Super-Resolution Microscopy in the Structural Analysis and Assembly Dynamics of HIV

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have done an excellent job in organizing this review and providing a clear account of how super-resolution microscopy (SRM) has been utilized over the past decade to investigate structural components of HIV, with a particular focus on assembly dynamics and the spatial organization of viral proteins. The manuscript effectively summarizes the applications of four major SRM techniques PALM, STORM, STED, and SIM alongside various labeling strategies, highlighting their utility in imaging key HIV components such as Gag, Env, capsid, and RNA. The review is further strengthened by its systematic literature coverage, clear structure, and visually informative figures and tables, which collectively enhance its value to researchers in the field. I have outlined below a set of key points that should be addressed to improve the manuscript prior to publication.

Major Comments

1. Limited Scope of Functional Insights: The review focuses heavily on structural and assembly dynamics but misses the opportunity to discuss how SRM has contributed to understanding virus-host interactions, immune evasion, or antiretroviral targeting. A brief paragraph in the Conclusion reflecting these broader applications would make the review more comprehensive.
2. Underrepresentation of Emerging SRM Techniques: While the exclusion of methods like MINFLUX, RESOLFT, and LLS is understandable based on inclusion criteria, these techniques could be mentioned briefly in the Conclusion to guide readers toward future directions in high-resolution virology.
3. Methodological Bias Toward Gag and Env: The review correctly notes the frequent use of SRM to study Gag and Env but does not fully analyze the implications of this imbalance. A short reflection on how this may affect the generalizability of conclusions would strengthen the discussion.
4. Minimal Technical Critique of SRM Methods: Table 3 (pages 11-12) summarizes technical limitations, but the main text underemphasizes critical constraints such as phototoxicity (STED), limited live-cell compatibility (STORM), and reduced photon output (PALM). A more technical paragraph in Section 3.9 or Conclusion would benefit researchers planning SRM experiments.
5. Labeling Strategy Challenges Not Fully Explored: Although labeling methods are categorized (Figure 5A and Section 3.8), practical challenges like antibody penetration, protein tagging artifacts, and intracellular delivery are not sufficiently discussed. A few lines added to Section 3.8 would add important experimental context.
6. Lack of Quantitative Meta-Analysis: The review provides no quantitative synthesis of resolution gains, localization precision, or error margins across SRM methods. While this may be beyond the scope of a narrative review, even approximate resolution ranges or performance benchmarks (e.g., in Table 3) would increase the comparative rigor.
7. Potential Redundancy in Some Descriptions: The results section repeats similar findings, particularly on Env clustering and Gag lattice formation. These could be more succinctly summarized with a clearer synthesis of agreement vs. divergence across studies.
8. Formatting and Flow: Some section transitions are abrupt—especially between component-based discussions (Sections 3.1-3.5) and between SRM technique associations (Sections 3.6-3.9). Improved narrative bridges or summary sentences would help readers better connect imaging choices with biological insight.

Minor Comments

• Duplicate Data Clarification: Please clarify in Section 2 (lines 130-133) whether studies with reused datasets (e.g., by the same research groups) were excluded or flagged to avoid redundancy.
• Rationale for PubPeer Use: The mention of PubPeer (line 132) is a good practice but should be supported by a sentence explaining why this step was necessary (e.g., to screen for research integrity concerns).

Author Response

Thank you for your feedback. We appreciate you taking the time to read through and provide comments on our review.

• Comment 1: “Limited Scope of Functional Insights: The review focuses heavily on structural and assembly dynamics but misses the opportunity to discuss how SRM has contributed to understanding virus-host interactions, immune evasion, or antiretroviral targeting. A brief paragraph in the Conclusion reflecting these broader applications would make the review more comprehensive.”
• C1 Response: Thank you for your suggestion. While maintaining our focus on the context of HIV structure and assembly dynamics best kept with the theme of our paper, we have added an acknowledgment of other applications of SRM on page 14, lines 465-468 (paragraph 2). E.g., we stated that “While this review has centered on HIV structure and assembly, SRM techniques have also contributed, and will continue to contribute, to studies of HIV in broader contexts, including virus-host interactions, immune evasion, and antiretroviral targeting.”

• Comment 2: “Underrepresentation of Emerging SRM Techniques: While the exclusion of methods like MINFLUX, RESOLFT, and LLS is understandable based on inclusion criteria, these techniques could be mentioned briefly in the Conclusion to guide readers toward future directions in high-resolution virology.”
• C2 Response: We appreciate your comment and now also mention ExM and LLS, providing three relatively recent and distinct advances in SRM. We highlight an important advancement(s) from each method starting on page 13, lines 451-460 (paragraph 5). We kept this discussion succinct since we feel that speculation about the future and other methods not widely found in our analysis should be kept to a minimum.

• Comment 3: “Methodological Bias Toward Gag and Env: The review correctly notes the frequent use of SRM to study Gag and Env, but does not fully analyze the implications of this imbalance. A short reflection on how this may affect the generalizability of conclusions would strengthen the discussion.”
• C3 Response: Thank you for your comment. We agree that this is important, and we have added a discussion to section 3.6, based on Figure 4B. Specifically on page 11, lines 347-358 (paragraph 1): “Thus, recent SRM work has shifted toward probing Gag and Env, likely reflecting the biological and experimental relevance of these targets in HIV assembly, function, and interactions. Gag is the central structural protein driving viral assembly, making it important for imaging studies focused on particle formation. As derivatives of full-length Gag, Capsid (CA) and Matrix (MA) are less central to the primary virion assembly process. Similarly, Env plays a critical role in viral entry and is a key determinant of infectivity. Its position on the virion surface not only makes it highly accessible for various imaging labeling strategies but also marks it as a major target for therapeutic antibody development. Given their essential roles in virion structure, assembly, and host-cell entry, it is unsurprising that SRM studies have increasingly prioritized Gag and Env to further insight into HIV biology.”

• Comment 4: “Minimal Technical Critique of SRM Methods: Table 3 (pages 11-12) summarizes technical limitations, but the main text underemphasizes critical constraints such as phototoxicity (STED), limited live-cell compatibility (STORM), and reduced photon output (PALM). A more technical paragraph in Section 3.9 or Conclusion would benefit researchers planning SRM experiments.”
• C4 Response: This is a reasonable critique. We aimed to broadly state that each SRM method had strengths and limitations related to end goals. While there is no “right” or “wrong” combination of SRM method and HIV component, experimental contexts may make certain SRM methods less ideal. We mention some of these limitations throughout the paper and in Table 3, and we have expanded the discussion on page 13, lines 439-450 (paragraph 4).

• Comment 5: “Labeling Strategy Challenges Not Fully Explored: Although labeling methods are categorized (Figure 5A and Section 3.8), practical challenges like antibody penetration, protein tagging artifacts, and intracellular delivery are not sufficiently discussed. A few lines added to Section 3.8 would add important experimental context.”
• C5 Response: We agree with your comment and have expanded the discussion of practical challenges regarding different labeling techniques and how they some might be more practical than other when it comes to labeling the various components of HIV for SRM studies. Specifically, on page 12, lines 398-404 (paragraph 2), we further discussed “With surface-exposed epitopes, Env is more immediately accessible to antibody-based probes. For targets inside the virion or cell, such as Gag, a permeabilization step may be needed for antibody exposure and binding, which may be incompatible with live-cell imaging (i.e., live-cell PALM). Hence, an internal protein component such as Gag, Vpr, or Vpx would favor a labeling strategy that incorporates a fluorescent protein label into its structure, especially if live-cell imaging is important for the context of the study.”

• Comment 6: “Lack of Quantitative Meta-Analysis: The review provides no quantitative synthesis of resolution gains, localization precision, or error margins across SRM methods. While this may be beyond the scope of a narrative review, even approximate resolution ranges or performance benchmarks (e.g., in Table 3) would increase the comparative rigor.”
• C6 Response: We agree with your assessment. We have noted the approximate resolution enhancement for each SRM in Table 3, which we have now expanded with more specific details distinguishing the four SRM methods discussed in the context of HIV studies. While there are many more SRM methods, we included strengths, limitations, and resolution only for those that appeared in our review (i.e, those used with HIV studies) to remain in scope. Other parameters, such as localization precision, can depend on the specific equipment set-up or localization algorithm (such as with dSTORM), which can vary from machine to machine and company to company. We only included hard numbers for more standardized values and those of greatest interest (i.e., resolution).

• Comment 7: “Potential Redundancy in Some Descriptions: The results section repeats similar findings, particularly on Env clustering and Gag lattice formation. These could be more succinctly summarized with a clearer synthesis of agreement vs. divergence across studies.”
• C7 Response: We appreciate your comment. The findings of each paper were made distinct to purposefully highlight the major outcomes of each study, which may have used different methodologies, even if the topic or outcome of the study was similar. We have expanded discussions and summary sentences at the ends of sections 3.1 through 3.5 to highlight similarities/ divergences across studies and address concerns about redundancy.

• Comment 8: “Formatting and Flow: Some section transitions are abrupt—especially between component-based discussions (Sections 3.1-3.5) and between SRM technique associations (Sections 3.6-3.9). Improved narrative bridges or summary sentences would help readers better connect imaging choices with biological insight.”
• C8 Response: Thank you for your insight. For better transitions, we have added expanded discussion and summary sentences for each section in sections 3.1-3.5 (pages 7 to 10).
• In sections 3.6-3.9, some distinction between sections was warranted to highlight the figures and data discussed in that part of the report. However, additional commentary has been added throughout sections 3.6-3.9 (pages 10-13) to improve the narrative and discussion points in those sections.

• Minor Comment 1: “Duplicate Data Clarification: Please clarify in Section 2 (lines 130-133) whether studies with reused datasets (e.g., by the same research groups) were excluded or flagged to avoid redundancy.”
• MC1 Response: Thank you for pointing out this omission. We have added a sentence on page 6, lines 144-146 (paragraph 1), which states the following: “Finally, if a publication relied on previously published data, rather than having obtained new data, only the original source study for those data was included to reduce redundancy.”

• Minor Comment 2: “The mention of PubPeer (line 132) is a good practice but should be supported by a sentence explaining why this step was necessary (e.g., to screen for research integrity concerns).”
• MC2 Response 2: We agree with the sentiment of this comment. We state on page 6, lines 147-149 (paragraph 1), that PubPeer is used to “… avoid including papers suspected of research misconduct or poor research practice.” We have added a citation addressing the need for avoiding publications with research integrity concerns and how PubPeer helps with this, while also specifying how we used it on page 6, lines 148-149 (paragraph 1).

Reviewer 2 Report

Comments and Suggestions for Authors

This article will be of great utility to researchers exploring methods to study HIV assembly and the interaction between the virus and the infected cell's components.

The article is clearly written and brings some very technical material down to a more understandable level.

Their differentiation of the SRM techniques and showing how each is used in different research contexts will guide researchers and in how SRM can be applied to HIV research. 

I have two questions, both on the minor side.

First, the sentence from lines 124 - 7 is confusing because it is too long. Describing what articles you want to eliminate from the search by describing what you want to include (first part of the sentence) ensures that one needs to read it multiple times to make sense of it. Try cutting it down.

The second point relates to Figure 3. First, why do A and B have different total number of articles by year? For example, in 2011-2013, the graph in A lists 10 publications, but in B somewhere in the high teens. The other years seem to have similar inconsistencies. Second, I'm not sure what Figure 3B contributes to the paper. Figure 4 covers the same material more clearly.

Otherwise, an admirable job.

Author Response

Thank you for your feedback. We appreciate you taking the time to read through and provide comments.

• Comment 1: “First, the sentence from lines 124 - 7 is confusing because it is too long. Describing what articles you want to eliminate from the search by describing what you want to include (first part of the sentence) ensures that one needs to read it multiple times to make sense of it. Try cutting it down.”
• Response 1: Thank you for catching this. We agree and have revised the sentence by breaking it in two to more clearly emphasize: first, inclusion/exclusion criteria; and second, which specific, related topics we excluded. We also revised the second paragraph for concision (page 6, paragraph 1, lines 139-143): “We focused on the structural and assembly dynamics of HIV, as driven by HIV native components. With this scope in mind, we excluded papers that focused mainly on the pathology of HIV in human cells, hijacked host cell proteins, HIV drugs, extracellular vesicles, and virus-like particles (VLPs).”

 

• Comment 2: “The second point relates to Figure 3. First, why do A and B have different total number of articles by year? For example, in 2011-2013, the graph in A lists 10 publications, but in B somewhere in the high teens. The other years seem to have similar inconsistencies. Second, I'm not sure what Figure 3B contributes to the paper. Figure 4 covers the same material more clearly.”
• Response 2: The different totals in Figure 4 (previously called Figure 3) are addressed on page 7, lines 183-187 (paragraph 4). Some articles either used more than one SRM method to study the same HIV component or studied multiple HIV components using the same SRM method, accounting for the differences. To make this clearer to readers, we have added an additional clarification to the Figure 4 caption (page 10, lines 325-328), stating: “Note that since some research studies have used more than one SRM method to study a component of HIV, or may have used one SRM method to study multiple HIV components, the total number of papers in a year bracket may not match between Figure 4A and Figure 4B.”

• As for your comment on Figure 4B, our intent was to identify any notable trend in the component of HIV being studied over time (i.e., was Gag a focus in earlier SRM studies of HIV, but over time became less common?). Since we looked at the usage of each SRM method over time, we felt that it was also appropriate to see how the target HIV component(s) of those studies changed over time. Because Figure 5 (previously called Figure 4) focused more on the associations between each SRM method and each HIV component, we felt that showing the time association for each HIV component necessitated a separate plot.

Reviewer 3 Report

Comments and Suggestions for Authors

Summary: Jurcenko et al. performed a literature search and then use the output to compare how super-resolution imaging techniques including SIM, PALM, STORM, and STED have been used to study different aspects of HIV assembly from 2011-2023. They determine which techniques were mostly commonly utilized to study viral components including: Gag; Env; Env and Gag together; MA, CA, and IN; and PR, Vpx, and vRNA. Furthermore, they determined which labeling techniques were used most commonly to detect each protein or vRNA with each imaging modality.

Major comments:

1. Although this is a nice systematic review of the literature, the major weakness is that the review article provides a list of studies using SRM and the major finding of each study, but there were no conclusion drawn by synthesizing the results of each group of studies together to indicate how they increased understanding of HIV-1 replication. For example, for each viral protein, each study was described but there was no conclusion or unifying assessment of how the study provided important information. Were the results of each study/approach consistent with one another or were there contradictory findings? If there were contradictory findings, how can that be potentially explained: were different cell lines used, were different microscopy approaches used that allowed only a portion of the cell to be viewed, were tags added to the proteins potentially disrupting localization or trafficking? As it stands, the article provides a catalogue of previously published reports, but it would be improved by explaining how these studies provide conclusions about or yield insights into HIV replication.
2. The table of the imaging strengths and weaknesses focused on comparing the methods themselves and were not specific for how each approach was valuable or limited in understanding HIV biology. It would be more meaningful to provide pros/cons of each SRM approach as they were applied to specific aspects of the replication cycle or to individual viral proteins such as HIV Env, Gag, etc.
3. The authors of this review should strive to provide more useful information to assess how these imaging approaches have advanced understanding of HIV biology that goes beyond listing and superficially describing the papers that have used SRM in HIV biology. 

Author Response

Thank you for your careful review and thoughtful comments. 

• Comment1: Although this is a nice systematic review of the literature, the major weakness is that the review article provides a list of studies using SRM and the major finding of each study, but there were no conclusion drawn by synthesizing the results of each group of studies together to indicate how they increased understanding of HIV-1 replication. For example, for each viral protein, each study was described but there was no conclusion or unifying assessment of how the study provided important information. Were the results of each study/approach consistent with one another or were there contradictory findings? If there were contradictory findings, how can that be potentially explained: were different cell lines used, were different microscopy approaches used that allowed only a portion of the cell to be viewed, were tags added to the proteins potentially disrupting localization or trafficking? As it stands, the article provides a catalogue of previously published reports, but it would be improved by explaining how these studies provide conclusions about or yield insights into HIV replication.
• Response to C1: Our primary objective was to create a comprehensive and unbiased resource that accurately reflects the diversity and evolution of super-resolution microscopy (SRM) applications in HIV research. Given the heterogeneity in experimental design, imaging modalities, labeling strategies, and biological systems among the studies reviewed, we determined that imposing a unifying conclusion or synthesis could risk oversimplifying or misrepresenting the nuanced findings of individual papers. Where meaningful, we highlighted trends, such as the frequent use of PALM for Gag and STED for Env but deliberately avoided broad generalizations due to the limited number of directly comparable studies and the wide variability in experimental conditions. In many cases, differences in cell lines, labeling methods, or imaging parameters preclude direct comparison or synthesis. Rather than speculate, we chose to present the data transparently, allowing readers to draw informed conclusions based on the evidence. SRM applications in HIV biology are rapidly evolving, and a detailed catalogue of methods, targets, and findings provides a valuable reference for both newcomers and experienced researchers. This approach supports reproducibility and enables future meta-analyses as the literature matures.

 

• Comment 2: The table of the imaging strengths and weaknesses focused on comparing the methods themselves and were not specific for how each approach was valuable or limited in understanding HIV biology. It would be more meaningful to provide pros/cons of each SRM approach as they were applied to specific aspects of the replication cycle or to individual viral proteins such as HIV Env, Gag, etc.
• Response to C2: We appreciate the comment and have added additional information to Table 3 (pages 14/15). 

 

• Comment 3: The authors of this review should strive to provide more useful information to assess how these imaging approaches have advanced understanding of HIV biology that goes beyond listing and superficially describing the papers that have used SRM in HIV biology.
• Response to C3: We appreciate your comment. While it is important for us to continue to highlight the SRM method, HIV component(s), and main outcome of each study, we have now expanded the discussion in the relevant sections (sections 3.1-3.5), addressing how these SRM studies, and conversely, how SRM, have furthered our knowledge of HIV. An overarching concluding statement was also added at the end of section 3.5 to reinforce how SRM has advanced our understanding of HIV biology in ways other techniques cannot. These revisions are on pages 8-10, lines 211-217 in section 3.1, lines 232-237 in section 3.2, lines 252-258 in section 3.3, lines 275-285 in section 3.4, and lines 298-318 in section 3.5.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have responded to the critiques and expanded the description of how SRM has been used to advance the understanding of the mechanisms underlying HIV replication.