Review Reports - Early Transcriptomic Response of Human Iris Stromal Cells During Herpes Simplex Virus Entry Reveals Interplay Between Cell Glycocalyx and Viral Exploitation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an excellent article that talks about HSV-1 infection in iris cells. This is a unique study as no other reports have specifically done temporal transcriptomics on these cells and there is much to learn from it. It is true that the virus can infect these cells without involving the epithelium and cause significant changes to vision and hence this study is of high significance. That being said there are some minor changes or edits that I would suggest for better clarity.

The authors should explain the reasoning behind using 2 MOI and not 0.1 or 1 MOI. As a higher MOI will push cells towards cell death rather than productive infection.
Can the authors do a comparative study in viral productivity between epithelial cells and iris cells? If not, an explanation or discussion regarding how they might differ can be significant interest to readers.
The authors show that there is VP16 expression within 3h of viral entry. Is this just entered virus or is it actually newly produced VP16? Probing the RNA seq for alignment andn mapping with HSV genome can provide whether viral transcripts and hence proteins were made. Also providing a full list of viral transcripts may shed light on differences between epithelial and iris cells.
The authors show IL17 regulation and TGFb regulation at different times post infection but shift their focus to HS3ST and other genes of interest in the second half of the manuscript. Could there be a little discussion within the results to explain why this is?

Author Response

Reviewer 1

The authors should explain the reasoning behind using 2 MOI and not 0.1 or 1 MOI. As a higher MOI will push cells towards cell death rather than productive infection.

We appreciate the reviewer’s concern regarding potential cell toxicity at an MOI of 2.0. However, moderate to high MOIs are routinely employed in HSV-1 transcriptomic studies to achieve near-complete and synchronized infection, a prerequisite for robust host–virus transcriptional profiling. For instance, Benxia Hu et al. (2016, Scientific Reports) used an MOI of 5.0, while Hui-Lan Hu et al. (2021, EMBO Reports) employed an MOI of 1.5 per cell for single-cell transcriptomic analyses. Moreover, higher MOIs have been reported in independent studies published in Nature Communications (Emanuel Wyler et al., 2019) and iScience (Robert J.J. Jansens et al., 2023), where HSV-1 infections were performed at an MOI of 10.0 at early time points. In our study, early sampling enabled near-complete infection without substantial cell loss, supporting the use of an MOI of 2.0 as a biologically justified and experimentally appropriate choice. We agree that future dosage-dependent studies would be valuable to further delineate HSV-1-mediated effects on cellular transcription and signaling pathways. This limitation has now been explicitly addressed in the revised manuscript (page 20, lines 459–476).

Can the authors do a comparative study in viral productivity between epithelial cells and iris cells? If not, an explanation or discussion regarding how they might differ can be of significant interest to readers.

We appreciate the reviewer’s suggestion, as a direct comparison of viral productivity between epithelial and iris cells would indeed be of significant interest. However, quantitative viral productivity assays were beyond the scope of the present study, which was designed to focus on early host transcriptional responses rather than late-stage viral yield.

As per suggestion, we have discussed the possible cell type variability in the Discussion section (on page 20; line 467) since viral productivity is expected to differ between epithelial and iris cells due to intrinsic differences in cellular permissiveness, antiviral signaling, and innate immune responses. Epithelial cells are generally highly permissive to HSV-1 replication and support robust lytic infection, whereas ocular-derived cells, including iris cells, often exhibit enhanced intrinsic antiviral defenses, altered interferon signaling, and cell-type-specific restriction factors that can limit viral replication and spread. Additionally, ocular tissues are known to maintain immune-privileged environments, which may further influence HSV-1 replication kinetics and viral output.

Importantly, our transcriptomic analyses were performed at early time points prior to extensive viral replication, enabling us to capture host–virus interactions independent of differences in viral yield. Future studies directly comparing viral productivity across these cell types would be valuable to further elucidate how cell-specific antiviral programs shape HSV-1 replication and pathogenesis.

The authors show that there is VP16 expression within 3h of viral entry. Is this just entered virus or is it actually newly produced VP16? Probing the RNA seq for alignment and mapping with HSV genome can provide whether viral transcripts and hence proteins were made. Also providing a full list of viral transcripts may shed light on differences between epithelial and iris cells.

We thank the reviewer for this important point. At 3 h post-infection, VP16 detection likely reflects both incoming tegument-associated VP16 and the earliest stages of de novo viral gene expression; protein-based assays alone cannot distinguish between these sources, and we have interpreted VP16 presence accordingly as an early infection marker. This part has now been clarified in the result section on page 6 (line 130-134). Our RNA-seq analysis was intentionally designed to capture host transcriptional responses at early time points and was not optimized for comprehensive viral transcriptome profiling or quantitative comparison of viral gene expression between epithelial and iris cells. While HSV-1–aligned reads were detected, we did not perform a full viral transcript analysis, as differences in viral replication kinetics could confound interpretation at later stages. We agree that detailed mapping of viral transcripts and comparative viral gene expression analyses would be highly informative and represent an important direction for future studies.

The authors show IL17 regulation and TGFb regulation at different times post infection but shift their focus to HS3ST and other genes of interest in the second half of the manuscript. Could there be a little discussion within the results to explain why this is?

We thank the reviewer for this helpful suggestion. The early focus on IL-17 and TGF-β signaling reflects the immediate host immune and regulatory responses to HSV-1 infection, whereas the later emphasis on HS3ST and related genes highlights their role in HSV-1 entry, and downstream transcriptional programs associated with cellular remodeling. To clarify this progression, we have added brief explanatory text in the Discussion section on pages 19-20 (lines 431-440) to explicitly link early cytokine signaling events to later changes in gene expression. This revision is intended to improve narrative continuity and emphasize the temporal structure of the host response.

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the authors profile the early host transcriptional responses of primary human iris stromal (HIS) cells to HSV-1 infection. The study addresses a gap in the understanding of HSV-1-induced anterior uveitis, a relatively common vision-threatening condition with limited therapeutic options. The use of primary human cells, temporal transcriptomics, and complementary imaging data strengthens the findings. The work is presented and described clearly, is methodologically sound, and provides novel insights into the interplay between viral entry and host glycocalyx remodeling. Each timepoint is discussed in detail. Many of the genes exhibit patterns observed in other cell types in other studies. The manuscript is in good shape, pending a few comments and concerns below:

Supp. Table 1: it would be helpful to have the tables themselves of the relative gene expression for future studies. I suppose these would be the supplementary files mentioned in the text, but I was not able to access these files in the materials for review.

Can the authors comment as to why there is such low HS expression at the earlier timepoints in Fig. 7?

The alignment steps between demultiplexing and differential expression analysis should be described in the methods.

An MOI of 2 reaching nearly 100% infection is unexpected, typically going as high as MOI 10 is required for every cell to be infected. Can the authors comment on how the virus was titered? For example, is this an MOI of 2 Vero units, by TCID50, etc.?

Minor edits:
Remove "Figure X" from the figures themselves
Fig. 2C, KDa is the preferred abbreviation than Kds
Line 255: place a comma after "In this regard"
The first paragraph of the discussion is nearly two pages long, and it would greatly improve readability if this was broken up into smaller paragraphs
Line 533: please make the 2 in "mm2" superscript
Lines 538, 549: please make the u into a mu for SI units
Line 569: remove the the space in "12 mm" for consistency
Line 596-597: was the RNA fragmentation in a basic solution or in pure water?

Author Response

We thank the reviewer for noting this issue. The tables containing relative gene expression data were intended to be provided as supplementary files; however, we recognize that these files were not accessible during the review process. We have now uploaded complete gene expression Excel files as Supplementary Tables (1-3) and verified that they are fully accessible. These tables include normalized expression values and differential expression results to facilitate reuse in future studies.

Can the authors comment as to why there is such low HS expression at the earlier timepoints in Fig. 7?

We thank the reviewer for this observation. The relatively low levels of heparan sulfate (HS) expression detected at early time points (Figure 7) likely reflect the fact that HSV-1 entry primarily depends on pre-existing cell surface HS rather than immediate de novo synthesis. During the early stages of infection, host transcriptional responses are predominantly driven by innate immune activation and cellular stress signaling. In contrast, modulation of HS biosynthetic and modifying enzymes, including members of the HS3ST family, is more likely to occur at later stages as part of virus-induced cellular remodeling. Accordingly, future studies examining both dose- and time-dependent effects on HS and HS-related gene expression would help determine whether these changes represent secondary transcriptional responses rather than immediate entry-associated events. This issue has been addressed on page 22 (lines 484-494).

The alignment steps between demultiplexing and differential expression analysis should be described in the methods.

We thank the reviewer for this suggestion. We have now expanded the Methods section on page 27-28 to explicitly describe the alignment, read mapping, and quantification steps performed between demultiplexing and differential expression analysis.

An MOI of 2 reaching nearly 100% infection is unexpected, typically going as high as MOI 10 is required for every cell to be infected. Can the authors comment on how the virus was tittered? For example, is this an MOI of 2 Vero units, by TCID50, etc.?

We appreciate the reviewer’s comment. The apparent high infection efficiency at an MOI of 2.0 reflects the method used for virus titration and the high permissiveness of the target cells rather than an overestimation of infectivity. The virus stock was titrated using standard plaque assay, and MOI values were calculated based on infectious units rather than total physical particles. In highly permissive epithelial and iris cells, HSV-1 spreads rapidly and achieves near-complete infection even at moderate MOIs, particularly at early time points. We have now clarified the virus titration method in the Methods section (on page 25) to avoid confusion.

Minor edits:
Remove "Figure X" from the figures themselves

We thank the reviewer for this suggestion. The labels of the figures themselves have been removed, as suggested to improve clarity and consistency.

Fig. 2C, KDa is the preferred abbreviation than Kds

Abbreviation has been corrected to Kda in Figure 2.

Line 255: place a comma after "In this regard"

The comma has been placed before “In this regard”.

The first paragraph of the discussion is nearly two pages long, and it would greatly improve readability if this was broken into smaller paragraphs

We thank the reviewer for this helpful suggestion. The first paragraph of the Discussion has been divided into smaller, more focused paragraphs to improve readability and flow.

Line 533: please make the 2 in "mm2" superscript

We have corrected by making 2 as a superscript

Lines 538, 549: please make the u into a mu for SI unit

We have provided the values into the SI units

Line 569: remove the space in "12 mm" for consistency

We have removed the space

Line 596-597: was the RNA fragmentation in a basic solution or in pure water?

We thank the reviewer for pointing this out. The RNA fragmentation was carried out in a mildly basic buffer (e.g., 10 mM Tris-HCl, pH 8.0) rather than in pure water. We have updated the Methods section to clearly specify the buffer and conditions used for RNA fragmentation on page 27.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

In this resubmission, the authors have made several text corrections and included the supplementary data for review. My concerns have been largely addressed, and I believe that the manuscript has been significantly improved and provides valuable insight into ocular HSV infections.

The only comments I have below are relatively minor. While it be appreciated if they were addressed in the next stage of proofing, I do not believe that they are enough to warrant flagging for minor revision.

My comment on the low HS levels in Figure 7 is apparent to the timepoints themselves, even mock, rather than infection. It is interesting that even infected cells have low HS levels at 1 and 3 hpi. Though the authors attempt to address the comment through added text is appreciated.

I would also consider changing "absorption" in line 543 to "adsorption" and to address the lone mu in line 579 after 300 x 300. Is this also micrometer?