Bicistronic Lentiviral Architecture Increases Surface Density of Membrane-Associated HIV Entry Inhibitors and Confers Tropism-Independent Protection to Modified Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript, "Bicistronic lentiviral architecture increases surface density of membrane-associated HIV entry inhibitors and confers tropism-independent protection to modified cells," by Chervyakova et al. is a modest contribution to the field of HIV-1 antivirals. In particular, the authors have expressed gp41 heptad repeat 2 (HR2) peptides (C peptides) from monocistronic or bicistronic lentiviral vectors in a variety of cells. The cells have been subjected to infection with a variety of HIV-1 variants, and results indicate that the constructs allow surface expression of C peptides, protection from CCR5 and CXCR4 HIV-1 strains, and delivery to hematopoietic stem cells (HSCs). In general, the results represent a somewhat expected engineering report, with little new basic material. That being said, the data appear solid, and may be of interest to readers focused on gene therapy approaches for the treatment of HIV-1.

Author Response

Dear Reviewer,

Thank you very much for your careful reading of our manuscript and for taking the time to provide your assessment. We appreciate your recognition of the technical robustness of our data and your acknowledgment that the findings may be of interest to readers working in the field of HIV-1 gene therapy.

We also thank you for your constructive characterization of the study as an engineering contribution, and we agree that the work builds upon established paradigms in membrane-displayed fusion inhibitors. Your positive evaluation of the experimental rigor and the translational relevance of the bicistronic platform is greatly appreciated.

We are grateful for your recommendation to accept the manuscript for publication and for contributing to its dissemination.

With sincere gratitude,

Alexandra K. Maslennikova, PhD

On behalf of all authors

Reviewer 2 Report

Comments and Suggestions for Authors

The authors designed and transduced bicistronic constructs carrying two types of C-peptide HIV fusion inhibitors that were anchored by GPI on target cells.  They show that the bicistronic constructs significantly enhanced surface expression levels of the transgenes, which enhanced anti-viral protection in cell lines and primary CD4+ T cells in vitro.  I think the use of CEM over other T cell lines like Jurkat was a good approach.  I find the CEM T cells more physiologically similar to primary T cells.  Their system is particularly appealing because they can also relatively efficiently transduce HSC progenitors, and they have designed a construct to enhance engraftment of the transduced cells by transiently inducing CXCR4 upregulation.  The article is very clear and concise, and their reported results indicate a very promising approach in this early in vitro stage.  The finding that “replication-competent virus was completely eliminated from co-cultures before unmodified target cells were fully depleted” provides hope that full transduction efficiency will not be needed in these gene/cell therapy approaches to effectively cure HIV in vivo.The authors clearly delineate the caveats and necessary future directions in the Discussion.  I have no suggested changes for the article and support publication as is. 

 

A technical note for the authors:  the approach is quite interesting; however, the use of a GPI-anchored construct may direct the fusion inhibitor to the uropod of the T cell, rather than the leading edge where chemokine receptors will be scanning their environment.  The uropod may not be the most protective location for these fusion inhibitors at the early stages of infection.  Microscopic evaluation of C-peptide and co-receptor co-localization in migrating T cells may indicate a different anchor would be more ideal.  However, your results indicate that this may not be required for protection and your construct is working well independent of subcellular location.

Author Response

Dear Reviewer,

Thank you very much for the positive assessment of our work and for raising an important consideration regarding potential differences in the subcellular distribution of GPI-anchored molecules between static cell lines and migrating primary lymphocytes.

Comments: A technical note for the authors:  the approach is quite interesting; however, the use of a GPI-anchored construct may direct the fusion inhibitor to the uropod of the T cell, rather than the leading edge where chemokine receptors will be scanning their environment.  The uropod may not be the most protective location for these fusion inhibitors at the early stages of infection.  Microscopic evaluation of C-peptide and co-receptor co-localization in migrating T cells may indicate a different anchor would be more ideal.  However, your results indicate that this may not be required for protection and your construct is working well independent of subcellular location.

Response: We have expanded the Discussion (lines 636–655) to address this point directly. Specifically, we now cite recent evidence indicating that chemokine receptor distribution on migrating T cells is highly dynamic and condition-dependent — largely isotropic during constitutive migration, with only transient bias toward the uropod upon chemokine exposure. Such plasticity further complicates the assumption that precise subcellular positioning per se determines antiviral efficacy.

Importantly, this question was tested experimentally in our prior study, where we generated a construct (MT-C34-15D) in which the C-peptide was non-covalently tethered to HIV coreceptors via the gp120-derived 15D peptide, thereby forcing close proximity between the inhibitor and CXCR4/CCR5 (Maslennikova et al., 2022). Contrary to expectations, this modification did not improve — and sometimes reduced — protective activity relative to the standard GPI-anchored form expressed independently in lipid rafts. These data indicate that physical recruitment of the fusion inhibitor to the coreceptor site does not confer additional benefit, supporting our conclusion that protective capacity is governed primarily by surface density and accessibility at any site of membrane apposition, rather than by precise subcellular positioning.

These observations suggest that physically recruiting the fusion inhibitor to the co-receptor site does not confer additional antiviral benefit under the conditions tested. Taken together, these data support our conclusion that the protective capacity of the construct is determined primarily by its surface density and accessibility within any zone of membrane apposition, rather than by its precise subcellular localization.

We have expanded the discussion of these points in the revised manuscript (lines 637-656).

With sincere gratitude,

Alexandra K. Maslennikova, PhD

On behalf of all authors

Reviewer 3 Report

Comments and Suggestions for Authors

Journal: Cells (ISSN 2073-4409), Manuscript ID: cells-4540839

Type: Article, Title: Bicistronic Lentiviral Architecture Increases Surface Density of Membrane-Associated HIV Entry Inhibitors and Confers Tropism-Independent Protection to Modified Cells

Authors: Yaroslava V. Chervyakova , ... , Alexandra Konstantia Y. Maslennikova *

 

This manuscript investigates the anti‑HIV effects, including protection against viral infection, using cells engineered to express several HIV fusion‑inhibitory peptide fusion proteins through vector‑based expression systems. Although the conceptual framework is not entirely novel, the authors successfully obtain diverse insights by optimizing the expression system, and they demonstrate that these engineered cells can be used safely and stably at least in vitro. The interpretation of the results presented on pages 7–15 appears reasonable. The authors show that tandem expression of fusion proteins from a single ORF increases their surface density and results in additive anti‑HIV activity, taking receptor tropism into account. However, synergistic effects are not explained. The manuscript also partially examines the influence of promoters, expression control, and GPI anchoring.

This reviewer finds no major deficiencies in the manuscript. With the addition of brief clarifications on the points below, this reviewer considers the manuscript suitable for publication in Cells.

Minor points

1. Although this journal covers a broad range of biological topics, general readers may require short explanatory notes for the following terms. The journal is not specialized in retroviral infections.
   - L63: Please clarify what gp41 is, where it is located, and what function it serves.
   - L66–68: Provide brief information on the origin, sequence, and differences among T20, MT‑C34, and 2P23.
   - 2A: JRFL and ZM‑135 are CCR5‑tropic, and NL4‑3 is CXCR4‑tropic. These points should also be stated in the text (around L377) or in the figure legend.
   - L240: TCID50 has not been explained previously in the manuscript.
   - L311: Please provide a short explanation of the WPRE cassette (Woodchuck hepatitis virus posttranscriptional regulatory element).
2. L291–301: Please elaborate on the rationale behind the construct design. Specifically, explain why the authors selected the central region of CD52 as the insertion site, and how structural features of CD52 make this region suitable for presenting the inserted C‑peptide.

Author Response

Dear Reviewer,

We thank you very much for the careful reading of our manuscript and for the constructive comments that helped us improve the clarity and accessibility of the work. We appreciate your positive assessment of the study's contribution and the thoughtful suggestions regarding terminology and construct design rationale. Below we provide point-by-point responses to each comment.

Reviewer's remark: "The authors show that tandem expression of fusion proteins from a single ORF increases their surface density and results in additive anti‑HIV activity, taking receptor tropism into account. However, synergistic effects are not explained."

Response: We thank the reviewer for drawing attention to this important nuance. In the revised manuscript, we have added a dedicated paragraph to the Discussion section that clarifies the mechanistic basis of the enhanced protection conferred by bicistronic constructs. Specifically, we now explain that co-expression of two structurally distinct C-peptides establishes a dual-barrier architecture that substantially raises the mutational threshold required for viral escape. Because envelope mutations conferring resistance to one inhibitor typically do not compromise binding to the other, HIV must simultaneously acquire non-overlapping gp41 modifications to overcome protection — a markedly higher genetic burden than that imposed by any single-peptide construct. This principle parallels the clinical rationale underlying combination antiretroviral therapy and is expected to delay the emergence of resistant variants, including those selected under pressure from soluble fusion inhibitors such as enfuvirtide. (L657-665)

Comment 1: Although this journal covers a broad range of biological topics, general readers may require short explanatory notes for the following terms. The journal is not specialized in retroviral infections.

Response: We fully agree with the reviewer that concise explanations would benefit the broader readership of Cells. We have revised the manuscript accordingly:

Comment 1.1: Please clarify what gp41 is, where it is located, and what function it serves.

Response: We have added a brief description specifying that gp41 is the transmembrane subunit of the HIV envelope glycoprotein complex (Env), which together with gp120 mediates virus–host cell attachment and subsequent fusion of the viral envelope with the host cell plasma membrane. Binding to the receptor (CD4) and coreceptor (CCR5 or CXCR4) is mediated primarily by gp120, whereas gp41 drives the membrane fusion step itself. (L64-69)

Comment 1.2: Provide brief information on the origin, sequence, and differences among T20, MT‑C34, and 2P23.

Response: We have expanded the introduction to clarify that all three peptides are derived from the heptad repeat 2 (HR2) domain of gp41. Structurally, T20 comprises the full-length 36-amino-acid HR2 sequence; MT-C34 is a C34 variant bearing an N-terminal M-T hook (Met-Thr) that caps the deep hydrophobic pocket on the gp41 NHR trimer and stabilizes inhibitor binding; and 2P23 is a synthetic 23-residue peptide designed based on the M-T hook structure, whose shortened length confers enhanced proteolytic stability and improved membrane accessibility when displayed within GPI-anchored scaffolds. An alignment of the three sequences has been included as Supplementary Figure S1. (L69–83)

Comment 1.3: JRFL and ZM‑135 are CCR5‑tropic, and NL4‑3 is CXCR4‑tropic. These points should also be stated in the text (around L377) or in the figure legend.

Response: We have explicitly stated in both the main text and the figure legend that JRFL and ZM-135 are CCR5-tropic strains, whereas NL4-3 is CXCR4-tropic. This clarification now appears consistently throughout the relevant passages. (L401-402)

Comment 1.4: TCID50 has not been explained previously in the manuscript.

Response: We have added a definition at first mention: "TCID₅₀ (tissue culture infectious dose 50%) denotes the amount of virus required to produce detectable infection in 50% of target cultures."  (L260-261)

Comment 1.5:  Please provide a short explanation of the WPRE cassette (Woodchuck hepatitis virus posttranscriptional regulatory element).

Response: We have provided a brief explanation noting that WPRE is a cis-acting RNA element derived from Woodchuck hepatitis virus that increases mRNA stability and translational efficiency across diverse cell types. We also clarified that we used the oncogenicity-reduced variant, in which sequence elements associated with residual transforming potential have been mutated while the RNA-stabilizing and translational-enhancing functions are preserved. (L144-150)

Comment 2: Please elaborate on the rationale behind the construct design. Specifically, explain why the authors selected the central region of CD52 as the insertion site, and how structural features of CD52 make this region suitable for presenting the inserted C-peptide.

Response: We thank the reviewer for this important suggestion. We have expanded the Results section to explain that CD52 was chosen as the minimal GPI-anchored scaffold because it provides the optimal balance between compact size and robust surface expression among all tested GPI proteins, and, being a native human protein, is not expected to add immunogenicity. The C-peptide was substituted for the central region of CD52, positioned between the N-glycosylation site and the GPI-attachment signal. Substitution at this position preserves efficient export through the secretory pathway and membrane anchoring while displaying the inhibitory peptide in a conformation accessible for engagement with gp41. (L326–332)

We appreciate the opportunity to improve our manuscript and hope that these revisions adequately address your concerns.

With sincere gratitude,

Alexandra K. Maslennikova, PhD

On behalf of all authors

Author Response File: Author Response.pdf