Interaction between hTIM-1 and Envelope Protein Is Important for JEV Infection
Round 1
Reviewer 1 Report
Comments for author File: 
Comments.pdf
Author Response
Dear Editor，
Thank you for your decision letter concerning our manuscript (viruses-2518903) entitled "Interaction between hTIM-1 and Envelope protein is Important for JEV infection", and for taking the time to review our revision. We sincerely appreciate the valuable suggestions by both you and the reviewer. We have thoroughly considered all the comments and have made the necessary revisions to the manuscript. In light of these improvements, we believe that the current version now meets the publication standards of the Journal. Below, we have provided a brief response to each of the comments and a comprehensive list of the changes we have implemented in the manuscript.
All the comments raised by the reviewer has been revised and highlighted in yellow. In addition, my manuscript has also been revised by a professor with good English writing, and major changes have been noted in green.
Major comments,
- The manuscript in its current form requires a moderate but important level of English language editing (sentence structure and meaning, spellings etc). These corrections must be done before the manuscript is accepted for publication.
A: Thanks for the reasonable comments. Manuscript has been checked and revised by a professor with good English writing. I used green highlighting to highlight areas with significant changes of sentences and some words.
- References
a. References cited are minimal, some are too old and at least one in a foreign language. While original studies are beneficial, more recent research must be included where relevant and possible, and references cited are strongly suggested to be in English for the international readership of the Journal.
A: Thanks for the reasonable suggestions. I have updated the recent progress on JEV or TIM-1 research. I also supplied another 7 papers on the related research. Papers within five years account for 30%, and the one within eight years account for 50%.
b. Please add references after lines 25-27, 27-29, 62-63, 147; check whether #5 is the correct reference for the sentence.
A: Thanks for the reasonable suggestions. I added or replaced some references with those published in recent years, line 28, 30, 33, 37, 64, 80, 156, 401.
- In several places in the Discussion, it is suggested or speculated that hTIM-1 may act as a dual attachment factor and receptor as it interacts with JEV E and PS on viral surface. However, it would be useful to add that further studies are needed to confirm this role and the basis of interactions with PS in JEV.
A: Thanks for the reasonable comments. This is the third article about TIM-1 protein promoting JEV infection in our lab. Our previous study demonstrated that hTIM-1 mediated JEV infection is PS-independent [1]. sTIM-1 also promotes JEV infection via the PS-dependent pathway [2]. Therefore, I didn’t elucidate the interaction between TIM-1 protein and PS in detail.
Minor comments,
- Figures
a. Please consider following a consistent and consecutive order for sub-figures (clockwise, left to right etc) and the corresponding legend (in some legends “C” appears before “BD”.
A: Thanks for the reasonable comments. Figure 1 and Figure 2 have been rearranged in consecutive order. Due to the fluorescence diagram take much space in Figure4/5 (A and C), the order was unchanged.
b. Fig 3A: Please check labels for rows, and match with text in line 257.
A: Thanks for the reasonable comments. I have revised the labels in the Figure 4A.
- Methods
a. Please include specifics and catalog numbers for materials used (eg lines 86, 112, 114, 115, 140, 153, 154)
A: Thanks for the reasonable comments. I have added the catalog numbers for some materials highlighting in yellow, line 93, 90,105, 111-114, 121, 123, 124, 148, 152-153, 176, 190.
b. Please mention the centrifuge type and model used for different spins in the methods section, and for the microscopes (lines 156, 157).
A: Thanks for the reasonable comments. The centrifuge type was supplied at line 121, and all the experiments used the same centrifuge. The microscopes type and model have also been revised at line 172-173.
c. What’s the composition of the elution buffer in line 134?
A: Thanks for the reasonable comments. The composition of the elution buffer was supplied at line 145-146.
d. Line 165- where is the mouse anti-JEV E Ab acquired from?
A: The mouse anti-JEV E antibody was kept by our lab. It has been revised at line 105-106.
e. Please mention controls used in each experiment in the text.
A: Thanks for the reasonable comments. The 2.5 GST-pull down assays, 2.6 plaque assay, 2.8 IFA, and 2.9 Protein neutralization assays have been revised and supplied the controls at line 141-142, 150, 164-165, and 178. However, 2.4 Co-IP assays were performed in different cells and conditions, so it’s a little complex to mention various controls, but it was clarified in the figure legend with different assays.
- General
a. Define abbreviations in the first instance. For eg, FBS in lines 87 and 91, PtdSer in line 54, GP and PS in line 67 etc. Check for consistency (E K38 or E-EK38? NJ2008 or NJ08? ImageJ or imageJ, also check line 332)
A: Thanks for the reasonable comments. The full name of FBS was added in the first instance at line 90. K38 of E/ E(K38R), NJ2008, and ImageJ was revised and in consistency.
b. Line 295 “.infected with mixture”- at what MOI?
A: Thanks for the reasonable comments. I have revised it at line 307.
c. In the Discussion, it would be helpful to add more information in text on the PS binding sites and cavity, as this is discussed in multiple places (lines 337-346, 363), and also on the hTIM variants. Does the capacity to promote infection differ across individual flaviviruses or between serogroups, and does this correlate with conservation of specific residues?
A: Thanks for the reasonable comments. I have added some relevant research on how this cavity is formed and how PS binds to this conformation at line 54-58. Researches have demonstrated that TIM-1 protein can effectively promote different flaviviruses, including ZIKV, DENV (including different serogroups), YFV, WNV, and JEV, which was all PS-dependent [1,3,4]. The PS-TIM-1 binding sites were conserved residues in TIM family. Later, Live imaging and immunoelectron microscopy indicated that TIM-1 and DENV co-internalized into cells and promote DENV entry [5]. Some researchers also found that ZIKV envelope protein interacts with TIM-1 protein to mediate ZIKV entry [6]. Therefore, we would like to know if TIM-1 protein interacts with JEV E protein to mediate JEV infection.
Reference
- Niu, J., Jiang, Y., Xu, H., Zhao, C., Zhou, G., Chen, P., Cao, R. TIM-1 Promotes Japanese Encephalitis Virus Entry and Infection. Viruses.2018; 10. doi:10.3390/v10110630
- Jiao, W., Xie, S., Liang, Z., Pan, J., Yang, X., Tong, H., Zhao, Y., Cao, R. P34L Mutation of swine TIM-1 enhances its ability to mediate Japanese encephalitis virus infection. Vet Microbiol.2022; 274: 109555. doi:10.1016/j.vetmic.2022.109555
- Jemielity, S., Wang, J.J., Chan, Y.K., Ahmed, A.A., Li, W., Monahan, S., Bu, X., Farzan, M., Freeman, G.J., Umetsu, D.T., Dekruyff, R.H., Choe, H. TIM-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine. Plos Pathog.2013; 9: e1003232. doi:10.1371/journal.ppat.1003232
- Meertens, L., Carnec, X., Lecoin, M.P., Ramdasi, R., Guivel-Benhassine, F., Lew, E., Lemke, G., Schwartz, O., Amara, A. The TIM and TAM families of phosphatidylserine receptors mediate dengue virus entry. Cell host & microbe.2012; 12: 544-557. doi:10.1016/j.chom.2012.08.009
- Dejarnac, O., Hafirassou, M.L., Chazal, M., Versapuech, M., Gaillard, J., Perera-Lecoin, M., Umana-Diaz, C., Bonnet-Madin, L., Carnec, X., Tinevez, J.Y., Delaugerre, C., Schwartz, O., Roingeard, P., Jouvenet, N., Berlioz-Torrent, C., Meertens, L., Amara, A. TIM-1 Ubiquitination Mediates Dengue Virus Entry. Cell reports.2018; 23: 1779-1793. doi:10.1016/j.celrep.2018.04.013
- Giraldo, M.I., Xia, H., Aguilera-Aguirre, L., Hage, A., van Tol, S., Shan, C., Xie, X., Sturdevant, G.L., Robertson, S.J., McNally, K.L., Meade-White, K., Azar, S.R., Rossi, S.L., Maury, W., Woodson, M., Ramage, H., Johnson, J.R., Krogan, N.J., Morais, M.C., Best, S.M., Shi, P.Y., Rajsbaum, R. Envelope protein ubiquitination drives entry and pathogenesis of Zika virus. Nature.2020; 585: 414-419. doi:10.1038/s41586-020-2457-8
Thank you very much for your attention and time. Look forward to hearing from you.
Author Response File: Author Response.pdf
Reviewer 2 Report
Liang et al., examined the influence of binding of hTIM1 cell surface protein to JEV envelop protein to replication efficiency of the virus. They proved, that the two molecules are interact, determined the smaller parts (down to amino acid level) of the molecules which interact with the other protein. They also showed, that both solubile hTIM1 molecules and anti hTIM1 IgG antibodies are inhibit viral binding to sensitive cells, and such decrease virus replication. These results could be basis for further vaccine developments. The applied methods are relevant, the results are well-documented, the English usage is fine (some little errors are given below).
Abstract – abbreviations are not given in the Abstract, but in the text at the first appearance (usually in the introduction).
line 175 And ?????
line 190 287, 310, 320, 353, 394, 402 – in vitro/vivo in italics
line 201 – thses???
line 230 - and is not necessary here, please omit
line 258 - mpi is given somewhere in the text? or only the abbreviation is given?
line 285 - "to a certain extent" Could you give here at least an approxinate value in numbers (e.g. %)? like in lines 314/315.
line 339 – „there is a” is not necessary + tick-borne encephalitis virus is also a very important vector-borne Flavivirus
line 353 – The in vitro binding affinity
good, with some minor spelling mistakes, indicated
Author Response
Dear Editor，
Thank you for your decision letter concerning our manuscript (viruses-2518903) entitled "Interaction between hTIM-1 and Envelope protein is Important for JEV infection", and for taking the time to review our revision. We sincerely appreciate the valuable suggestions by both you and the reviewer. We have thoroughly considered all the comments and have made the necessary revisions to the manuscript. In light of these improvements, we believe that the current version now meets the publication standards of the Journal. Below, we have provided a brief response to each of the comments and a comprehensive list of the changes we have implemented in the manuscript.
All the comments raised by the reviewer has been revised and highlighted in yellow. In addition, my manuscript has also been revised by a professor with good English writing, and major changes have been noted in green.
Reviewer 2
1.line 175 And ?????
A: It has been revised into “the commercial Mouse IgG (A7028, Beyotime, China)”, line 175.
- line 190 287, 310, 320, 353, 394, 402 – in vitro/vivo in italics
A: “in vitro/vivo” in the manuscript has all been changed into italics, line 190, 284, 307, 317, 347, 396.
3.line 201 – thses???
A: It has been revised into “these”, line 201.
- line 230 - and is not necessary here, please omi
A: Thanks for this reviewer’s suggestion. The reference was added here so that we can better understand the reason why I directly mutated the amino acid at the position 38 of E protein, making context more coherent.
5.line 258 - mpi is given somewhere in the text? or only the abbreviation is given
A: “mpi” represents “minutes post infection”, line 255.
6.line 285 - "to a certain extent" Could you give here at least an approxinate value in numbers (e.g. %)? like in lines 314/315.
A: The comments are well taken. It has been revised into “Furthermore, the hTIM-1 ECD protein could also prevent JEV from entering A549 cells and hTIM-1-expressing 293T cells with the inhibition rate at about 50% and 40%, respectively”, line 282,283.
7.line 339 – „there is a” is not necessary + tick-borne encephalitis virus is also a very important vector-borne Flavivirus
A: Thanks for this reviewer’s suggestion. “there is a” has been deleted and “Tick-borne Encephalitis virus (TBEV)” has been added into the line 333 of the new manuscript.
8.line 353 – The in vitro binding affinity
A: It has been revised into “the in vitro binding affinity”, line 347.
Author Response File: Author Response.pdf
Reviewer 3 Report
In this manuscript, based on the previous finding that TIM-1 can promote JEV infection, Liang et al. further found that TIM-1 can directly interact with JEV E protein. Moreover, the IgV domain of TIM-1 and the K38 site of E protein were determined to play an important role in the interaction between TIM-1 and E protein. They also found that soluble TIM-1 ectodomain domain protein, as well as TIM-1 specific antibodies, could inhibit JEV infection in cells. This study provides new information about the replication mechanism of JEV and provides theoretical support for potential therapeutic target of JEV infection. After reviewing the paper, I think it is acceptable for publication in this journal but needs minor revision.
Fig.1B the band names in blots were not properly labeled. In Fig.1B and Fig.1C, the IP were conducted with anti-TIM-1 antibody, not anti-His-tag antibody.
Fig.1D: In the first and second rows, the fluorescent figures are not clear, please adjust the brightness and contrast and use appropriate magnification figure.
Line 232: “E-K38” should be “E-k38R”.
Fig.3 A The first row should be Mock infection, and the second row should be 25 min infection group. Please check and revise the labels in the figure.
In Figure 5, mouse IgG control and anti-TIM-1 antibody should be labeled, not just the antibody concentration.
Line 272: “6*His tag” should be “6×His tag”.
Line 332: “ImageL” should be “ImageJ”. The full name of the abbreviation MFI should be given.
Line 341: “hhTIM-1” should be “hTIM-1”.
Line 350-351: “interacts” should be “interact”.
English writing needs to be improved.
Author Response
Dear Editor，
Thank you for your decision letter concerning our manuscript (viruses-2518903) entitled "Interaction between hTIM-1 and Envelope protein is Important for JEV infection", and for taking the time to review our revision. We sincerely appreciate the valuable suggestions by both you and the reviewer. We have thoroughly considered all the comments and have made the necessary revisions to the manuscript. In light of these improvements, we believe that the current version now meets the publication standards of the Journal. Below, we have provided a brief response to each of the comments and a comprehensive list of the changes we have implemented in the manuscript.
All the comments raised by the reviewer has been revised and highlighted in yellow. In addition, my manuscript has also been revised by a professor with good English writing, and major changes have been noted in green.
Reviewer 3
- Fig.1B the band names in blots were not properly labeled. In Fig.1B and Fig.1C, the IP were conducted with anti-TIM-1 antibody, not anti-His-tag antibody.
A: Thanks for the reasonable comments. I have revised the “His” into “TIM-1”.
2.Fig.1D: In the first and second rows, the fluorescent figures are not clear, please adjust the brightness and contrast and use appropriate magnification figure.
A: Thanks for the reasonable comments. I also think the clarity of this image is not enough, so I replaced a new one, Figure 1D.
- Line 232: “E-K38” should be “E-k38R”.
A: It has been revised into “E-K38R”, line 231.
4.Fig.3 A The first row should be Mock infection, and the second row should be 25 min infection group. Please check and revise the labels in the figure.
A: Thanks for this reviewer’s suggestion. The labels has been revised in Fig.3A.
5.In Figure 5, mouse IgG control and anti-TIM-1 antibody should be labeled, not just the antibody concentration.
A: Thanks for the reasonable comments. All of the labels in Figure 5 have been revised.
6.Line 272: “6*His tag” should be “6×His tag”.
A: It has been revised into “6×His tag”, line 269.
7.Line 332: “ImageL” should be “ImageJ”. The full name of the abbreviation MFI should be given.
A: It has been revised into “ImageJ”. And the full name of the abbreviation MFI was “Mean Fluorescent Intensity” and has been added into line 326.
8.Line 341: “hhTIM-1” should be “hTIM-1”.
A: It has been revised into “hTIM-1”, line 335.
9.Line 350-351: “interacts” should be “interact”.
A:It has been revised into “interact”, line 351.
10.Comments on the Quality of English Language: English writing needs to be improved.
A: Thanks for the reasonable comments. Manuscript has been checked and revised by a professor with good English writing. I use green highlighting to highlight areas with significant changes of sentences. There are still some word modifications that I have not marked.
Author Response File: Author Response.pdf
