Structural and functional definition of human astrovirus-receptor interactions - PROJECT SUMMARY/ABSTRACT Human astroviruses (HAstVs) are a global cause of pediatric gastroenteritis, and can cause disseminated infection in immunocompromised hosts. Seroprevalence studies indicate almost universal HAstV infection during childhood. Despite their clinical importance, HAstVs are highly understudied. Little is known about the host factors needed for infection or their mechanisms of viral entry into cells, and no proteinaceous viral receptor has been identified to date. Beyond providing critical insights into virus-host interactions, identification of host proteins sufficient and/or necessary for HAstV infection is key for creating small animal models permissive to infection and to revealing antiviral strategies to combat infection. Leveraging both genome-wide knock-out CRISPR and surfaceome-specific CRISPR activation screening, novel proteinaceous host receptors for HAstV were identified. These factors, neonatal Fc receptor (FcRn) and dipeptidyl peptidase 4 (DPP4), have also been implicated in viral infection for echovirus and coronavirus infection respectively. Both were confirmed as necessary for in vitro HAstV infection of Caco2 cells and as sufficient to permit HAstV infection of normally non-permissive human cells when overexpressed. Additionally, use of biolayer interferometry confirmed direct physical interactions between both FcRn and DPP4 with the HAstV capsid. These preliminary data serve as the critical foundation for the extensive structural and functional studies proposed here. In Aim 1, the specific protein domains and amino acid residues of FcRn and DPP4 that functionally interact with the HAstV capsid will be identified, as will the viral capsid residues critical for binding these host factors. Cryogenic electron microscopy will be leveraged to reveal how binding to these factors affects the HAstV virion. These genetic and biophysical analyses will provide much-needed insight into HAstV entry biology and form the basis of future pharmacological screens for identification of entry inhibitors. In Aim 2, permissive human intestinal enteroid cultures will be used to determine the necessity of identified entry factors for ex vivo HAstV infection. Blocking of infection via addition of soluble entry factors, anti-factor antibodies, inhibitors, and CRISPR-mediated disruption of factors in these cultures will be performed. Additionally, existing mouse models expressing human FcRn and DPP4 will be used to develop mice permissive to HAstV infection and to evaluate whether these factors are critical for murine astrovirus infection. These studies will reveal the species-specificity of receptor use and provide powerful new tools for study of HAstV pathogenesis and immune response as well as for preclinical testing of vaccines and antivirals. The use of numerous orthogonal approaches, including mechanistic studies of critical receptor domains, state-of-the-art structural techniques, and physiologically-relevant models, will yield a comprehensive picture of how these novel entry factors promote HAstV infection.
