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.