amount of work before it would be suitable for publication.

Major concerns

The most pressing concern with the manuscript is that is has a large number of spelling and grammatical errors that make many ideas or topics difficult to understand or interpret. The author should consider having the paper copy edited. Additionally, there are a number of mistakes in the descriptions of mitochondria and mitochondrial processes as well as protein localization. The author should be clear on the literature surrounding mitochondrial function so as to not make misleading in incorrect statements. Additionally, the focus of the manuscript should be shifted more towards how the virus affects mitochondrial dynamics and function rather than localization of viral proteins to the mitochondria (see specific points for details). The author should also refrain from making broad conclusions that are not supported by the evidence in the literature they are reviewing.

Specific points

The first sentence in the introduction (line 27) starts by stating that “Mitochondria are self-replicating double membrane organelles”. Though the Mito have their own genome, they require many factors coded from the nuclear DNA in order to grow and divide. Therefore, mitochondria should not be described as “self-replicating”. In line 63, the author states that “more or less all HB viral proteins localize into mitochondria”. This is a confusing statement especially since the papers cited do not show localization of HBV proteins to the mitochondria. In fact Kim et al. show that HBsAg is not localized to the mitochondria. Though there is some evidence that a subpopulation of HBx and HBV polymerase might localize to the mitochondria, there is little evidence that core or HBsAgs reside there. In the sentence starting on line 120, the author states that HBx-mediated down regulation of MAVS “confirms the localization of HBx onto the OMM”. This does not confirm localization to the OMM. This is a phenotypic and physical connection with MAVS which says nothing about HBx protein localization or where this interaction occurs. In line 183 the author claims that the viral polymerase might go to the mitochondrial matrix for proper folding. Knowing what we do about protein folding and protein transport into the mitochondrial matrix, this is not a feasible model. There is little evidence for protein export from the Mitochondria. The author should provide more evidence for this type of conclusions before making them in a review article. In line 204 the author states that HBsAgs might concentrate around the mitochondria (MAMs or OMMs); however, the data provided by Kim et al. (Ref #6) do not see any HBsAg accumulation in mitochondrial fractions. Additionally, none of the references provided in the following paragraph (lines 205-213, ref #60-63) show HBsAg at the mitochondrial. Rather they suggest HBsAg is responsible for directing several factors away from the mitochondria. The author needs to address this discrepancy.

Reviewer 2 Report

The review aims at describing the effects of the HBV proteins on mitochondria. 

A review on a topic like this should include a more systematic review of the literature available, preferably it should be described how the author gathered this literature by a systematic search approach

The title does not cover the contents, the review does not focus on the effect of HBV proteins on mitochondrial dynamics, but on the effects of HBV proteins interacting with the mitochondria and associated proteins.

The abstract suggests that mitochondria can be targeted to reduce HBV replication and/or pathology, but this is not supported by the review. only one study is mentioned (ref. 30), this is insufficient.

The review seems to 'push' the notion that HBx localises to the outer mitochondrion membrane, but this notion is insufficiently supported by the referenced literature.

The canonical function of HBx, to drive HBV RNA transcription, is not introduced. Interactions witht eh mitochondria, and the relation to HBV replication, should be considered in this perspective.

The interactions between HSPs and HBV proteins do not involve mitochondria or mitochondria function (!). HSPs are NOT mitochondrial chaperones, as stated by the author (line 162, 177). The review critically depends on this 'notion'.

Some sentences cannot be interpreted (e.g. 111-112), and if the author wishes to make such statements they should be supported by literature. 

lines 121-122: this statement is false, one cannot conclude this from the data referenced.

All in all, it is speculative if the effects of HBx on mitochondria depend on interactions with specific proteins on the mitochondria, as the author suggests, or stem from another interaction that results in the modulations described.

Reviewer 3 Report

The main topic of this review is of great interest since a review summarizing the direct impact of HBV proteins on mithocondria functionality and signaling pathways is still missing. This issue is particularly relevant since several papers suggest that altered mithocondria dynamics can be involved in mechanisms underlying HCC onset. However, the review is difficult to follow particularly for the pletora of English mistakes, disseminated throughout the whole text. Some sentences are meaningless. The different data from the already published studies are reported in the text without a precise order and in absence of a valid interpretation. For all the aspects I retain that the review is not suitable for publication in Viruses.

Reviewer 4 Report

The manuscript by Hossain aims to summarize current data on interaction of hepatitis B virus (HBV) and its proteins with mitochondria and its consequences. The topic is very interesting indeed. However, the manuscript has a number of drawbacks that make it not acceptable in its current form.

First of all, English is quite poor. The whole manuscript has to be edited by a native-speaker. In the current version, numerous flaws take all attention of the reader from scientific ideas. I hate to comment the text of manuscripts but here numerous phrases have to be rewritten.. Most of research has been carried out in cells overexpression individual virus proteins. So, it should be acknowledged that most of the data discussed in the review still have to be validated in infectious models. And the author needs to carefully show which data were obtained in which system. The text makes the reader think that HBV proteins are localized solely in mitochondria. However, this is not true. For example, there are data showing that HBx can be found in the ER and even nucleus, and HBe as well is an ER-residing/associated protein. So again, protein localization should be discussed more carefully. The title of the review declares that it should discuss mitochondria dynamics. But there are only scarce words about fusion/fission and mitophagy. The author should consider expanding the topic by discussing what is known about association of fusion/fission with metabolism and respiration in general, how dynamics is affected by interaction of mitochondria with lipid droplets etc. And probably how this knowledge outside virology field may impact research for HBV. Poor quality (resolution) of the Figure 1, especially of its panel A. Human VDAC should be abbreviated as (hVDAC).