Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection.

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. 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].

登革病毒（Dengue virus, DENV）可劫持非中和性IgG抗体，促进其被表达Fcγ受体（Fc gamma receptors, FcγR）的靶细胞摄取——这一过程被称为感染的抗体依赖性增强（antibody-dependent enhancement, ADE）。除FcγR的介导作用外，该非经典感染途径的宿主依赖因子仍未明确。为鉴定ADE专属必需的细胞因子，本研究在仅在IgG抗体存在时才允许感染的体外系统中开展了CRISPR敲除筛选（CRISPR knockout screen）。实验验证显示，排名靠前的候选因子为FcγRIIa，其可介导结合IgG的登革病毒的结合与内化，但对于经典感染并非必需。此外，我们还鉴定出此前未被报道参与登革病毒感染的宿主因子，包括TBC1D24与SV2B，二者均在调控性分泌中具有已知功能。通过基因敲除与反式互补细胞（trans-complemented cells）实验，我们在多种细胞系中，使用单克隆抗体及多克隆血清、针对全部4种登革病毒血清型开展的ADE实验中，验证了这些宿主因子的功能必要性。研究证实，TBC1D24与SV2B可促进IgG-登革病毒复合物与细胞的结合，且不影响FcγRIIa的表达水平。据此，我们鉴定出除FcγR之外，登革病毒感染ADE所必需的细胞因子。本研究结果为推进ADE生物学的基础认知迈出了第一步，这些认知最终可用于指导疫苗开发与治疗策略。全基因组筛选及后续靶向筛选均以K562细胞（货号CCL-243，美国典型培养物保藏中心ATCC）为研究对象，分别采用人类GeCKO v2文库（#1000000048与#1000000049，Addgene），或自定义子文库（靶向全基因组筛选中排名前500的候选基因）对细胞进行诱变。其中，自定义子文库通过CHOPCHOP与Guides工具（文献PMIDs 31106371、28858339）为每个基因设计6条向导RNA（small guide RNA, sgRNA），并设置200条非靶向对照（non-targeting controls, NTC）。全基因组筛选仅开展1次，设置2个技术重复（分别对应GeCKO v2文库A与B）；靶向筛选则使用3批独立诱变的文库开展3次生物学重复。每轮筛选结束后，使用QIAamp DNA Blood Mini Kit（货号51185，Qiagen）提取基因组DNA，扩增sgRNA序列并制备用于下一代测序（Illumina）。通过MAGeCK工具（文献PMID: 25476604），计算筛选获得的细胞中每条sgRNA相对于平行培养并收获的未筛选文库的富集程度。