Functional characterization of DPP4 and FcRn as receptor and coreceptor for classical human astroviruses in Caco-2 cells

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.