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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 27, 2024 |
| Title |
Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection. |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
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 TBC1D24 and SV2B promote 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.
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| Overall design |
Genome-wide and follow-up targeted CRISPR screens were performed on K562 cells (Cat #CCL-243, ATCC) mutagenized with either the human GeCKO v2 library (#1000000048 and #1000000049, Addgene) or a custom sub-library targeting the 500 highest-ranking candidates from the genome-wide screen, respectively. For the latter, we designed six guides per gene plus 200 non-targeting controls (NTC) using CHOPCHOP and Guides [PMIDs 31106371, 28858339]. The genome-wide screen was performed once in two technical replicates (one for each GeCKO v2 library A or B) and targeted screens were performed in biological triplicate using three independently mutagenized libraries. Following each screen, genomic DNA was isolated using the QIAamp DNA Blood Mini Kit (Cat #51185, Qiagen), and sgRNA sequences were amplified and prepared for next-generation sequencing (Illumina). The enrichment of each sgRNA in the selected cells relative to the unselected libraries cultured and harvested in parallel was calculated using MAGeCK [PMID: 25476604].
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| Web link |
https://journals.asm.org/doi/10.1128/jvi.01582-24
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| Contributor(s) |
Belmont L, Contreras M, Cartwright-Acar CH, Marceau CD, Agrawal A, Levoir LM, Lubow J, Goo L |
| Citation(s) |
39377586 |
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| Submission date |
Apr 23, 2024 |
| Last update date |
Oct 22, 2024 |
| Contact name |
Laura Belmont |
| Organization name |
Fred Hutchinson Cancer Research Center
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| Street address |
1100 Fairview Ave N
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| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platforms (2) |
| GPL15520 |
Illumina MiSeq (Homo sapiens) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA1103674 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE264625_gecko_WG_counts.tsv.gz |
2.5 Mb |
(ftp)(http) |
TSV |
| GSE264625_targeted_counts.tsv.gz |
55.9 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
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