Enterovirus A71 adaptation to heparan sulfate comes with capsid stability tradeoff

Because of high mutation rates, viruses constantly adapt to new environments. When propagated in cell lines, certain viruses acquire positively charged amino acids on their surface proteins, enabling them to utilize negatively charged heparan sulfate (HS) as an attachment receptor. In this study, we used enterovirus A71 (EV-A71) as model and demonstrated that unlike the parental MP4 variant, the cell-adapted strong HS-binder MP4-97R/167G does not require acidification for uncoating and releases its genome in the neutral or weakly acidic environment of early endosomes. We experimentally confirmed that this pH-independent entry is not associated with the use of HS as an attachment receptor but rather with compromised capsid stability. We then extended these findings to another HS-dependent strain, suggesting that adaptation to HS generally modifies capsid stability and alters entry mechanism. Our data show EV-A71 pH-independent entry for the first time and, more importantly, highlight the intricate interplay between HS-binding, capsid stability, and viral fitness, wherein enhanced multiplication in cell lines leads to attenuation in hostilein vivoenvironments such as the gastrointestinal tract.