Review 4: "Functional Genomics Screens Reveal a Role for TBC1D24 and SV2B in Antibody-dependent Enhancement of Dengue Virus Infection"

RR:C19 Evidence Scale rating by reviewer:

• Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review: The study “Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection” carried out by Belmont and group provides an insight into the molecular underpinnings of antibody-dependent enhancement (ADE) in dengue virus infection, a complex and well-documented phenomenon. The researchers conducted a high-throughput genetic knockout screen to explore the roles of the novel proteins TBC1D24 and SV2B, as well as FcγRIIa. They further investigated ADE using K562 and U937 cell lines. We have a few questions that might help clarify certain aspects of the study and enhance its readability.

• The literature suggests that the dengue virus (whether clinical or laboratory-adapted) can infect K562 cells even in the absence of IgG at an MOI of 1.

• The current study used a high MOI for dengue infection (MOI 24), which seems excessively high and may not accurately reflect physiological conditions in humans.

• For genomic screens related to antibody-dependent enhancement (ADE), it would be better to use U397 cells rather than K562 cells, as U397 cells express different receptors (FCγRI) and could provide more insightful results.

• Additionally, including a control scenario with virus infection in the absence of antibodies could offer better insights by comparing differentially expressed genes.

• Since ADE involves the enhancement of the virus due to antibody effects at various levels or concentrations of the same antibody, it may be interesting to examine genomics at the same concentration or dilution at which the virus is enhanced. Different host genes might be involved when virus infection levels are high.

• During the discussion, it was mentioned that HELZ2 has two isoforms. Are the identified genes (SV2B and TBC1D24) also isoform-dependent?

• In Figure 2B, the ADE efficiency in the SV2B knockout was not restored to the same level when trans-complemented with the same gene of interest, unlike in the TBC1D24 case. This could have been explained in the discussion.

• It was observed that HELZ2 had a high score in both genome-wide and targeted screenings, and its role in virus infection was discussed in the manuscript. The rationale for not investigating its further role in dengue ADE could have been discussed.

• The present study explored the association of TBC1D24 and SV2B with DENV ADE. However, these proteins also play crucial roles in synaptic vesicle trafficking in the brain. It would be valuable if the authors could study their role in ADE in primary cultures of immune cells involved in ADE to gain a clearer understanding of their physiological functions related to virus infection. If CRISPR knockout is not feasible in primary cultures, as discussed by the authors, a gene silencing approach could achieve similar effects.

• This study provides valuable insights into the involvement of TBC1D24 and SV2B in DENV ADE. It would be beneficial if the authors could investigate the protein expression profiles of TBC1D24 and SV2B in immune cells involved in ADE under normal physiological conditions and examine any changes in their expression during ADE. This could be performed in primary cultures and would offer a clearer understanding of their roles in ADE.