Discovery of a Heparan-Sulfate Binding Domain in Monkeypox Virus H3 Protein as a New Anti-poxviral Drug Target

Reviewer #1 (Public Review):

Summary:

The study aimed to better understand the role of the H3 protein of the Monkeypox virus (MPXV) in host cell adhesion, identifying a crucial α-helical domain for interaction with heparan sulfate (HS). Using a combination of advanced computational simulations and experimental validations, the authors discovered that this domain is essential for viral adhesion and potentially a new target for developing antiviral therapies.

Strengths:

The study's main strengths include the use of cutting-edge computational tools such as AlphaFold2 and molecular dynamics simulations, combined with robust experimental techniques like single-molecule force spectroscopy and flow cytometry. These methods provided a detailed and reliable view of the interactions between the H3 protein and HS. The study also highlighted the importance of the α-helical domain's electric charge and the influence of the Mg(II) ion in stabilizing this interaction. The work's impact on the field is significant, offering new perspectives for developing antiviral treatments for MPXV and potentially other viruses with similar adhesion mechanisms. The provided methods and data are highly useful for researchers working with viral proteins and protein-polysaccharide interactions, offering a solid foundation for future investigations and therapeutic innovations.

Weaknesses:

However, some limitations are notable. Despite the robust use of computational methodologies, the limitations of this approach are not discussed, such as potential sources of error, standard deviation rates, and known controls for the H3 protein to justify the claims. Additionally, validations with methodologies like X-ray crystallography would further benefit the visualization of the H3 and HS interaction.

Reviewer #2 (Public Review):

Summary:

The manuscript presenting the discovery of a heparan-sulfate (HS) binding domain in monkeypox virus (MPXV) H3 protein as a new anti-poxviral drug target, presented by Bin Zhen and co-workers, is of interest, given that it offers a potentially broad antiviral substance to be used against poxviruses. Using new computational biology techniques, the authors identified a new alpha-helical domain in the H3 protein, which interacts with cell surface HS, and this domain seems to be crucial for H3-HS interaction. Given that this domain is conserved across orthopoxviruses, authors designed protein inhibitors. One of these inhibitors, AI-PoxBlock723, effectively disrupted the H3-HS interaction and inhibited infection with Monkeypox virus and Vaccinia virus. The presented data should be of interest to a diverse audience, given the possibility of an effective anti-poxviral drug.

Strengths:

In my opinion, the experiments done in this work were well-planned and executed. The authors put together several computational methods, to design poxvirus inhibitor molecules, and then they test these molecules for infection inhibition.

Weaknesses:

One thing that could be improved, is the presentation of results, to make them more easily understandable to readers, who may not be experts in protein modeling programs. For example, figures should be self-explanatory and understood on their own, without the need to revise text. Therefore, the figure legend should be more informative as to how the experiments were done.

Reviewer #3 (Public Review):

Summary:

The article is an interesting approach to determining the MPOX receptor using "in silico" tools. The results show the presence of two regions of the H3 protein with a high probability of being involved in the interaction with the HS cell receptor. However, the α-helical region seems to be the most probable, since modifications in this region affect the virus binding to the HS receptor.

Strengths:

In my opinion, it is an informative article with interesting results, generated by a combination of "in silico" and wet science to test the theoretical results. This is a strong point of the article.

Weaknesses:

Has a crystal structure of the H3 protein been reported?

The following text is in line 104: "which may represent a novel binding site for HS". It is unclear whether this means this "new binding site" is an alternative site to an old one or whether it is the true binding site that had not been previously elucidated.