Mutation of the highly conserved amino acids in the N-terminal region of the EV-A71 VP1 protein impairs viral RNA release during virus entry.

Enterovirus A71 (EV-A71) causes hand-foot-mouth disease, which may progress to severe neurological and systemic complications in young children and infants. To deliver viral genomic RNA into the target cell for replication, the mature EV-A71 capsid particle undergoes drastic structural changes to become an expanded A particle, which is marked by the movement of the N-terminal region of viral protein VP1 from the interior to the capsid surface and the dislodgement of the pocket factor. It is not completely understood which amino acids support these key activities of VP1 during EV-A71 entry. To answer this question, we investigated the functions of amino acids that are highly conserved in VP1 across all genotypes of EV-A71 by performing reverse genetic mutagenesis and functional experiments. Among the 21 highly conserved amino acids, 20 affect the replication of EV-A71. Notably, 7 of the 21 highly conserved amino acids are located in the 71-amino-acid N-terminal region, and mutation of any of these seven amino acids is lethal to EV-A71. Further mechanistic studies showed that the most detrimental defect occurred at the step of viral capsid uncoating and viral RNA release during virus entry, which is supported by the results of structural analyses showing the disruption of complex hydrogen bonding and hydrophobic networking due to mutating these amino acids. Our data have thus unraveled the genetic determinants that govern the key function of the N-terminal region of VP1 during EV-A71 entry and viral RNA release. IMPORTANCE: Hand, foot, and mouth disease (HFMD) annually affects millions of children worldwide. EV-A71 is a major cause of HFMD in the Asia-Pacific region. Notably, EV-A71 infection can lead to severe neurological syndromes and even death in young children; however, no specific vaccines or drugs are available. Among picornavirus capsid proteins, VP1 is the most external, surface-accessible, and immunodominant protein. However, it is not completely understood which amino acids support these key activities of VP1 during EV-A71 entry. Here, we report that 7 of the 21 highly conserved amino acids within the 71-amino-acid N-terminal region of VP1, and mutation of any of these 7 amino acids is lethal to EV-A71 by disrupting capsid uncoating and RNA release during entry. Overall, our findings highlight that the highly conserved N-terminal region residues of VP1 play a crucial role in viral infectivity and may contribute to the development of broad-spectrum anti-EV-A71 vaccines and drugs.