Flexible changes to the Heliothis virescens ascovirus 3h (HvAV-3h) virion components affect pathogenicity against different host larvae species

ABSTRACT The pathogenicity of a virus to a specific host species is an inerratic and describable ability of a virus to cause infection but is generally shaped by a variety of abiotic and biotic factors. In this investigation, the variations in pathogenicity of Heliothis virescens ascovirus 3h (HvAV-3h) to five noctuid pests were assessed based on mass spectrometry analysis on the virion compositions. Twenty-nine common HvAV-3h proteins were shared across all hosts, and different flexible proteins were identified in the virions of each specific host. Different host proteins were identified as HvAV-3h virion-associated proteins, including different detoxification enzyme proteins. Furthermore, a relatively fixed relationship between viral replication and changes in host detoxification enzyme activity caused by deficiencies in various viral structural proteins was found in the host larvae using a correlation matrix analysis: the host larval carboxylesterase and cytochrome P450 monooxygenases generally had highly similar responses to the viruses blocked by different structural proteins’ antisera and their effects on viral DNA replication. Different interaction patterns for the virion structural proteins were found in different host larvae-produced virions, and the interactions between Spodoptera litura glutathione S-transferases and viral structural proteins were confirmed. The different host responses after viral infection could be the reason for the changes in viral pathogenicity, while the virus responses gradually adapted to the different hosts and there were flexible changes in the virion structures. IMPORTANCE Different pathogenic processes of a virus in different hosts are related to the host individual differences, which makes the virus undergoes different survival pressures. Here, we found that the virions of an insect virus, Heliothis virescens ascovirus 3h (HvAV-3h), had different protein composition when they were purified from different host larval species. These “adaptive changes” of the virions were analyzed in detail in this study, which mainly included the differences of the protein composition of virions and the differences in affinity between virions and different host proteins. The results of this study revealed the flexible changes of viruses to help themselves adapt to different hosts. Also, these interesting findings can provide new insights to improve our understanding of virus adaptability and virulence differentiation caused by the adaptation process.

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Sincerely, Clinton Jones
Editor, Microbiology Spectrum Reviewer comments: Reviewer #1 (Comments for the Author): Dear Authors, The Authors summitted a detailed research on the " Flexible changes to the Heliothis virescens ascovirus 3h (HvAV-3h) virion components affects pathogenicity against different host larvae species."I appreciate the valuable contributions of the study in the field by exploring the virus-host interaction mysteries between different hosts in one of the lesser-known virus group Ascoviridae.
After a thorough review of the work, I have two key suggestions to improve the manuscript.: the co-IP Assay section and the presentation of proteomics data in the supplementary tables.

Co-IP Assay Elaboration and Controls:
Please provide a more detailed description of the co-IP Assay methodology.Elaborate on the experimental setup, including the specific antibodies used for immunoprecipitation and the detection of interacting proteins.Additionally, clarify the controls used in the assay to ensure the reliability of your results, especially concerning false positive detection.Describing negative controls and how they help identify non-specific interactions will strengthen the validity of your findings.Proteomics Data and Supplementary Tables: In the supplementary tables presenting the proteomics data, ensure that the information is comprehensive and well-organized.Provide additional relevant details on the identified proteins, such as their functional annotations and potential roles in immune responses.If any of the detected proteins are not unique to your study but are closely related to previously reported data in related virus groups, make a clear comparison to highlight any similarities or differences.This will help readers contextualize your findings within the existing knowledge on these proteins.Overall, these revisions will enhance the clarity and robustness of your study, enabling readers to understand better your experimental approach and the significance of your results.Addressing these suggestions will significantly improve the manuscript's quality and its impact on the scientific community.
Reviewer #2 (Comments for the Author): Ascoviruses are a group of large, double-stranded DNA viruses that mainly infect insects of the family Noctuidae.In this manuscript submitted by Yu et al. entitled "Flexible changes to the Heliothis virescens ascovirus 3h (HvAV-3h) virion components affects pathogenicity against different host larvae species", the authors found that the virus (HvAV-3h) infection had significant effects on the life span of five different lepidopteran insects, although the morphology of the virions purified from the infected insects was similar.Mass spectrometry analysis revealed that the virions produced by different host insects share 29 common viral proteins but also contain some distinct viral components and host proteins, particularly of some detoxification enzymes (P450, GST).Western blotting and immuno-electronic microscopy confirmed the presence of some viral proteins (3H-13, 27, 55, 56, 57, 58, 152) on the purified virions.Blocking/neutralizing the virions with the antibodies of those viral proteins reduced the viral DNA replication and virus infectivity, and to a certain extent, affected the host detoxification enzymes activities.Overall, the manuscript provided a large body of data, however, the results were not well organized and described or interpreted.It is expectable that the components of virions purified from different host insects have subtle difference, but the functional links of those components, particularly of those detoxification enzymes, with the virus infectivity are not clear.
Major points: 1. Lines 140-147, the morphology of the purified virions has no obvious difference.This section shouldn't stand alone.It could be combined with the section "Different host larvae produced HvAV-3h virions had similar......" in lines 148-149.Also, Lines 220-233, they compared the host proteins associated with the HvAV-3h virion, this section should be combined with those in lines 148-149.Additionally, the description and interpretation of the components of virions purified from different hosts are not clear.2. Lines 250-251, they used the inhibitors of the detoxification enzymes to evaluated the relationship of the virus infectivity and the activity of enzymes.The inhibitory effect of each inhibitor on the specific enzyme of insects are not clear.3. Lines 262-296.Since the antibody block/neutralization substantially reduced the viral DNA replication and infectivity, it is not conceivable to assess the relationship of the viral structural proteins and host detoxification enzyme activity by using the antibody blocked/neutralized virus to infect the insect.

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Response letter for Spectrum02488-23
Dear editor Clinton Jones, Thank you for your decision letter concerning our manuscript (ID Spectrum02488-23) entitled "Flexible changes to the Heliothis virescens ascovirus 3h (HvAV-3h) virion components affects pathogenicity against different host larvae species" and your time regarding for our revision.I also appreciate all the critical comments from you and reviewers.We have carefully considered the comments and revised the manuscript based on your comments and suggestions.With these improvements, we hope that the current version can meet the Journal's standards for publication.The following is a point-by-point response to all those comments and a list of changes we have made to the manuscript.
Reviewer: 1 1.Co-IP Assay Elaboration and Controls: Please provide a more detailed description of the co-IP Assay methodology.Elaborate on the experimental setup, including the specific antibodies used for immunoprecipitation and the detection of interacting proteins.Additionally, clarify the controls used in the assay to ensure the reliability of your results, especially concerning false positive detection.Describing negative controls and how they help identify non-specific interactions will strengthen the validity of your findings.Response: Thank you for your suggestion.We had revised the method of Co-IP according to your suggestion.Please see the revised manuscript.
2. Proteomics Data and Supplementary Tables: In the supplementary tables presenting the proteomics data, ensure that the information is comprehensive and well-organized.Provide additional relevant details on the identified proteins, such as their functional annotations and potential roles in immune responses.If any of the detected proteins are not unique to your study but are closely related to previously reported data in related virus groups, make a clear comparison to highlight any similarities or differences.This will help readers contextualize your findings within the existing knowledge on these proteins.
Response: Thank you for your suggestion.We had 24 supplementary Tables to present the proteomic data, 12 for the viral proteins and 12 for host larval proteins.All them contained annotation information.Considering we do not detect the immune responses, such as melanization or other humoral immunity or cellular immunity pathways, we did not revise these tables.Please excuse us for we did not adding any highlights.

Reviewer: 2
Comments: Overall, the manuscript provided a large body of data, however, the results were not well organized and described or interpreted.It is expectable that the components of virions purified from different host insects have subtle difference, but the functional links of those components, particularly of those detoxification enzymes, with the virus infectivity are not clear.Response: Thank you for your valuable comments.The study on ascovirus is relatively backward, and there are many unknowns about their pathogenesis and the structure of virions, which brings great difficulties to our work.In this study, we mainly aim to demonstrate the mutual "adaptation" between the ascovirus and the host by verifying the variability of the structure of virions.There are indeed some aspects that cannot be explained clearly in this study, and this is also what we want to study in the future.At present, we are preparing antibodies against the host detoxification enzyme proteins associated to the virions, and we will use these antibodies to further study the relationship between virus infection and host detoxification enzyme proteins and activity.Specific points: 1. Lines 140-147, the morphology of the purified virions has no obvious difference.This section shouldn't stand alone.It could be combined with the section "Different host larvae produced HvAV-3h virions had similar......" in lines 148-149.Also, Lines 220-233, they compared the host proteins associated with the HvAV-3h virion, this section should be combined with those in lines 148-149.Additionally, the description and interpretation of the components of virions purified from different hosts are not clear.Response: Thank you for your suggestion.We had rearranged the sections in the RESULTS according to your suggestion.The description of the mass spectrum results about the different host produced virions were separated into two parts: the protein encoded by the virus and the protein encoded by host larvae.The had been combined into a same subsection according to your suggestion.And we had added some general description about the host larval coded proteins identified from the MS data.Hope the revised manuscript could meet your requirements.
2. Lines 250-251, they used the inhibitors of the detoxification enzymes to evaluated the relationship of the virus infectivity and the activity of enzymes.The inhibitory effect of each inhibitor on the specific enzyme of insects are not clear.
Response: Thank you.PBO, DEM, and TPP were commonly used as inhibitors to the insect detoxification enzymes, thus we did not detect their inhibitory effects in this study.We had added several references in the M&M sections.Please see the revised manuscript.
3. Lines 262-296.Since the antibody block/neutralization substantially reduced the viral DNA replication and infectivity, it is not conceivable to assess the relationship of the viral structural proteins and host detoxification enzyme activity by using the antibody blocked/neutralized virus to infect the insect.
Response: As we had discussed in the DISCUSSION, the viral DNA replication and infectivity might be directly resulted from the reduced invading of the viruses due to the blocked structural protein.But how does the blocked structural protein in the virions affect the invading of ascovirus was unknown.From the MS data we can see that the virions might carry the host detoxification enzyme protein, which suggested that the host detoxification enzyme protein should interacted with the virion structural proteins (virus coded ones).These interactions might happen in the late stage of the infection of ascovirus (the stage of assemble of virions), but this still indicates the infection of ascovirus are associated to the host larval detoxification enzyme activity.On the other side, as shown in Fig. 5B, the host larval detoxification enzyme activity changed a lot from 3-24 hpi, and this stage was the invading stage of the ascovirus, which indicates that the invading of HvAV-3h is related to the host larval detoxification enzyme activity.To reveal whether the selected viral structural protein had any functions to stimulate the host larval detoxification enzyme activity, so as to influence the invading of ascovirus, we performed the experiments of Fig. 6A (Lines 262-296).4. Lines 158-160, the proteins associated with the virions purified from each insect species are varied remarkably.
Response: Many thanks.To identify the virion components, the MS analysis of virion protein samples purified from each larval species were performed with 3 biological repeats.As you see there are differences between the 3 repeats.The Veen analysis of the 3 repeats of each insect species were performed, and to avoid the inaccuracies caused by these differences, we used those proteins commonly identified in all the 3 repeats.We hope that the conclusion obtained in this way will be more reliable.5. Lines 226,228,[231][232], the difference of "P450" and "P450s" is not clear.P450 is a large protein family, which specific member(s) of P450 were detected in those purified virions?
Response: Sorry for our careless.We had uniform the "P450s" into "P450", "GSTs" into "GST", please see the revised manuscript.The specific numbers of P450 were provided in the supplementary Tables.There are too many identified P450, and it is not appropriate to list their numbers one by one.For example, 15 P450 proteins (CYP324A6, CYP4L7, CYP6AE97, CYP4S8, CYP6AE10, CYP354A14, CYP6AB61, CYP332A1, CYP6B68, CYP339A1, CYP9A21v4, CYP6AB31, CYP306A1, CYP4M15, CYP321A9) were identified from the virions purified from S. exigua.Furthermore, CarE and GST also are protein families, if we list the numbers of P450, we also have to separate CarE and GST proteins by their subgroups.So, we didn't add the specific member(s) of P450 in the revised manuscript.The readers can find and select the detailed information in our provided supplementary data.6. Line 231, "S.frugiperda" should be "S.litura".
Response: Sorry for our careless.We had corrected the mistakes.Please see the revised manuscript.
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Thank you for submitting your paper to Spectrum.

Sincerely, Clinton Jones
Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred -23R1 (Flexible changes to the Heliothis virescens ascovirus 3h (HvAV-3h) virion components affects pathogenicity against different host larvae species) Dear Dr. Guo-Hua Huang: