Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Center, Seattle, Washington, USA.
Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, Washington, USA.
J Virol. 2024 Nov 19;98(11):e0158224. doi: 10.1128/jvi.01582-24. Epub 2024 Oct 8.
Under some conditions, dengue virus (DENV) can hijack 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 unusual IgG-mediated infection route remain unknown. To identify cellular factors exclusively required for ADE, here, we performed CRISPR knockout (KO) screens in an system poorly permissive to infection in the absence of IgG antibodies. Validating our approach, a top hit was FcgRIIa, which facilitates the 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, which have known functions in regulated secretion. Using genetic knockout and -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 the binding of IgG-DENV complexes to cells without affecting FcgRIIa expression levels. Thus, we identify cellular factors beyond FcgR that promote efficient ADE of DENV infection. Our findings represent a first step toward advancing fundamental knowledge behind the biology of a non-canonical infection route implicated in disease.IMPORTANCEAntibodies can paradoxically enhance rather than inhibit dengue virus (DENV) infection in some cases. To advance knowledge of the functional requirements of antibody-dependent enhancement (ADE) of infection beyond existing descriptive studies, we performed a genome-scale CRISPR knockout (KO) screen in an optimized system permissive to efficient DENV infection only in the presence of IgG. In addition to FcgRIIa, a known receptor that facilitates IgG-mediated uptake of IgG-bound, but not naked DENV particles, our screens identified TBC1D24 and SV2B, cellular factors with no known role in DENV infection. We validated a functional role for TBC1D24 and SV2B in mediating ADE of all four DENV serotypes in different cell lines and using various antibodies. Thus, we identify cellular factors beyond Fc gamma receptors that promote ADE mechanisms. This study represents a first step toward advancing fundamental knowledge beyond a poorly understood non-canonical viral entry mechanism.
在某些条件下，登革热病毒（DENV）可以劫持 IgG 抗体，使其更容易进入表达 Fcγ受体（FcgR）的靶细胞——这一过程被称为感染的抗体依赖性增强（ADE）。除了需要 FcgR 之外，宿主对这种不寻常的 IgG 介导的感染途径的依赖性因素仍然未知。为了确定这种独特的 IgG 依赖性感染途径所需要的细胞因子，我们在此在一个在没有 IgG 抗体的情况下对感染的自然感染率很低的系统中进行了 CRISPR 敲除（KO）筛选。通过验证一种主要的命中物是 FcgRIIa，它促进了 IgG 结合的 DENV 的结合和内化，但对经典感染并不是必需的。此外，我们还鉴定了宿主因子，这些因子在 DENV 感染中没有先前描述的作用，包括 TBC1D24 和 SV2B，它们在调节分泌中具有已知的功能。使用遗传敲除和互补细胞，我们验证了这些宿主因子在使用单克隆抗体和多克隆血清在多种细胞系中进行的 ADE 测定中的功能需求，并且使用了所有四种 DENV 血清型。我们表明，TBC1D24 或 SV2B 的敲除会损害 IgG-DENV 复合物与细胞的结合，而不影响 FcgRIIa 的表达水平。因此，我们确定了除 FcgR 之外的促进 DENV 感染的有效 ADE 的细胞因子。我们的研究结果代表了在疾病中涉及的非经典感染途径的生物学背后的基本知识的一个进步。
在某些情况下，抗体可以促进而不是抑制登革热病毒（DENV）的感染。为了在现有描述性研究的基础上进一步了解抗体依赖性增强（ADE）感染的功能要求，我们在一个优化的系统中进行了全基因组 CRISPR 敲除（KO）筛选，该系统仅在存在 IgG 的情况下才允许高效感染 DENV。除了已知的受体 FcgRIIa 外，它促进 IgG 介导的 IgG 结合但不结合裸露的 DENV 颗粒的摄取，我们的筛选还鉴定了 TBC1D24 和 SV2B，它们是 DENV 感染中没有已知作用的细胞因子。我们验证了 TBC1D24 和 SV2B 在不同细胞系和使用各种抗体介导所有四种 DENV 血清型的 ADE 中的功能作用。因此，我们确定了除 Fcγ受体之外的促进 ADE 机制的细胞因子。这项研究代表了在一个理解甚少的非经典病毒进入机制之外推进基本知识的一个第一步。
