Aguilera-Flores Catalina, Valencia-Morales María Del Pilar, López Tomás, Moreno-Contreras Joaquín, Lentz Adam, Espinoza Marco A, DuBois Rebecca M, López Susana, Arias Carlos F
Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico.
Department of Biomolecular Engineering, University of California, Santa Cruz, California, United States of America.
PLoS Pathog. 2025 Jul 18;21(7):e1013316. doi: 10.1371/journal.ppat.1013316. eCollection 2025 Jul.
Classical human astroviruses (HAstV) are a global cause of viral gastroenteritis, particularly in children and immunocompromised individuals. Despite their clinical significance, the biology of HAstV remains poorly understood. In particular, the identification of cellular receptors and coreceptors has been elusive. Recent studies have identified the human neonatal Fc receptor (FcRn) as a functional receptor and dipeptidyl peptidase IV (DPP4) as an entry factor for HAstV. However, the precise roles of FcRn and DPP4 during HAstV infection are unknown. To learn about their function, we used FcRn-knockout (KO), DPP4-KO, and FcRn/DPP4 double-KO Caco-2 cells generated via CRISPR/Cas9. Our results showed that DPP4 serves as the receptor for classical HAstV. In contrast, infectious virus assays and confocal fluorescence microscopy revealed that FcRn acts as a coreceptor, facilitating viral internalization and the release of the RNA genome. The half-time for HAstV-1 genome uncoating was delayed threefold in FcRn-KO Caco-2 cells compared to WT cells. Additionally, the characterization of HAstV-8 variants with reduced FcRn binding capacity allowed the identification of two amino acids in the viral capsid spike protein, D471 and N512, critical for the spike-FcRn interaction. These amino acid residues are part of the epitope footprint of neutralizing monoclonal antibodies (Nt-MAbs) to HAstV previously mapped by X-ray crystallography. Further experiments using virus infectivity and attachment assays, along with Nt-MAbs targeting HAstV-1, suggest that the binding sites for FcRn and DPP4 are spatially proximal on the viral spike, defining a functional domain for cell infection. Notably, the infectivity of the divergent HAstV-VA1 was independent of these two proteins, highlighting the receptor variability across HAstV clades. These findings provide new insights into the mechanism of HAstV infection, offering relevant implications for the development of antiviral therapies and vaccines targeting this significant human pathogen.
经典人类星状病毒（HAstV）是引起病毒性肠胃炎的一个全球性病因，在儿童和免疫功能低下的个体中尤为常见。尽管其具有临床重要性，但人们对HAstV的生物学特性仍知之甚少。特别是，细胞受体和共受体的鉴定一直难以实现。最近的研究已将人类新生儿Fc受体（FcRn）鉴定为功能性受体，并将二肽基肽酶IV（DPP4）鉴定为HAstV的一个进入因子。然而，FcRn和DPP4在HAstV感染过程中的精确作用尚不清楚。为了解它们的功能，我们使用了通过CRISPR/Cas9技术构建的FcRn基因敲除（KO）、DPP4基因敲除和FcRn/DPP4双基因敲除的Caco-2细胞。我们的结果表明，DPP4是经典HAstV的受体。相比之下，感染性病毒检测和共聚焦荧光显微镜显示，FcRn作为共受体，促进病毒内化和RNA基因组的释放。与野生型（WT）细胞相比，在FcRn基因敲除的Caco-2细胞中，HAstV-1基因组脱壳的半衰期延迟了三倍。此外，对FcRn结合能力降低的HAstV-8变体的特性分析，使得能够鉴定出病毒衣壳刺突蛋白中的两个氨基酸D471和N512，它们对刺突蛋白与FcRn的相互作用至关重要。这些氨基酸残基是先前通过X射线晶体学绘制的针对HAstV的中和单克隆抗体（Nt-MAbs）表位足迹的一部分。使用病毒感染性和附着检测以及靶向HAstV-1的Nt-MAbs进行的进一步实验表明，FcRn和DPP4的结合位点在病毒刺突上在空间上是近端的，从而确定了细胞感染功能域。值得注意的是，不同的HAstV-VA1的感染性与这两种蛋白无关，这突出了HAstV各进化枝间受体的变异性。这些发现为HAstV感染机制提供了新的见解，对针对这种重要人类病原体的抗病毒疗法和疫苗的开发具有相关意义。
