Abstract
Despite their worldwide prevalence and association with human disease, the molecular bases of human as-trovirus (HAstV) infection and evolution remain poorly characterized. Here, we report the structure of the capsid protein spike of the divergent HAstV MLB clade (HAstV MLB). While the structure shares a similar folding topology with that of classical-clade HAstV spikes, it is otherwise strikingly different. We find no ev-idence of a conserved receptor-binding site between the MLB and classical HAstV spikes, suggesting that MLB and classical HAstVs utilize different receptors for host-cell attachment. We provide evidence for this hypothesis using a novel HAstV infection competition assay. Comparisons of the HAstV MLB spike structure with structures predicted from its sequence reveal poor matches, but template-based predictions were sur-prisingly accurate relative to machine-learning-based predictions. Our data provide a foundation for under-standing the mechanisms of infection by diverse HAstVs and can support structure determination in similarly unstudied systems.