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Review 2: "Functional Genomics Screens Reveal a Role for TBC1D24 and SV2B in Antibody-dependent Enhancement of Dengue Virus Infection"

Reviewers found the preprint to be potentially informative, with evidence suggesting a role for both TBC1D24 and SV2B in increasing antibody-dependent enhancement (ADE) for Dengue virus (DENV) infection.

Published onJun 18, 2024
Review 2: "Functional Genomics Screens Reveal a Role for TBC1D24 and SV2B in Antibody-dependent Enhancement of Dengue Virus Infection"
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key-enterThis Pub is a Review of
Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection
Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection

Abstract Dengue virus (DENV) can hijack non-neutralizing IgG antibodies to facilitate its uptake into target cells expressing Fc gamma receptors (FcgR) - a process known as antibody-dependent enhancement (ADE) of infection. Beyond a requirement for FcgR, host dependency factors for this non-canonical infection route remain unknown. To identify cellular factors exclusively required for ADE, here, we performed CRISPR knockout screens in an in vitro system permissive to infection only in the presence of IgG antibodies. Validating our approach, a top hit was FcgRIIa, which facilitates binding and internalization of IgG-bound DENV but is not required for canonical infection. Additionally, we identified host factors with no previously described role in DENV infection, including TBC1D24 and SV2B, both of which have known functions in regulated secretion. Using genetic knockout and trans-complemented cells, we validated a functional requirement for these host factors in ADE assays performed with monoclonal antibodies and polyclonal sera in multiple cell lines and using all four DENV serotypes. We show that knockout of TBC1D24 or SV2B impaired binding of IgG-DENV complexes to cells without affecting FcgRIIa expression levels. Thus, we identify cellular factors beyond FcgR that are required for ADE of DENV infection. Our findings represent a first step towards advancing fundamental knowledge behind the biology of ADE that can ultimately be exploited to inform vaccination and therapeutic approaches.

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.


Review: This is an interesting paper that looks to identify host factors responsible for DENV ADE, beyond the FcgR. The authors utilise K562 cell line, whose infection with DENV is enhanced in the presence of antibody, and a CRISPR and secondary targeted screen to select for non-infected cells following gene KO. The screen identifies several genes, and the authors select two genes, TBC1D24 and SV2B for further study. The authors show that KO of these genes reduced DENV-ADE and that this is reversed by adding back TBC1D24 or SV2B. The outcomes are suggestive for a role of TBC1D24 and SV2B in DENV infection, the binding analysis suggests that this is at the binding and entry stage, and the conclusions are largely substantiated. The conclusion, however, would be more compelling if impact of the actual KO was shown, if the supplementary data was available for viewing, and if some primary data visualising the infected cells was shown to support changes in % infection. Specific comments that would strengthen the substantiation of the conclusions are expanded on below


  1. The initial ADE for the CRISPR screen is not well designed. mouse IgG antibody is used by the authors to complex with DENV. However, mouse IgG reportedly binds poorly to human FcgR (DOI: 10.1016/j.molimm.2020.08.015), so the infection would be expected to not be very efficient. Given the selection is a negative ‘survive’ infection, it is hard to know how many of the resultant hits reflect cells that missed getting infection, due to low efficiency or due to the KO phenotype. Some indication of the % infection in these screens with WT cells would be useful to add to strengthen confidence in this data.

  2. The supplementary files were not available for review. Thus, it was not possible to review evidence of KO cells, and evidence of dose response to ADE KO. It would be useful to critique what fold increase is afforded by ADE of infection. Additionally, it would be useful to have data validating that these KO clones still have comparable growth characteristics, and that the reduced infection is not associated with a lower rate of cell growth.

  3. Discussion – is focused on neuronal proteins, and the results suggest several neuronal proteins were identified in the screen. I am not sure about the biological relevance of this?

  4. Fig 1 – selected genes were identified based on ranking, and comparison to MAGeCK score cut off for known DENV targets? Not sure how these are determined to be significantly different, or of the rigour around this analysis – eg. values for n for sequencing – I assume it is just one DENV and one DENV-ADE? Is there also a comparator for uninfected cells (relates to the first comment about % infection).

  5. siRNA knockdown in primary mphage +/- ADE would be informative. The authors state that these experiments were not able to be done with CRISPR KO and also not able to demonstrate WB KO of protein. I feel this is a drawback – perhaps siRNA knockdown in mphage could be undertaken, some support for impact on the gene of interest (RT-PCR data? Immunostaining in situ? Functional change in endocytic pathway?

  6. These findings would be more compelling if some microscopy was shown, as a secondary measure to support the flow cytometry, and demonstrating the % of infected cells in the KO vs WT ADE-DENV-infected lines.


  1. Not correct that K562 and U937 are infectable only in the presence of ADE – there is some infection and it is certainly enhanced with antibody

  2. It would be interesting to look at FcgRIIa levels on the cell surface after ADE-DENV infection in the two KO lines.

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