Abstract
Coxsackievirus B3 (CVB3), a member of the Enterovirus genus within the Picornaviridae family, has emerged as a key model for studying viral evolution and pathogenesis. Although traditionally considered obligate lytic viruses, recent research reveals that enteroviruses can also be released non-lytically within extracellular vesicles (EVs). This study explores the impact of mutations at position 63 of the VP3 capsid protein on CVB3 fitness and release mechanisms by substituting asparagine at this position with aromatic, charged, and aliphatic amino acids. We show that mutations at position 63 significantly affect viral release mechanisms and viral spread in cell culture. Specifically, aromatic mutations (N63H, N63Y, N63F, N63W) and the N63D mutation reduce the release of membrane-associated viral particles, while aromatic residues increase viral spread in cell culture and plaque size under specific conditions. These findings suggest that N63 mutations alter protomer interactions, influencing viral release, spread, and plaque formation, providing insights into the molecular mechanisms of CVB3 egress.