Male-Biased Cyp17a2 Governs Antiviral Sexual Dimorphism in Fish via STING Stabilization and Viral Protein Degradation

Reviewer #1 (Public review):

Summary:

In this manuscript Lu & Cui et al. observe that adult male zebrafish are more resistant to infection and disease following exposure to Spring Viremia of Carp Virus (SVCV) than female fish. The authors then attempt to identify some of the molecular underpinnings of this apparent sexual dimorphism and focus their investigations on a gene called cytochrome P450, family 17, subfamily A, polypeptide 2 (cyp17a2) because it was among genes that they found to be more highly expressed in kidney tissue from males than in females. Their investigations lead them to propose a direct connection between cyp17a2 and modulation of interferon signaling as the key underlying driver of difference between male and female susceptibility to SVCV.

Strengths:

Strengths of this study include the interesting observation of a substantial difference between adult male and female zebrafish in their susceptibility to SVCV, and also the breadth of experiments that were performed linking cyp17a2 to infection phenotypes and molecularly to the stability of host and virus proteins in cell lines. The authors place the infection phenotype in an interesting and complex context of many other sexual dimorphisms in infection phenotypes in vertebrates. This study succeeds in highlighting an unexpected factor involved in antiviral immunity that will be an important subject for future investigations of infection, metabolism, and other contexts.

Weaknesses:

Weaknesses of this study include a proposed mechanism underlying the sexual dimorphism phenotype based on experimentation in only males, and widespread reliance on over-expression when investigating protein-protein interaction and localization. Additionally, a minor weakness is that the text describing the identification of cyp17a2 as a candidate contains errors that are confusing. For example:

- Lines 139-140 describe the data for Figure 2 as deriving from "healthy hermaphroditic adult zebrafish". This appears to be a language error and should be corrected to something that specifies that the comparison made is between healthy adult male and female kidneys.

- In Figure 2A and associated text cyp17a2 is highlighted but the volcano plot does not indicate why this was an obvious choice. For example, many other genes are also highly induced in male vs female kidneys. Figure 2B and line 143 describe a subset of "eight sex-related genes" but it is not clear how these relate to Figure 2A. The narrative could be improved to clarify how cyp17a2 was selected from Figure 2A and it seems that the authors made an attempt to do this with Figure 2B but it is not clear how these are related. This is important because the available data do not rule out the possibility that other factors also mediate the sexual dimorphism they observed either in combination, in a redundant fashion, or in a more complex genetic fashion. The narrative of the text and title suggests that they consider this to be a monogenic trait but more evidence is needed.

Reviewer #2 (Public review):

This study conducted by Lu et al. explores the molecular underpinnings of sexual dimorphism in antiviral immunity in zebrafish, with a particular emphasis on the male-biased gene cyp17a2. The authors demonstrate that male zebrafish exhibit stronger antiviral responses than females, and they identify a teleost-specific gene cyp17a2 as a key regulator of this dimorphism. Utilizing a combination of in vivo and in vitro methodologies, they demonstrate that Cyp17a2 potentiates IFN responses by stabilizing STING via K33-linked polyubiquitination and directly degrades the viral P protein via USP8-mediated deubiquitination. The work challenges conventional views of sex-based immunity and proposes a novel, hormone- and sex chromosome-independent mechanism.

Strengths:

(1) The following constitutes a novel concept, sexual dimorphism in immunity can be driven by an autosomal gene rather than sex chromosomes or hormones represents a significant advance in the field, offering a more comprehensive understanding of immune evolution.

(2) The present study provides a comprehensive molecular pathway, from gene expression to protein-protein interactions and post-translational modifications, thereby establishing a link between Cyp17a2 and both host immune enhancement (via STING) and direct antiviral activity (via viral protein degradation).

(3) In order to substantiate their claims, the authors utilize a wide range of techniques, including transcriptomics, Co-IP, ubiquitination assays, confocal microscopy, and knockout models.

(4) The utilization of a singular model is imperative. Zebrafish, which are characterized by their absence of sex chromosomes, offer a clear genetic background for the dissection of autosomal contributions to sexual dimorphism.

Weaknesses:

(1) Limited discussion on whether this mechanism extends beyond Cyprinidae and its implications for teleost adaptation.

Comments on revisions:

The authors successfully achieved their primary aim, which was to identify and characterize a male-biased gene governing antiviral sexual dimorphism in fish. The data provide robust support for the conclusion that Cyp17a2 enhances antiviral immunity through dual mechanisms, STING stabilization and viral protein degradation, independent of classical sex-determining pathways. The findings are consistent across a range of experimental setups and are statistically robust. The revisions have significantly enhanced the clarity, depth, and overall quality of the manuscript. The authors have addressed each concern meticulously, resulting in a much-improved and robust article. No further suggestions are offered.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Weaknesses:

(1) Figure 10 outlines a mechanistic link between cyp17a2 and the sexual dimorphism the authors report for SVCV infection outcomes. The data presented on increased susceptibility of cyp17a2-/- mutant male zebrafish support this diagram, but this conclusion is fairly weak without additional experimentation in both males and females. The authors justify their decision to focus on males by stating that they wanted to avoid potential androgen-mediated phenotypes in the cpy17a2 mutant background (lines 152156), but this appears to be speculation. It also doesn't preclude the possibility of testing the effects of increased cyp17a2 expression on viral infection in both males and females. This is of critical importance if the authors intend to focus the study on sexual dimorphism, which is how the introduction and discussion are currently structured.

Thank you for your suggestion. We have revised the relevant statements in the introduction and discussion sections accordingly. The cyp17a2 overexpression experiments were not conducted in both male and female individuals was primarily based on two reasons. First, our laboratory currently lacks the technical capability to achieve cyp17a2 overexpression at the organismal level, existing methodologies are limited to gene knockout via CRISPR-Cas9. Second, even if overexpression were feasible, subsequent comparisons would need to be restricted within sexes (i.e., female vs. female controls or male vs. male controls) to eliminate potential confounding effects of sex hormones. Such experimental outcomes would only demonstrate the antiviral function of Cyp17a2 itself rather than directly elucidate mechanisms underlying sexual dimorphism, which diverges from the central objective of this study.

We fully agree with your perspective and have accordingly refined relevant discussions in the revised manuscript. Our conclusions now emphasize that "cyp17a2 is one of the factors contributing to sex-based differences in antiviral immunity" rather than implying that it "solely mediates the entire phenotypic divergence." These modifications have been incorporated into the resubmitted version (Lines 112-115).

(2) The authors present data indicating an unexpected link between cyp17a2 and ubiquitination pathways. It is unclear how a CYP450 family member would carry out such activities, and this warrants much more attention. One brief paragraph in the discussion (starting at line 448) mentions previous implications of CYP450 proteins in antiviral immunity, but given that most of the data presented in the paper attempt to characterize cyp17a2 as a direct interactor of ubiquitination factors, more discussion in the text should be devoted to this topic. For example, are there any known domains in this protein that make sense in this context? Discussion of this interface is more relevant to the study than the general overview of sexual dimorphism that is currently highlighted in the discussion and throughout the text.

We are grateful to the reviewer for their suggestion to elaborate on this novel finding. The discussion on this point has been expanded significantly (Lines 448-460). It is acknowledged that Cyp17a2 is devoid of the canonical domains that are typically associated with the ubiquitination machinery (e.g., RING, U-box). The present study proposes that the endoplasmic reticulum (ER) localization of Cyp17a2, in conjunction with its capacity to function as a scaffold protein, is of paramount significance. By residing in the ER, Cyp17a2 is strategically positioned to interact with key immune regulators such as STING, which also localizes to the ER. It is hypothesized that Cyp17a2 facilitates the recruitment of E3 ligases (btr32) and deubiquitinates (USP8) to their substrates (STING and SVCV P protein, respectively) by providing a platform for protein-protein interactions, rather than directly catalyzing ubiquitination. This noncanonical, scaffolding role for a cytochrome P450 (CYP450) enzyme represents an exciting evolutionary adaptation in teleost immunity.

(3) Figures 2-9 contain information that could be streamlined to highlight the main points the authors hope to make through a combination of editing, removal, and movement to supplemental materials. There is a consistent lack of clarity in these figures that could be improved by supplementing them with more text to accompany the supplemental figures. Using Figure 2 and an example, panel (A) could be removed as unnecessary, panel (B) could be exchanged for a volcano plot with examples highlighting why cyp17a2 was selected for further study and also the full dataset could be shared in a supplemental table, panel (C) could be modified to indicate why that particular subset was chosen for plotting along with an explanation of the scaling, panel (D) could be moved to supplemental because the point is redundant with panels (A) and (C), panel (E) could be presented as a heatmap, in panels (G) and (H) data from EPC cells could be moved to supplemental because it is not central to the phenotype under investigation, panels (J) to (L) and (N) to (P) could be moved to supplemental because they are redundant with the main points made in panels (M) and (Q). Similar considerations could be made with Figures 3-9.

We thank the reviewer for these excellent suggestions to improve the clarity and focus of our figures. A comprehensive review of all figures has been conducted in accordance with the recommendations made. Figure 2A has been removed. Figure 2B (revised Figure 2A) has been replaced with a volcano plot highlighting cyp17a2 and the full dataset has been provided as supplementary Table S2. Figure 2C (revised Figure 2B) is now a heatmap with eight sex-related genes and an explanation of the scaling has been added to the revised figure legends. Several panels (D, G, H, J-L, N-P) have been moved to the supplementary information (now Figure S1). Figure 2E has been presented as a heatmap. The same approach to streamlining has been applied to Figures 3-9, with confirmatory or secondary data being moved to supplements in order to better emphasize the main conclusions. The figure legends and main text have been updated accordingly.

(4) The data in Figure 3 (A)-(C) do not seem to match the description in the text. That is, the authors state that cyp17a2 overexpression increases interferon signaling activity in cells, but the figure shows higher increases in vector controls. Additionally, the data in panel (H) are not described. What genes were selected and why, and where are the data on the rest of the genes from this analysis? This should be shared in a supplemental table.

We apologize for the lack of clarity. In Figures 3A-C, the vector control shows baseline activation due to the stimulants (poly I:C/SVCV), but the fold-increase is significantly greater in the Cyp17a2-overexpressing groups. We have re-plotted the data to more clearly represent the stimulant-induced activation over baseline and added statistical comparisons between the Vector and Cyp17a2 groups under each condition to highlight the enhancing effect of Cyp17a2. For Figure 3H (revised Figure 3F), the heatmap shows a curated set of IFN-stimulated genes (ISGs) most significantly regulated by Cyp17a2 based on our RNA-seq analysis. We have added a description in the revised figure legend and in the results section (Lines 837-840). The full list of differentially expressed genes from this analysis is now provided in Supplementary Table S3.

(5) Some of the reagents described in the methods do not have cited support for the applications used in the study. For example, the antibody for TRIM11 (line 624, data in Figures 6 & 7) was generated for targeting the human protein. Validation for use of this reagent in zebrafish should be presented or cited. Furthermore, the accepted zebrafish nomenclature for this gene would be preferred throughout the text, which is bloodthirsty-related gene family, member 32.

We thank the reviewer for raising this important point regarding reagent specificity. To address the concern about antibody validation in zebrafish, we performed the following verification steps. First, we aligned the antigenic sequence targeted by the Abclonal btr32 antibody (ABclonal, A13887) with orthologous sequences from zebrafish, which showed 45% protein sequence similarity (Author response image 1). More importantly, we conducted experimental validation by expressing Myc-tagged btr32 in EPC cells. Both the anti-Myc and the anti-btr32 antibodies detected a protein band at the same molecular weight. Furthermore, when a btr32-specific knockdown plasmid was introduced, the band recognized by the anti-btr32 antibody was significantly reduced (Author response image 2). These results support the specificity of the antibody in recognizing fish btr32. In accordance with the reviewer’s suggestion, we have also updated the gene nomenclature to “bloodthirsty-related gene family, member 32 (btr32)” throughout the manuscript.

Author response image 1.

Author response image 2.

Reviewer #2 (Public review):

Weaknesses:

(1) Colocalization analyses (Figures 4G, 6I, 9D) require quantitative metrics (e.g., Pearson's coefficients) rather than representative images alone.

We concur with the reviewer's assessment. We have now performed quantitative colocalization analysis (Pearson's coefficients) for all indicated figures (4G, 6I, 9D). The quantitative results are now presented within the figures themselves and described in the revised figure legends.

(2) Figure 1 survival curves need annotated statistical tests (e.g., "Log-rank test, p=X.XX")

The survival curves have now been annotated with the specific p-values from the Log-rank (Mantel-Cox) test (see revised Figures 1A, 2E).

(3) Figure 2P GSEA should report exact FDR-adjusted *p*-values (not just "*p*<0.05").

Figure 2P (revised Figure S1J) has been updated to include the exact FDR p-values for the presented GSEA plots.

(4) Section 2 overextends on teleost sex-determination diversity, condensing to emphasize relevance to immune dimorphism would strengthen narrative cohesion.

The section on teleost sex-determination diversity in the Discussion (lines 357-365) has been condensed, with a more direct focus on how this diversity provides a unique context for studying immune dimorphism independent of canonical sex chromosomes, as exemplified by the zebrafish model.

(5) Limited discussion on whether this mechanism extends beyond Cyprinidae and its implications for teleost adaptation.

The discussion has been expanded (lines 375-386) to address the potential conservation of this mechanism. It is acknowledged that cyp17a2 is a teleost-specific gene, and it is hypothesized that its function in antiviral immunity may signify an adaptive innovation within this extensively diverse vertebrate group. It is suggested that further research in other teleost families will be essential to ascertain the broader evolutionary significance of the present findings.

Reviewer #2 (Recommendations for the authors):

(1) Expand the Discussion to address why teleosts may have evolved male-biased immunity. Consider: pathogen pressure differentials in aquatic vs. terrestrial environments; trade-offs between immune investment and reproductive strategies (e.g., male-male competition); comparative advantages in external fertilization systems.

We have expanded the discussion on lines 412-430, to address the potential conservation of this mechanism. We note that Cyp17a2 is a teleost-specific gene and speculate that its role in antiviral immunity represents an adaptive innovation within this highly diverse group of vertebrates. We propose that future studies of other teleost families are crucial for determining the broader evolutionary significance of our findings.