Elucidation of antiviral mechanisms of natural therapeutic molecules Herbacetin and Caffeic acid phenethyl ester against chikungunya and dengue virus

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   Review Commons

   Apr 9, 2024

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   Reply to the reviewers

   1. General Statements__: __The manuscript entitled "Dual antiviral mechanisms of Herbacetin and Caffeic acid phenethyl ester against Chikungunya and Dengue viruses with insights into Dengue methyltransferase-CAPE crystal structure" is the first report of broad spectrum alphavirus and flavivirus inhibitors with dual roles that efficiently inhibit virus replication by diminishing the levels of polyamines in the host cells as well as inhibit the enzymatic activity of the virus-specific methyltransferase (MTases). Chikungunya virus (CHIKV) and Dengue virus (DENV) are re-emerging alpha- and flaviviruses respectively. Until now, no antivirals are commercially …

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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   Reply to the reviewers

   1. General Statements__: __The manuscript entitled "Dual antiviral mechanisms of Herbacetin and Caffeic acid phenethyl ester against Chikungunya and Dengue viruses with insights into Dengue methyltransferase-CAPE crystal structure" is the first report of broad spectrum alphavirus and flavivirus inhibitors with dual roles that efficiently inhibit virus replication by diminishing the levels of polyamines in the host cells as well as inhibit the enzymatic activity of the virus-specific methyltransferase (MTases). Chikungunya virus (CHIKV) and Dengue virus (DENV) are re-emerging alpha- and flaviviruses respectively. Until now, no antivirals are commercially available to combat these two viral infections. This study delves into the antiviral mechanisms of Herbacetin (HC) and Caffeic acid phenethyl ester (CAPE) against DENV and CHIKV. Treatment of Vero cells with these compounds resulted in polyamine depletion. However, adding exogenous polyamines did not completely rescue the virus, suggesting alternative antiviral mechanisms. Interestingly, these compounds exhibited anti-MTase activity against purified viral MTases of CHIKV and DENV. The crystal structure of the DENV 3 MTase in complex with CAPE revealed its binding site within the GTP-binding region of DENV MTase. This study presents the novel dual inhibition mechanism of HC and CAPE, offering promising prospects for developing broad-spectrum antivirals.

   2. Point-by-point description of the revisions

   We express our gratitude to the reviewers for their time and insightful comments, which have significantly contributed to the improvement of the manuscript. We believe that the thoughtful critiques and suggestions have significantly enhanced the overall quality of our work. Below, we provide a point-by-point response to each comment, addressing the concerns raised by the reviewers.

   Reviewer 1: -

   Comment 1: My main concern is that the depletion of polyamines is likely to have broad implications for host cell metabolism. Polyamines are critical for genome folding and stability. Hence, polyamine depletion will likely compromise cellular metabolic homeostasis. My suggestion is to perform a literature survey on this topic, identify appropriate assays of cellular homeostasis, and add at least one such assay in the relevant HC and CAPE concentration range to address my question..

   I also suggest adding the potential negative effects of polyamine depletion on host cell metabolism in the discussion section

   • Response: *We appreciate the reviewer's constructive feedback for their insightful remarks on the potential extensive influence of polyamine depletion on host cell metabolism. We acknowledge the critical role polyamines play in genome folding and stability, and their depletion could indeed disrupt cellular homeostasis. In response to this valuable feedback, we conducted a comprehensive literature review. This literature review uncovered studies investigating the targeting of the polyamine biosynthetic pathway as a potential therapeutic strategy for combating various infections and diseases. Additionally, **DFMO , a drug that targets polyamine biosynthetic pathway enzyme is an FDA-approved drug for African sleeping sickness and high-risk neuroblastoma (Bouteille & Dumas, 2003; Nazir et al., 2024) indicating that despite the critical role of polyamines in cellular metabolic homeostasis, the host polyamine pathway can also be successfully targeted for antiviral drug discovery. As recommended, we have added this information in the revised manuscript. * *Additionally, ribavirin, an FDA-approved antiviral agent, employs various mechanisms to inhibit viral replication, including the reduction of polyamine levels (Tate et al., 2019). Furthermore, we have also examined the protocols available in the literature for CAPE, HC, and DFMO treatment. Most of these studies have employed MTT assay, as illustrated in the research conducted by Arisan et al. 2012 and Shen et al. 2013 (Arisan et al., 2012; Shen et al., 2013). Notably, Aljabr et al.,2016 also employed the MTT assay for viability testing, underscoring its relevance (Aljabr et al., 2016). Similarly, our manuscript employed the MTT assay at various compound concentrations to ensure the utilization of non-cytotoxic concentrations for antiviral activity testing. *

   As per reviewer's recommendation, we have discussed the potential adverse effects of polyamine depletion on cellular processes in the revised manuscript's discussion section.

   *Line no.s 513 – 523 of the revised manuscript have the revised text as per the suggestion. *

   Reviewer 2:-

   Comment 1:- Authors describe anti-CHIKV and anti-DENV activities of herbacetin and caffeic acid phenyl ester (CAPE). The antiviral effect is not reversed buy exogenous polyamines suggesting multiple mechanisms of action. NS5-Met complex with caffeic acid phenyl ester was obtained and its structure resolved at high resolution. The resolved structure reveals two binding sites for antiviral compound overlapping with that of GTP and possibly with a site involved in binding of RNA

   Other than analysis of crystal structure of NS5/CAPE complex the provided data is of low quality and is not analyzed properly. There is no evidence that data is reproducible. Authors have calculated significance from "experimental repeats" which, based on the description of experiments, are not independent experiments but technical replicates. Some key technical details are missing and some experiments are not described at all. The writing can be vastly improved and figures be made a lot more easier to understand.

   • *Response :-We appreciate the reviewer's positive feedback of on the crystal structure and as pointed out towards data quality and analysis, we have tried and made significant improvements, including enhancing data representation and providing detailed protocols in the supplementary materials where necessary. Additionally, we have addressed key technical details that were previously missing and ensured that all experiments are described adequately. We acknowledge the need for clearer writing and have now mentioned clearly that independent experiments have been carried out in the study. We have made suggested revisions to the revised manuscript. *

   Comment 2:- Bad writing lines 64-65 . Viral genomes lack protein synthesis machinery. Basically correct but no genome has protein synthesis machinery

   • Response:-We thank the reviewer for pointing this out. We have modified the text as follows: lines 64-65 "*Viral genomes lack protein synthesis machinery, and the ability to hijack the host cell's resources for replication is crucial for all viruses". to *lines 65-67 "Viral particles lack essential protein synthesis machinery. Consequently, viruses rely on the host cell's resources to replicate effectively."

   Comment 3:- line 137 flavonoids play a role in reducing the levels of nsP1 in CHIKV - what can this possibly mean? Are shown to reduce the level of nsP1 in CHIKV-infected cells?

   • *Response: We appreciate the reviewer for bringing this to our attention, and we acknowledge that it was due to a writing issue in English. This has now been rectified. A dose-dependent reduction of the CHIKV E2, nsP1, and nsP3 proteins was observed upon treatment with baicalein and fisetin. This finding would suggest that baicalein and fisetin might inhibit the production of CHIKV protein, especially the proteins involved in the negative-strand synthesis and part of the replicase unit (Lani et al., 2016). To account for this suggestion, we have modified the text in the revised manuscript to (line 145-147): " Moreover, flavonoids treatment has demonstrated the dose-dependent decrease in CHIKV titer due to reduced levels of CHIKV viral proteins, including nsP1. *

   __Comment 4 :-__line 250-251 - RNA was isolated from the infected cells' supernatant, used for cloning, and inserted between the NheI and XhoI restriction sites... …..It should be impossible as one cannot insert RNA into bacterial plasmid DNA.

   • Response:- *We thank the reviewer for pointing this out. line 250-251 – "RNA was isolated from the infected cells' supernatant……..". This **has been **changed to line 267-271 *" RNA was isolated from the supernatant of the cells infected with DENV 3, and used for cDNA preparation, cloning of the MTase gene fragment into the pET28c (+) vector using NheI and XhoI restriction sites."

   __Comment 5 :-__Missing parts. Examples

   the source of nsP1 of CHIKV is not indicated, True, there are references to previous studies, but this is extremely important point and it should have been clearly stated that it was obtained from E. coli. The issue is that authors made some predictions and modelling based on structure of nsP1 from eukaryotic expression system. It is not known does the enzyme purified from bacteria have similar structure (actually, in cited Nature paper - doi: 10.1038/s41586-020-3036-8 - attempts to purify nsP1 from bacteria were made. The protein was monomeric and had no activity)

   • Response:- We thank the reviewer for the comments. In response to the reviewer's concern regarding the source of the nsP1 protein from CHIKV, we would like to clarify that the recombinant protein was expressed and purified from E. coli Rossetta cells in our laboratory. We acknowledge the importance of this point and apologize for any oversight in not explicitly stating it in the manuscript. In response to the reviewer's suggestion, we have incorporated a detailed expression and purification protocol into the manuscript supplementary methodology (line number 1068-1091).
   • Response:- Alphaviruses share a high degree of sequence similarity (>80%), particularly within the nsP1 protein, with conserved active site residues (Supplementary Figure 2). Several studies investigating nsP1 proteins from alphaviruses, including Sindbis virus, Semliki Forest virus, and Venezuelan equine encephalitis virus, have successfully employed E. coli Rosetta cells for protein expression, followed by enzyme activity assays (Abdelnabi et al., 2020; Li et al., 2015; Tomar et al., 2011). Our laboratory is working on this protein for more than a decade and have conducted extensive assays on the activity of nsP1 protein purified from bacterial expression system. Our results are reproducible. These studies have been published in reputed peer reviewed research articles, including (Kaur et al., 2018; Mudgal et al., 2020). Additionally, similar assays have been demonstrated in the study by Bullard-Feibelman et al., 2016. We trust that this clarification resolves the reviewer's concern, and we are delighted to address any further inquiries.

   Comment 6:- Figure lacks quality (and figure legends are unclear) Examples:

   • it is impossible to understand what exactly is shown in Figure 1J

   • important information is missing, for example, it is not clear what were concentrations of antiviral compounds for panels 1F and 1I

   • Response :- We thank the reviewer for the constructive comments that has helped us to improve the revised manuscript. We have revised Figure 1J and as suggested we have updated the legends accordingly. Similar revisions have been made in the revised manuscript to the TLC protocol and results to ensure clarity. We thank the reviwer for pointing out the missing information regarding the concentrations of the antiviral compounds used in panels 1F and 1I. As per your suggestion, we added the antiviral compounds concentrations for these experiments in figure legends.

   Comment 7:- 4. wrong data

   • line 478 it is stated that there is no vaccine for DENV or CHIKV. It is correct, DENV vaccine has been in use for several years and CHIKV vaccine was approved at 2023
   • line 476 refers to family alphaviridae. This does not exist, family is Togaviridae

   • Response:- We appreciate the reviewer for bringing this to our attention. We have accordingly revised the sentences for accuracy. "Although human viruses belong to several viral families, Alphaviridae and Flaviviridae are the most significant burden on public health" changed to line number 505-506 "Although human viruses belong to several viral families, Togaviridae and Flaviviridae impose one of the most significant burdens on public health"

   Line no.. 478 “ Neither commercially available drugs nor vaccines are available for these viruses.” Changed to line number 508 to 509 “Although FDA-approved vaccines for Dengue and Chikungunya viruses are available, no antiviral therapies have been approved against these viral infections.”

   Comment 8: ____5. unjustified conclusions. Example

   • authors have analyzed sequences of nsP1 of alphaviruses and made conclusions regarding conservation of active site. It is probably correct but the analyzed viruses do not represent all diversity of alphaviruses, insect specific members and aquatic alphaviruses should also be analyzed (same problem with analysis performed for flaviviruses)

   • Response:-Following the reviewer's recommendation, we have included Salmonid alphavirus, an aquatic virus, and Eilat virus, an insect-specific virus, in our comparison along with other human-infecting alphaviruses. Additionally, for flaviviruses, we have incorporated Palm Creek virus, an insect-specific virus, and Wenzhou shark flavivirus, an aquatic virus. As suggested, the relevant modifications have been done to the MSA protocol, results, and figure legends.

   Comment 9:- 6. Insufficient analysis of data. In some cases, there is a significant discrepancy between the results of different assays. For example, CAPE inhibits DENV at 2.5 microM (Fig 1H) but in test tube assay only small inhibition was observed even at 1000 microM. Authors should provide plausible explanation for this and similar discrepancies.

   (CE and ELISA-based assays shown on figure 6 also resulted in drastically different inhibitions). It is expected assays would produce different results but there should also be explanation for this. If this is not provided one can assume that it is due to experimental errors.

   • Response:- *We thank the reviewers for their valuable comments. We acknowledge the importance of providing plausible explanations for such variations and are committed to addressing these concerns in our revised analysis. * *Our explanation: Capillary electrophoresis (CE) offers a direct approach for detecting S-adenosylhomocysteine (SAH), the product of the methyltransferase reaction. However, this assay has a limitation in sensitivity, it is only able to detect SAH concentrations above ~ 300 µM. A previously validated CE-based assay for Chikungunya virus (CHIKV) nsP1 by Mudgal et al.,2020 addresses this limitation. Their work demonstrates that using specific concentrations of S-adenosylmethionine (SAM) at 0.3 mM and guanosine triphosphate (GTP) at 4 mM enables reliable detection of SAH in the reaction. However, *CAPE is observed to inhibit DENV at ~2.5 micro, supporting that viral inhibition not only is due to MTase inhibition but through other mechanism i.e. host cells polyamine depletion.

   • Therefore, this presents one plausible explanation, although we cannot currently dismiss the possibility of other mechanisms that could also contribute to viral inhibition by CAPE.*

   The established ELISA assay of nsP1 utilizes an indirect detection method, which exhibits higher sensitivity. Additionally, previously published studies on alphaviral nsP1 inhibitors also report nsP1 enzyme activity inhibition by compounds at concentrations several folds higher than their respective active doses in cell culture-based studies (Delang et al., 2016; Mudgal et al., 2020; Kovacikova et al., 2020).Therefore, differing substrate concentrations and CE-based assay limitations may be attributed to discrepancies between the capillary electrophoresis (CE) and ELISA assays. Numerous studies have utilized the CE-based assay or equivalent assays based on similar principles as qualitative tools for evaluating enzyme activity.

   In the revised manuscript, Figures 6B and 6C graphical representation has been transitioned from a dose-response curve IC50 format to a bar chart for enhanced clarity. This bar chart effectively conveys the key finding of a dose-dependent decrease in activity observed for both HC and CAPE.

   *Similarly, we again tried to reoptimize the MTase CE-based assay by reducing the GTP concentration in enzyme reaction from 4 mM to 0.3 mM. This modification resulted in slight improvement and shows clear (~50%) decrease in enzyme activity at the highest concentration, as shown in Fig. 6 F and G. Furthermore, *our approach with CE based assay is centered around detecting inhibition rather than conducting quantitative analyses.

   *The discrepancy in the in vitro vs the enzyme test tube assay could be attributed to HC and CAPE's multifaceted mechanism of action when used in vitro (i.e polyamine depletion and anti methyltransferase activity). However, only methyltransferase inhibition has been assessed in enzymatic assay. *Following the reviewer's suggestion, we have revised the methyltransferase assay protocol, results, and figure legends for clarifications. Additionally, the results have been appropriately discussed in the discussion section.

   Comment 10 :-6. Discussion is essentially missing, it is just list of statements mostly repeating what was said in other sections

   *> *Response: We appreciate the reviewer's suggestion regarding the discussion section; we have incorporated a comprehensive discussion in the revised manuscript.

   3rd reviewer :-

   The manuscript submitted by Bhutkar M. et al. details the antiviral properties of two compounds, herbacetin (HC) and caffeic acid phenethyl ester (CAPE), against Chikungunya virus (CHIKV) and Dengue virus (DENV) through cellular, bioinformatics, biochemical, biophysical, and structural studies. The authors propose a dual antiviral mechanism of action exhibited by these compounds, beginning with an evaluation of their cytotoxicity. Subsequent assessments of their antiviral efficacy against CHIKV and DENV are addressed using plaque reduction assay and other orthogonal assays such as qRT-PCR, and Immunofluorescence assay (IFA). Further, authors performed thin layer chromatography (TLC) to monitor polyamine levels in the cells treated with these compounds and concluded that these compounds leads to polyamine depletion which is also supported by previous studies. These experiments included DFMO as a control which is well established for its role in this regulation. Beyond their impact on cellular polyamine levels, the authors propose a role for these compounds in the inhibition of MTase domains in CHIKV and DENV, supported by the crystal structure of the DENV-3 NS5 MTase domain in complex with CAPE.

   Comment 1:-

   __Major points:- __ While the manuscript presents promising findings regarding the dual antiviral effects of the tested compounds, the authors fall short of demonstrating direct inhibition of MTase activity as a meaningful and complementary effect to polyamine depletion. Being only indirect, the enzyme inhibition data is not convincing, and the measured indirect inhibition is not precise enough in the case of CHIK nsp1 and too weak in the case of DENV NS5 (detailed below).

   Conceptually, the organization of the results should be changed to first data (structural data of DENV MTase in complex with CAPE, which is a significant achievement), then interpretation/discussion with modeling, and not the other way around.

   The discussion section requires more elaborate scientific justification than simply re-reporting the results.

   • Response:- We express our gratitude to the reviewers for their time and insightful comments, which have significantly contributed to in the improvement of our manuscript. We believe that the thoughtful critiques and suggestions have substantially improved the overall quality of our work. The changes made in the revised manuscript are highlighted in red. Below, we provide a point-by-point response to each comment, addressing the concerns raised by the reviewers.

   Comment 2:-

   It would be best to organize the ms as follows:

   • Crystal structure of DENV MTase in complex with CAPE
   • Building of a model of nsp1 by superimposition with NS5 MTase
   • Modeling compound binding
   • Inhibition assays using enzyme assays at least in the case of NS5 MTase. The direct enzyme assays are well described in the literature.

   • Response :- We appreciate the reviewer's suggestion regarding the manuscript organization. We understand the value of presenting the data in a logical flow. For this study, our initial investigations focused on the polyamine depletion ability of HC and CAPE, followed by antiviral activity assays. Based on the preliminary data from cell-based polyamine depletion assay and antiviral assays, the identified molecules were used for in silico investigations, followed by biochemical and biophysical validation. the crystal structure studies were performed to gain a deeper understanding of the inhibition mechanism. Therefore, we believe this flow, approach and the current structure have merit and is request to be considered.

   Comment 3:- Inhibition assays using enzyme assays at least in the case of NS5 MTase. The direct enzyme assays are well described in the literature.

   • If there is no inhibition, then discussion about possible reasons would be interesting and help the AV field. For example, CAPE could bind to other enzyme or sites, etc...

   Figure 5 is problematic.

   • When presenting an y IC50 data, care should be taken that the IC50 inflexion point is preceded and followed by at least two experimental points, which is not the case. The IC50 value of 7.082 and 5.156 µM are too imprecise (and there is no need to give digits after the value). Please add more low concentration experimental points.

   • Panel F and G: A reduction of 25 % at the highest inhibitor concentration is a strong indication that there is no effect.

   • Response:- *We sincerely thank the reviewers for their valuable comments and insights regarding the discrepancies observed in our data. We acknowledge the importance of providing plausible explanations for such variations and are committed to addressing these concerns in our revised analysis. * *Capillary electrophoresis (CE) offers a direct approach for detecting S-adenosylhomocysteine (SAH), the product of the methyltransferase reaction. However, this assay has a limitation in sensitivity, typically only detecting SAH concentrations exceeding ~300 µM. *

   *A previously validated CE-based assay for Chikungunya virus (CHIKV) nsp1 by Rajat et al. addresses this limitation and has been mentioned in the revised manuscript with the reference. Their work demonstrates that using specific concentrations of S-adenosylmethionine (SAM) at 0.3 mM and guanosine triphosphate (GTP) at 4 mM enables reliable detection of SAH in the reaction. The established ELISA assay utilizes an indirect detection method and exhibits higher sensitivity. Also, previous studies on alphaviral nsP1 inhibitors have also reported nsP1 enzyme activity inhibition by compounds at concentrations several folds higher than their respective active doses in cell culture-based studies (Delang et al., 2016; Mudgal et al., 2020; Kovacikova et al., 2020). *

   Hence, differing substrate concentrations may be attributed to discrepancies between the capillary electrophoresis (CE) and ELISA assays.* Numerous studies have utilized the CE-based assay or equivalent assays based on similar principles as qualitative tools for evaluating enzyme activity.*

   •         **In response to the reviewer's suggestion to test compounds at lower dilutions, we acknowledge that we are currently unable to perform an assay for lower dilutions as recommended due to time constraints and limited availability (screen shot below) of "MABE419 Sigma-Aldrich (Merk), Anti-m3G-cap, m7G-cap Antibody, clone H-20 antibody" used as the primary antibody (Kaur et al., 2018). Our attempts to procure this antibody from Sigma were unsuccessful.For India it shows limted availability and the vendor has given the estimated shipment time of more than 7 weeks. As per reviewers suggestion and the current limitations in the IC50 data, we have revised the graphical representation from a non-linear regression format (which estimates IC50) to a bar chart format. In the revised manuscript, Figures 6B and 6C graphical representation has been transitioned from a dose-response IC50 format to a bar chart for clarity. This bar chart effectively conveys the key finding of inhibitory activity observed for both HC and CAPE.*
     

   *We tried to reoptimize the Dengue virus MTase CE-based assay by reducing the GTP concentration from 4 mM to 0.3 mM. **This modification resulted in slight improvement and shows clear (~50%) decrease in enzyme activity at the highest concentration, as shown in Fig. 6 F and G. **The CE-based assay for HC and CAPE data clearly indicates inhibition above >50%. Our approach with this assay is centered around detecting inhibition rather than conducting quantitative analyses. *Following the reviewer's suggestion, we have revised the methyltransferase assay protocol, results, and figure legends. Additionally, the results have been appropriately discussed in the discussion section.

   Comment 4- Please describe more panel D in the legend.

   • Response :-We sincerely appreciate your suggestion and wish to express our gratitude. We have revised figure legend 6 D from. Line no. 791 "The CE based HC and CAPE Methyltransferase inhibition activity assay CHIKV nsP1" changed to line no. 884 to 886 "CE-based nsP1 MTase activity inhibition assay as described previously by Mudgal et al. 2020". HC and CAPE compounds were tested at a concentration of 200 µM and CAPE 1000 µM respectively.

   Minor Points/Comments/ Suggestions:

   Comment 1:-

   In the Introduction section, line 58: Are DENV infection numbers representative of worldwide distribution, please clarify. Also, in the case of CHIKV infection, the most affected countries are mentioned, why not follow the same pattern for DENV, please consider homogenizing the text.

   Response:-* Thank you for your suggestion; we have revised the text accordingly. Line no. 58 "It is estimated that ~100-400 million DENV infections occur annually" changed to line no. 58 to 61 "It is estimated that annually ~100-400 million DENV infections occur worldwide. The Philippines and Vietnam are among the most affected countries. Moreover, dengue is endemic in India, Indonesia, Myanmar, Sri Lanka, and Thailand (Bhatt et al., 2013; Lobo et al., 2011, National Center for Vector Borne Diseases Control Report 2022 (NCVBDC)."*

   __Comment 2:- __B. Before p. 4 (line 91), alphaviruses were not introduced. Please consider introducing them.

   Response :- Thank you for your feedback; brief introduction of alphaviruses have been added.

   1. • 4 (line 92) Alphaviruses belonging to the Togaviridae family include viruses such as Chikungunya, Eastern equine encephalitis, Venezuelan equine encephalitis, etc.*

   Comment 3:- C. Consider introducing Dengue serotypes to help readers understand the significance of DENV-2 and DENV-3.

   Additionally, ensure uniformity by referring to these serotypes as DENV-2, DENV-3 throughout. There are inconsistencies in the current text, such as 'DENV 3' in lines 39 and 152, and 'DENV3' in lines 249 and 250, among others.

   • Response:-Thank you for your valuable input. Dengue serotypes have been introduced, and we have meticulously reviewed and rectified all inconsistencies regarding their nomenclature. Line no. 120 to 123 "Flaviviruses are classified within the Flaviviridae family and encompass viruses like Dengue, Zika, Japanese encephalitis, etc. Dengue virus consists of four distinct antigenic types: DENV 1, DENV 2, DENV 3, and DENV 4. DENV 2 has been India's most prevalent serotype for the past 50 years, however serotypes 3 and 4 have also appeared in some recent epidemics (Kalita et al., 2021)."

   Comment 4:- D. P. 4, 5 lines 91-134: Consider rephrasing/reorganizing the methylation process: conventional and unconventional. The current introduction doesn't clearly indicate the difference between the cap-0 capping in alphaviruses and cap-1 in flaviviruses.

   • Response:-Line 100 changed from "Cellular enzyme capping mechanisms usually involve the methylation of guanosine triphosphate (GTP) after transferring it to the 5' end of the RNA. However, the molecular mechanism of viral mRNA capping in alphaviruses is distinct." To line no. 102 to 108 "Cellular enzymes use conventional capping mechanisms, usually where GTP is first transferred to RNA's 5' end, followed by its methylation. On the other hand, viral capping in the case of alphaviruses is unconventional, where GTP is first methylated, followed by the guanyltion of viral RNAs. Furthermore, Cap 0 alphaviruses feature monomethylation at the N7 position of the guanosine nucleotide, while Cap 1 in flaviviruses has additional methylation at both the N7 and 2'O positions."

   Comment 5:-

   Please consider citing the article instead of the referred link, wherever possible, for e.g., for ref. 22 PMID: 28218572 (a more recent reference for Flaviviridae taxonomy available than that mentioned in the current manuscript.)

   • Response :- We have addressed the reviewer's insightful suggestion regarding the citation and included the references accordingly.

   Comment 6:- F. Homogenize the writing of taxonomic names (viral families) in the text. For example, in line 126 change Flaviviridae to Flaviviridae, and line 476 (Discussion section), alphaviridae to Alphaviridae, flaviviridae to Flaviviridae and so on. For further clarification on addressing this, one can also refer to https://ictv.global/faq/names.

   • Response :-We sincerely appreciate the reviewer's input. We have incorporated the suggested changes as follows : In line 126, we changed "Flaviviridae" to "Flaviviridae".

   In line 476 (Discussion section), we corrected "alphaviridae" to "Togaviridae".

   We ensured consistency in the formatting of taxonomic names throughout the manuscript.

   Comment 7:-

   1. Please make sure to appropriately reference the corresponding supplementary information (text or figures) in the main text wherever necessary to avoid the impression of missing information. For instance, in none of the sub-sections of Materials and Methods (M&M), it is being indicated to refer to the suppl. experimental procedures for more details. Also consider not repeating the same information between the main experimental procedures text and the supplementary text.

   • Response :-The reviewer's feedback has been invaluable, and we've acted upon it accordingly. In response to the suggestion, we've made it clear in the manuscript to refer to the supplementary experimental procedures for detailed protocols where appropriate. Additionally, we've listed certain protocols exclusively in the supplementary material to enhance clarity and avoid repetition.

   Comment 8:-

   M&M sub-section. 2, line 163: Which specific culture media is being referred to here? Could you provide additional details? On line 164, it mentions that polyamines were diluted in water. Is this water sterile tissue culture-grade water as indicated in line 161?

   • Response :-We appreciate the reviewer's attention to detail. At the time of usage, further dilutions were prepared in 2% DMEM media. Additionally, individual polyamines (putrescine, spermidine, and spermine) stocks were diluted in sterile tissue culture-grade water from Alfa-Aeser, USA, and used as indicated. As such, we have revised the sentence to enhance clarity. *Line number 173 to 175 "At the time of usage, further dilutions were prepared in culture media. Similarly, individual polyamines (put, spm, and spd) (Alfa-Aeser, USA) stocks were diluted in water and used as designated." *changed to this "At the time of usage, further dilutions were prepared in 2 % DMEM media. Similarly, individual polyamines (put, spm, and spd) (Alfa-Aeser, USA) stocks were diluted in sterile tissue culture grade water and used as designated."

   Comment 9:-

   M&M, line 274: What is CE? Please expand the term before using the abbreviation.

   • Response :- Thank you for bringing that to our attention. CE mentioned in line 294 stands for Capillary electrophoresis__.__

   Comment 10:-

   line 306. Ref. 53: This is not a reference.

   • Response :-Thank you for bringing this to our attention. We understand that reference 53 does not correspond to a valid source. We acknowledge this and want to clarify that due to the unavailability of the proper reference, we included this reference. We have now changed the reference to the Crysalis Pro software.

   Comment 11:-

   Results. 1: Didn't understand the relevance of Fig. 1C, as this data is already included in Fig. 1B.

   • Response :-Thank you for bringing this to our attention. We apologize for any confusion caused by including Fig. 1C, especially since the data it presents overlaps with that of Fig. 1B. To ensure clarity, we have made modifications accordingly. Figures (A) and (C) depict the viability of Vero cells measured by an MTT assay after a total incubation of 134 hours. This protocol involved a 12-hour pre-treatment with either HC (A) or CAPE (C), followed by additional incubation steps as detailed in the legend. In contrast, figure (B) shows the cell viability of Vero cells treated with CAPE only, measured after a total incubation of 38 hours.
   • To avoid further confusion figure legend has been changed from "(A) and (C) depicts the percent cell viability of Vero cells treated with HC and CAPE for 12 hr pre-treatment and 24 hr post-treatment and incubated in maintenance media for 4 days, (B) shows the percent cell viability of Vero cells treated with CAPE for 12 hr pre-treatment and 24 hr post-treatment. " to "(A) and (C) depicts the percent cell viability of Vero cells treated with HC and CAPE for 12 hr pre-treatment followed by a 2-hour incubation with maintenance media, 24 hr post-treatment, and incubated in maintenance media for 4 days, (B) shows the percent cell viability of Vero cells treated with CAPE for 12 hr pre-treatment, followed by a 2-hour incubation with maintenance media and 24 hr post-treatment."

   Comment 12:-

   Fig. 1G and H are not referred to in the result text.

   • Response :-Thank you for pointing out the oversight regarding Fig. 1G and H not being referred to in the results text. We have added following statement Results p.1 Line no. 354 "Likewise, HC and CAPE treatment to Vero cells has shown a decrease in viral titer DENV-infected cells in a dose-dependent manner (Figure 1 G-H)."

   Comment 12:-

   Lines 342, 343: 'At the mentioned concentrations', where are these concentrations mentioned?

   • Response:-*Thank you for bringing this to our attention. We acknowledge this mistake regarding the mentioned concentrations at lines 342 and 343. RT-PCR was conducted for CHIKV using concentrations of 200 µM for HC, 25 µM for CAPE, and 1000 µM for DFMO. Similarly, for DENV, RT-PCR was performed with concentrations of 200 µM for HC, 2.5 µM for CAPE, and 1000 µM for DFMO. To avoid further confusion, Figure legends were revised and line no. 846 to 848 "(1F) RT-PCR for CHIKV with HC 200 µM, CAPE 25 µM, DFMO 1000 µM concentration (1I) RT-PCR for DENV with HC 200 uM, CAPE 2.5 uM and DFMO 1000 µM" *

   Comment 13:-

   qRT-PCR data is not very clear. Please consider elaborating on some details. Why were the statistics only performed between HC and DFMO and not with CAPE? How the fold reduction is being calculated? For example, the fold difference of 97 is not visibly evident.

   • Response:- *We regret that the clarity of the qRT-PCR data was not satisfactory. We acknowledge your feedback and understand the importance of elaborating on certain details. *The statistics were performed for all treatment groups, including HC, CAPE, and DFMO. However, the representation in the graph was adjusted by replacing the "top square bracket" with a "line" to avoid confusion. The y-axis of the graph depicts the log10 fold change in target gene expression relative to a designated virus control (VC). A value of ~ -2 on this axis corresponds to a significant downregulation, reflecting a 97-fold decrease in expression compared to the VC. A comparable graphical depiction is also evident in the work by Mudgal et al. (2022).

   Comment 14:-

   Line 375: 'SAM is lined by residues ... would be more appropriate than 'formed'

   • Response :-Done as suggested. We have revised the sentence in question and similar ones accordingly. "In CHIKV nsP1, SAM is formed by residues Gly65, Ser66, Ala67, Pro83, Arg85, Ser86, Asp89, Thr137, Asp138…" changed to line no. 393 "In CHIKV nsP1, SAM binding site is lined by,….."

   Comment 15:-

   Fig. 1J. For TLC results, consider using the term panel (left, center, right) to navigate within this figure. The representation of this result is not uniform, as the time course is shown for HC while it is not shown for DFMO and CAPE. The treatment time is not indicated for DFMO and CAPE. For better representation and significant differences, one can consider quantifying these TLC results.

   • Response:- *Thank you for bringing these points to our attention. Done as suggested. We have simplified the presentation of the TLC results to enhance clarity and revised the methodology, results, figure, and legend accordingly. Also, we have quantified the TLC results. * -Polyamine determination by Thin-layer chromatography (TLC)

   -Vero cells were treated with HC, CAPE and DFMO, as mentioned in the antiviral assay protocol. Similarly, HC-treated cells were collected after 12, 24, and 36 hr of treatment." Revised to " *Vero cells were treated with CAPE (25 µM), HC (200 µM), and DFMO (1000 µM) for 36 hr …… *Further, TLC images were quantified utilizing ImageJ software." *Figure legend 1:- (J) depicts the effect of polyamines level after treating with HC (200 µM) and CAPE (25 µM). Polyamine level of Vero treated cells at 12, 24, and 36 hr for HC and pre (12 hr) and post-treatment (24 h) for CAPE and DEMO, using untreated cells as a cell control (CC) for both of the conditions. 0.1 μM putrescine (put), spermine (spm), and spermidine (spd) as a positive control marker. changed to *

   *"(J) the chromatographic analysis of polyamine levels in Vero cells after 36 hr treatment with (from left) CAPE (25 µM), HC (200 µM), DFMO(1000 µM), and cell control (CC), 0.1 μM putrescine (Put), spermine (Spm), and spermidine (Spd) as a positive control marker. *"

   Results: Line no. 351 "Polyamine levels in cells treated with CAPE were significantly lower as compared to DFMO treatment (Figure 1J). Meanwhile, HC showed a reduction in polyamine levels with the initial 12 hr treatment; later, polyamine levels elevated gradually with time."

   Revised to line no. 371 to 373"After treatment with CAPE, HC, and DFMO to Vero cells, overall residual polyamine levels are 28.33%, 29.67 %, and 46 %, respectively, compared to cell control."

   Comment 16:-

   Fig. 1, figure legend, lines 750-751: instead of 'Panels D-G depicts the inhibitory effect of CHIKV and DENV infected cells on different concentrations of HC and CAPE' should be

   'Panels D-G* depicts the inhibitory effect of different concentrations of HC and CAPE on CHIKV and DENV infected cells'*

   • Response:-Thank you for the suggestion. We have updated the figure legend to ensure clarity based on your recommendation. (D,E,G,H) depicts the inhibitory effect of different concentrations of HC and CAPE on CHIKV and DENV infected cells'.

   Comment 17:-

   Line 755: DFMO is wrongly written as 'DEMO'

   • Response:- Thank you for bringing that to our attention. We have corrected the typo, changing Line 845 'DEMO' to 'DFMO' as appropriate.

   Comment 18:-

   Fig.2. IFA. Authors must consider on elaborating the IFA data. One can also consider quantifying these data for better comparison with other assays.

   • Response:- We thank reviewer for your input. As per the suggestion we have elaborated the results on IFA. The qualitative application of IFA was chosen because of the absence of dedicated paid software/hardware for image quantification on the Thermofisher EVOS platform, thereby impeding our quantification efforts.

   Comment 19:-

   Result 1 (Suppl. Fig. 1). Line 359: 'After infection': please indicate the time here.

   • Response:- Thank you for the feedback. Line no. 377:We have updated the line to specify the time as “ after 2 h of virus infection," and we have also revised this in the methodology section for clarity.

   Comment 20:-

   Suppl. Fig.1: How was the concentration of these polyamines chosen to be 1µM?

   What will be the effect on increasing concentrations?

   Why were all these three polyamines added together?

   What is the effect of addition of individual polyamine in the rescue of viral titer?

   Will this effect vary if cells are pre-treated with these polyamines and compounds in question are added post viral infection or if both are added simultaneously?

   Response:- *We thank the reviewer, for raising these insightful questions. We performed an Exogenous polyamine addition assay as per Mounce et al. 2016 to maintain consistency with established practices and the research focus. The concentration of 1 µM biogenic polyamines (Putrescine, Spermidine, and Spermine) was chosen based on the findings of Mounce et al. (2016), where viral titers were restored to levels comparable to non-treated conditions at this concentration (Mounce, Cesaro, et al., 2016; Mounce, Poirier, et al., 2016). Furthermore, increasing the concentration of these polyamines did not yield significant additional effects on viral titer rescue, as observed in their study. *

   The potential influence of pre-treating cells with the biogenic polyamines (putrescine, spermidine, spermine) prior to viral infection, compared to simultaneous addition with the compound in question, is an interesting point. While Mounce et al. (2016) suggest this order may not significantly impact the rescue effect (Mounce, Poirier, et al., 2016)*. Further investigations are warranted to address this question definitively within the context of our specific experimental design. *

   Comment 20:-

   It is understandable that from the data of Suppl Fig.1, authors became keen on exploring the 'other' antiviral target, but then conclusions from Fig. 1J and Suppl. Fig. 1 are contradictory. As from Fig. 1J, it is being conveyed that the tested compounds depletes polyamines level better than the control. On the other hand, in suppl fig.1, when these polyamines are supplemented, the viral titer is not rescued. Of course this might be related to the time of addition of polyamines and compounds. Authors should consider discussing these results in details.

   • Response:-Thank you for your insightful suggestion. We have addressed these results in detail in the discussion section of the manuscript. We conducted an Exogenous Polyamine Addition Assay following the methodology outlined by Mounce et al. (2016) to adhere to established procedures and align with our research objectives. Treatment with DFMO in the presence of exogenous polyamines, as well as treatment with DFMO followed by polyamine addition, led to the rescue of virus titers, as indicated by Mounce et al. (2016). Therefore, according to the data, the timing of exogenous polyamine addition may not be a significant factor. In our manuscript, the timing of polyamine and compound addition was consistent across all treatments (HC, CAPE, and DFMO).

   Comment 21:-

   Result 2. Suppl fig. 2. MSA. Provide complete information in the figure legend: indicate virus names to the corresponding Accession numbers and GenBank ID.

   • Response:-Thank you for bringing this to our attention. We have updated the figure legend in Supplementary Figure 2 to include complete information, indicating the virus names corresponding to the Accession numbers and GenBank IDs.

   Comment 22:-

   Line 392: '2 dimensions' ?

   • Response:-Thank you for bringing this to our attention. As suggested, we have made the change, replacing "2 dimensions" with "2D" for clarity.

   Comment 23:-

   Result 3. Authors didn't comment/discuss on the significance of these tests with GTP, SAM and difference in the Kd values: for CHIKV and DENV and other details

   • Response:- We appreciate the reviewer's feedback. We have expanded upon these results in more detail in the discussion section. Discussion p.4 line no. 512 "Biophysical interactions by TFS indicate distinct red shift for nsP1 and NS5 MTase, with each compound displaying specific affinities toward the target proteins." revised to line no. 551 to 557 "The binding affinities of SAM and GTP with CHIKV nsP1 and DENV NS5 MTase were investigated and used as a reference to compare with HC and CAPE. HC has a high binding affinity for both enzymes, as evidenced by the Kd values. Conversely, CAPE demonstrates a more selective binding profile, exhibiting a significantly stronger affinity towards nsP1 than NS5 MTase. Significantly, both HC and CAPE have demonstrated a dose-dependent red shift, indicating structural changes upon interaction (Figure 5 and Supplimentary figure 5)."

   Comment 25 Result 4. Fig. 6A and 6E: The text does not report this result (SDS-PAGE). Fig. 6

   • Response We appreciate the reviewer for bringing this to our attention. As per suggestion, we have incorporated the SDS-PAGE results in Fig. 6 in the text.line no. 467 to 468 "Single band at ~ 56 and ~ 32 kDa was observed in 12% SDS-PAGE for purified nsP1 and NS5 MTase, respectively ( Figure 6A and 6E)."

   Comment 24:-

   Did authors also perform the enzymatic assays (inhibition assays) with DFMO?

   • Response:- Thank you for your intriguing question. We appreciate the reviewer's interest. We opted not to conduct enzymatic assays (inhibition assays) with DFMO, as it is a known analog of ornithine, a well-established inhibitor of the polyamine pathway (ornithine decarboxylase inhibitor). This decision was made as it was deemed outside the scope of our study.

   Comment 25:-

   Typographic errors: ml to mL, µl to µL, E. coli to E. coli (line 956), in multiple figures: chose titre or titer

   • Response:- We thank the reviewer for their meticulous attention to detail. As per your observation, we have carefully reviewed the manuscript and made the necessary corrections, including changing "ml" to "mL", "µl" to "µL", and "E. coli" to " coli" (line no.. 1042). Additionally, we have standardized the usage of "titre" to "titer" across multiple figures. __References: __

   Aljabr, W., Touzelet, O., Pollakis, G., Wu, W., Munday, D. C., Hughes, M., Hertz-Fowler, C., Kenny, J., Fearns, R., Barr, J. N., Matthews, D. A., & Hiscox, J. A. (2016). Investigating the Influence of Ribavirin on Human Respiratory Syncytial Virus RNA Synthesis by Using a High-Resolution Transcriptome Sequencing Approach. Journal of Virology, 90(10), 4876. https://doi.org/10.1128/JVI.02349-15

   Arisan, E. D., Obakan, P., Coker, A., & Palavan-Unsal, N. (2012). Inhibition of ornithine decarboxylase alters the roscovitine-induced mitochondrial-mediated apoptosis in MCF-7 breast cancer cells. Molecular Medicine Reports, 5(5), 1323–1329. https://doi.org/10.3892/MMR.2012.786

   Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF. The global distribution and burden of dengue. Nature. 2013 Apr 25;496(7446):504-7.

   Bouteille, B., & Dumas, M. (2003). Human African Trypanosomiasis. Encyclopedia of the Neurological Sciences, 587–594. https://doi.org/10.1016/B0-12-226870-9/00431-7

   DENGUE/DHF SITUATION IN INDIA :: National Center for Vector Borne Diseases Control (NCVBDC). [cited 2022 Jan 15].

   https://nvbdcp.gov.in/index4.php?lang=1&level=0&linkid=431&lid=3715

   Kalita, J. M., Aggarwal, A., Yedale, K., Gadepalli, R., & Nag, V. L. (2021). A 5-year study of dengue seropositivity among suspected cases attending a teaching hospital of North-Western region of India. Journal of Medical Virology, 93(6), 3338–3343. https://doi.org/10.1002/JMV.26592

   Lani, R., Hassandarvish, P., Shu, M. H., Phoon, W. H., Chu, J. J. H., Higgs, S., Vanlandingham, D., Abu Bakar, S., Zandi, K., R, L., P, H., MH, S., WH, P., JJ, C., S, H., D, V., S, A. B., & K, Z. (2016). Antiviral activity of selected flavonoids against Chikungunya virus. Antiviral Research, 133, 50–61. https://doi.org/10.1016/J.ANTIVIRAL.2016.07.009

   Lobo DA, Velayudhan R, Chatterjee P, Kohli H, Hotez PJ. The neglected tropical diseases of India and South Asia: review of their prevalence, distribution, and control or elimination. PLoS neglected tropical diseases. 2011 Oct 25;5(10):e1222.

   Logiudice, N., Le, L., Abuan, I., Leizorek, Y., & Roberts, S. C. (2018). medical sciences Alpha-Difluoromethylornithine, an Irreversible Inhibitor of Polyamine Biosynthesis, as a Therapeutic Strategy against Hyperproliferative and Infectious Diseases. https://doi.org/10.3390/medsci6010012

   Mounce, B. C., Cesaro, T., Moratorio, G., Hooikaas, P. J., Yakovleva, A., Werneke, S. W., Smith, E. C., Poirier, E. Z., Simon-Loriere, E., Prot, M., Tamietti, C., Vitry, S., Volle, R., Khou, C., Frenkiel, M.-P., Sakuntabhai, A., Delpeyroux, F., Pardigon, N., Flamand, M., … Vignuzzi, M. (2016). Inhibition of Polyamine Biosynthesis Is a Broad-Spectrum Strategy against RNA Viruses. Journal of Virology, 90(21), 9683–9692. https://doi.org/10.1128/JVI.01347-16

   Mounce, B. C., Poirier, E. Z., Passoni, G., Simon-Loriere, E., Cesaro, T., Prot, M., Stapleford, K. A., Moratorio, G., Sakuntabhai, A., Levraud, J. P., & Vignuzzi, M. (2016). Interferon-Induced Spermidine-Spermine Acetyltransferase and Polyamine Depletion Restrict Zika and Chikungunya Viruses. Cell Host & Microbe, 20(2), 167–177. https://doi.org/10.1016/J.CHOM.2016.06.011

   Mudgal, R., Bharadwaj, C., Dubey, A., Choudhary, S., Nagarajan, P., Aggarwal, M., Ratra, Y., Basak, S., & Tomar, S. (2022). Selective Estrogen Receptor Modulators Limit Alphavirus Infection by Targeting the Viral Capping Enzyme nsP1. Antimicrobial Agents and Chemotherapy, 66(3). https://doi.org/10.1128/AAC.01943-21/ASSET/CFFD4322-C11C-41A1-9D51-7D0C3242FA63/ASSETS/IMAGES/LARGE/AAC.01943-21-F006.JPG

   Nazir, A., Nazir, A., & Kandel, K. (2024). Advancing neuroblastoma care: Future horizons after approval of eflornithine by FDA. International Journal of Surgery (London, England). https://doi.org/10.1097/JS9.0000000000001182

   Pegg, A. E., & Casero, R. A. (n.d.). Current Status of the Polyamine Research Field. https://doi.org/10.1007/978-1-61779-034-8_1

   Rao, J. N., Rathor, N., Zhuang, R., Zou, T., Liu, L., Xiao, L., Turner, D. J., & Wang, J. Y. (2012). Polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca2+ signaling by differentially modulating STIM1 and STIM2. American Journal of Physiology - Cell Physiology, 303(3), C308. https://doi.org/10.1152/AJPCELL.00120.2012

   Shen, H., Yamashita, A., Nakakoshi, M., Yokoe, H., & Sudo, M. (2013). Inhibitory Effects of Caffeic Acid Phenethyl Ester Derivatives on Replication of Hepatitis C Virus. PLoS ONE, 8(12), 82299. https://doi.org/10.1371/journal.pone.0082299

   Tate, P. M., Mastrodomenico, V., & Mounce, B. C. (2019). Ribavirin Induces Polyamine Depletion via Nucleotide Depletion to Limit Virus Replication. Cell Reports, 28(10), 2620-2633.e4. https://doi.org/10.1016/J.CELREP.2019.07.099

   Terui, Y., Yoshida, T., Sakamoto, A., Saito, D., Oshima, T., Kawazoe, M., Yokoyama, S., Igarashi, K., & Kashiwagi, K. (2018). Polyamines protect nucleic acids against depurination. The International Journal of Biochemistry & Cell Biology, 99, 147–153. https://doi.org/10.1016/J.BIOCEL.2018.04.008

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   Referee #3

   Evidence, reproducibility and clarity

   The manuscript submitted by Bhutkar M. et al. details the antiviral properties of two compounds, herbacetin (HC) and caffeic acid phenethyl ester (CAPE), against Chikungunya virus (CHIKV) and Dengue virus (DENV) through cellular, bioinformatics, biochemical, biophysical, and structural studies. The authors propose a dual antiviral mechanism of action exhibited by these compounds, beginning with an evaluation of their cytotoxicity. Subsequent assessments of their antiviral efficacy against CHIKV and DENV are addressed using plaque reduction assay and other orthogonal assays such as qRT-PCR, and Immunofluorescence assay (IFA). …

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   Referee #3

   Evidence, reproducibility and clarity

   The manuscript submitted by Bhutkar M. et al. details the antiviral properties of two compounds, herbacetin (HC) and caffeic acid phenethyl ester (CAPE), against Chikungunya virus (CHIKV) and Dengue virus (DENV) through cellular, bioinformatics, biochemical, biophysical, and structural studies. The authors propose a dual antiviral mechanism of action exhibited by these compounds, beginning with an evaluation of their cytotoxicity. Subsequent assessments of their antiviral efficacy against CHIKV and DENV are addressed using plaque reduction assay and other orthogonal assays such as qRT-PCR, and Immunofluorescence assay (IFA). Further, authors performed thin layer chromatography (TLC) to monitor polyamine levels in the cells treated with these compounds and concluded that these compounds leads to polyamine depletion which is also supported by previous studies. These experiments included DFMO as a control which is well established for its role in this regulation. Beyond their impact on cellular polyamine levels, the authors propose a role for these compounds in the inhibition of MTase domains in CHIKV and DENV, supported by the crystal structure of the DENV-3 NS5 MTase domain in complex with CAPE.

   Major points

   While the manuscript presents promising findings regarding the dual antiviral effects of the tested compounds, the authors fall short of demonstrating direct inhibition of MTase activity as a meaningful and complementary effect to polyamine depletion. Being only indirect, the enzyme inhibition data is not convincing, and the measured indirect inhibition are not precise enough in the case of CHIK nsp1, and too weak in the case of DENV NS5 (detailed below).

   Conceptually, the organization of the results should be changed to first data (structural data of DENV MTase in complex with CAPE, which is a significant achievement), then interpretation/discussion with modeling, and not the other way around.

   The discussion section requires more elaborate scientific justification rather than simply re-reporting the results.

   Specific major remarks:

   It would be best to organize the ms as follows:

   • Crystal structure of DENV MTase in complex with CAPE
   • Building of a model of nsp1 by superimposition with NS5 MTase
   • Modeling compound binding
   • Inhibition assays using enzyme assays at least in the case of NS5 MTase. The direct enzyme assays are well described in the literature.
   • If there is no inhibition, then discussion about possible reasons would be interesting and help the AV field. For example, CAPE could bind to other enzyme or sites, etc...

   Figure 5 is problematic.

   • When presenting an y IC50 data, care should be taken that the IC50 inflexion point is preceded and followed by at least two experimental points, which is not the case. The IC50 value of 7.082 and 5.156 µM are too imprecise (and there is no need to give digits after the value). Please add more low concentration experimental points.
   • Please describe more panel D in the legend.
   • Panel F and G: A reduction of 25 % at the highest concentration of inhibitor is a strong indication that there is no effect.

   Minor Points/Comments/ Suggestions:

   A. In the Introduction section, line 58: Are DENV infection numbers representative of worldwide distribution, please clarify. Also, in the case of CHIKV infection, the most affected countries are mentioned, why not follow the same pattern for DENV, please consider homogenizing the text.

   B. Before p. 4 (line 91), alphaviruses were not introduced. Please consider introducing them.

   C. Consider introducing Dengue serotypes to help readers understand the significance of DENV-2 and DENV-3. Additionally, ensure uniformity by referring to these serotypes as DENV-2, DENV-3 throughout. There are inconsistencies in the current text, such as 'DENV 3' in lines 39 and 152, and 'DENV3' in lines 249 and 250, among others.

   D. P. 4, 5 lines 91-134: Consider rephrasing/reorganizing the methylation process: conventional and unconventional. The current introduction doesn't clearly indicates the difference between the cap-0 capping in alphaviruses and cap-1 in flaviviruses.

   E. Please consider citing the article instead of the referred link, wherever possible, for e.g., for ref. 22 PMID: 28218572 (a more recent reference for Flaviviridae taxonomy available than that mentioned in the current manuscript.)

   F. Homogenize the writing of taxonomic names (viral families) in the text. For example, in line 126 change Flaviviridae to Flaviviridae, and line 476 (Discussion section), alphaviridae to Alphaviridae, flaviviridae to Flaviviridae and so on. For further clarification on addressing this, one can also refer to https://ictv.global/faq/names.

   G. Please make sure to appropriately reference the corresponding supplementary information (text or figures) in the main text wherever necessary to avoid the impression of missing information. For instance, in none of the sub-sections of Materials and Methods (M&M), it is being indicated to refer to the suppl. experimental procedures for more details. Also consider not repeating the same information between the main experimental procedures text and the supplementary text.

   H. M&M sub-section. 2, line 163: Which specific culture media is being referred to here? Could you provide additional details? On line 164, it mentions that polyamines were diluted in water. Is this water sterile tissue culture-grade water as indicated in line 161?

   I. M&M, line 274: What is CE? Please expand the term before using the abbreviation.

   J. line 306. Ref. 53: This is not a reference.

   K. Results. 1: Didn't understand the relevance of Fig. 1C, as this data is already included in Fig. 1B. Fig. 1G and H are not referred to in the result text. Lines 342, 343: 'At the mentioned concentrations', where are these concentrations mentioned? qRT-PCR data is not very clear. Please consider elaborating on some details. Why the statistics were only performed between HC and DFMO and not with CAPE? How the fold reduction is being calculated? For example, the fold difference of 97 is not visibly evident. Line 375: 'SAM is lined by residues ... would be more appropriate than 'formed' Fig. 1J. For TLC results, consider using the term panel (left, center, right) to navigate within this figure. The representation of this result is not uniform, as the time course is shown for HC while it is not shown for DFMO and CAPE. The treatment time is not indicated for DFMO and CAPE. For better representation and significant differences, one can consider quantifying these TLC results. Fig. 1, figure legend, lines 750-751: instead of 'Panels D-G depicts the inhibitory effect of CHIKV and DENV infected cells on different concentrations of HC and CAPE' should be 'Panels D-G depicts the inhibitory effect of different concentrations of HC and CAPE on CHIKV and DENV infected cells'. Line 755: DFMO is wrongly written as 'DEMO' Fig.2. IFA. Authors must consider on elaborating the IFA data. One can also consider quantifying these data for better comparison with other assays.

   Result 1 (Suppl. Fig. 1). Line 359: 'After infection': please indicate the time here. Suppl. Fig.1: How was the concentration of these polyamines chosen to be 1µM? What will be the effect on increasing concentrations? Why were all these three polyamines added together? What is the effect of addition of individual polyamine in the rescue of viral titer? Will this effect vary if cells are pre-treated with these polyamines and compounds in question are added post viral infection or if both are added at the same time? It is understandable that from the data of Suppl Fig.1, authors became keen on exploring the 'other' antiviral target, but then conclusions from Fig. 1J and Suppl. Fig. 1 are contradictory. As from Fig. 1J, it is being conveyed that the tested compounds depletes polyamines level better than the control. On the other hand, in suppl fig.1, when these polyamines are supplemented, the viral titer is not rescued. Of course this might be related to the time of addition of polyamines and compounds. Authors should consider discussing these results in details.

   Result 2. Suppl fig. 2. MSA. Provide complete information in the figure legend: indicate virus names to the corresponding Accession numbers and GenBank ID. Line 392: '2 dimensions' ?

   Result 3. Authors didn't comment/discuss on the significance of these tests with GTP, SAM and difference in the Kd values: for CHIKV and DENV and other details

   Result 4. Fig. 6A and 6E: This result (SDS-PAGE) is not reported in the text. Fig. 6

   Did authors also perform the enzymatic assays (inhibition assays) with DFMO?

   Typographic errors: ml to mL, µl to µL, E. coli to E. coli (line 956), in multiple figures: chose titre or titer

   Significance

   This a body of work that is very interesting and has good potential, however it lacks the correct demonstration of the additive effect of MTase inhibition to polyamine depletion.

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   Referee #2

   Evidence, reproducibility and clarity

   Authors describe anti-CHIKV and anti-DENV activities of herbacetin and caffeic acid phenyl ester (CAPE). The antiviral effect is not reversed buy exogenous polyamines suggesting multiple mechanisms of action. NS5-Met complex with caffeic acid phenyl ester was obtained and its structure resolved at high resolution. The resolved structure reveals two binding sites for antiviral compound overlapping with that of GTP and possibly with site involved in binding of RNA

   Other than analysis of crystal structure of NS5/CAPE complex the provided data is of low quality and is not analyzed properly. There is no evidence that data is …

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   Referee #2

   Evidence, reproducibility and clarity

   Authors describe anti-CHIKV and anti-DENV activities of herbacetin and caffeic acid phenyl ester (CAPE). The antiviral effect is not reversed buy exogenous polyamines suggesting multiple mechanisms of action. NS5-Met complex with caffeic acid phenyl ester was obtained and its structure resolved at high resolution. The resolved structure reveals two binding sites for antiviral compound overlapping with that of GTP and possibly with site involved in binding of RNA

   Other than analysis of crystal structure of NS5/CAPE complex the provided data is of low quality and is not analyzed properly. There is no evidence that data is reproducible. Authors have calculated significance from "experimental repeats" which, based on the description of experiments, are not independent experiments but technical replicates. Some key technical details are missing and some experiments are not described at all. The writing can be vastly improved and figures be made a lot more easier to understand.

   There are several points that need to be addressed, so here I provide some examples:

   1. Bad writing lines 64-65 . Viral genomes lack protein synthesis machinery. Basically correct but no genome has protein synthesis machinery line 137 flavonoids play role in reducing of the levels of nsP1 in CHIKV - what this can possibly mean? Are shown to reduce level of nsP1 in CHIKV infected cells? line 250-251 - RNA was isolated from the infected cells' supernatant, used for cloning, an inserted between the NheI and XhoI restriction sites... It should be impossible as one cannot insert RNA into bacterial plasmid DNA
   2. Missing parts. Examples
      • the source of nsP1 of CHIKV is not indicated, True, there are references to previous studies but this is extremely important point and it should have been clearly stated that it was obtained from E. coli. The issue is that authors made some predictions and modelling based on structure of nsP1 from eukaryotic expression system. It is not known does the enzyme purified from bacteria have similar structure (actually, in cited Nature paper - doi: 10.1038/s41586-020-3036-8 - attempts to purify nsP1 from bacteria were made. The protein was monomeric and had no activity)
      • description of experiment shown on Figure 4 is missing
   3. Figure lacks quality (and figure legends are unclear) Examples:
      • it is impossible to understand what exactly is shown on Figure 1J
      • important information is missing, for example it is not clear what were concentrations of antiviral compounds for panels 1F and 1I
   4. wrong data
      • line 478 it is stated that there is no vaccine for DENV or CHIKV. It is correct, DENV vaccine has been in use for several years and CHIKV vaccine was approved at 2023
      • line 476 refers to family alphaviridae. This does not exist, family is Togaviridae
   5. unjustified conclusions. Example
      • authors have analyzed sequences of nsP1 of alphaviruses and made conclusions regarding conservation of active site. It is probably correct but the analyzed viruses do not represent all diversity of alphaviruses, insect specific members and aquatic alphaviruses should also be analyzed (same problem with analysis performed for flaviviruses)
   6. Insufficient analysis of data. Some cases there is significant discrepancy between results of different assays. For example, CAPE inhibits DENV at 2.5 microM (Fig 1H) but in test tube assay only small inhibition was observed even at 1000 microM. Authors should provide plausible explanation for this and similar discrepancies (CE and ELISA based assays shown on figure 6 also resulted in drastically different inhibitions). It is expected assays would produce different results but there should also be explanation for this. If this is not provided on can assume that it is due to experimental errors.
   7. Discussion is essentially missing, it is just list of statements mostly repeating what was said in other sections

   The reviewer is sorry for not being able to provide more specific and useful comments and suggestions. To my opinion, manuscript should have been better prepared before submitting for review. Multiple mistakes, discrepancies and lack of clarity makes it difficult (for me nearly impossible) to focus on scientific value of study and provide constructive comments

   Significance

   It is difficult to assess the significance of the studys findings as the data presented and writing lacks sufficient quality and depth. While some experiments that can be understood (crystal structure, some antiviral assays) show potentially interesting scientific findings, the manuscript needs a major overhaul before it can be considered relevant for the scientific community.

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   Referee #1

   Evidence, reproducibility and clarity

   In this manuscript, the authors use structural and functional approaches to investigate the potential anti-DENV and anti-CHIKV activity of HC and CAPE, two naturally occurring compounds. They find that these compounds reduce cellular polyamine levels and specifically inhibit the viral methyltransferase (MTase) activity. Hence, the authors propose that HC and CAPE have anti-viral potential against DENV and CHIKV, which have been implicated in severe disease in humans.

   Overall, this is a straightforward investigation and is quite suitable for publication as a "first report" on the anti-MTase activity of these compounds. The data …

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   Referee #1

   Evidence, reproducibility and clarity

   In this manuscript, the authors use structural and functional approaches to investigate the potential anti-DENV and anti-CHIKV activity of HC and CAPE, two naturally occurring compounds. They find that these compounds reduce cellular polyamine levels and specifically inhibit the viral methyltransferase (MTase) activity. Hence, the authors propose that HC and CAPE have anti-viral potential against DENV and CHIKV, which have been implicated in severe disease in humans.

   Overall, this is a straightforward investigation and is quite suitable for publication as a "first report" on the anti-MTase activity of these compounds. The data support the conclusions. This will be of interest to researchers in the anti-virals field. A strength of this investigation is the multi-faceted approach to get to the target of these compounds, i.e., the viral MTase enzymes. This is commendable.

   My main concern is that the depletion of polyamines is likely to have broad implications for host cell metabolism. Polyamines are critical for genome folding and stability. Hence, polyamine depletion will likely compromise cellular metabolic homeostasis. My suggestion is to perform a literature survey on this topic, identify appropriate assays of cellular homeostasis, and add at least one such assay in the relevant HC and CAPE concentration range to address my question.

   I also suggest adding the potential negative effects of polyamine depletion on host cell metabolism in the discussion section.

   Significance

   Strengths- multi-faceted approach

   Target audience- researchers interested in anti-virals

   Read the original source
5. 

   Version published to 10.1101/2022.05.31.494145v4 on bioRxiv

   Apr 18, 2023
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   Version published to 10.1101/2022.05.31.494145v3 on bioRxiv

   Feb 27, 2023
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   Version published to 10.1101/2022.05.31.494145v2 on bioRxiv

   Jan 11, 2023
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   Version published to 10.1101/2022.05.31.494145v1 on bioRxiv

   May 31, 2022