An IRES-like cis-acting element located within the EV-A71 coding region drives translation independent of the 5'-IRES and modulates viral fitness through regulated binding of viral RNA to 3D polymerase.

Enterovirus A71 (EV-A71) is a leading cause of hand, foot, and mouth disease and is occasionally associated with severe neurological complications in young children. Previous studies of EV-A71 and related enteroviruses suggested that the virus possibly contains additional cis-acting elements (CAEs) within the major open reading frame (ORF) regulating viral protein translation. To systematically explore CAEs in the EV-A71 genome and characterize their functions, we utilized bicistronic reporter plasmids to screen the entire genome, identifying internal ribosome entry site (IRES)-like CAEs within the VP3-VP1 and 2A-2B coding regions. We demonstrate that EV-A71 translation can occur independently of the 5'-IRES and that the element within 2A-2B specifically facilitates this alternative initiation. Furthermore, the IRES-like CAE significantly modulates viral propagation by altering the expression of viral proteins including VP1 and 3D polymerase and shifting the ratio of positive-sense to negative-sense genomic RNA. Disruption of its RNA structure via synonymous mutations reduced viral genome replication, progeny production, and fitness. Mechanistically, these impairments correlated with decreased RNA binding to the viral 3D polymerase. These findings illuminate the multifaceted roles of coding-region IRES-like CAEs in viral infection and fitness, reveal a noncanonical mechanism for enteroviral protein synthesis independent of the 5'-IRES, and provide a framework to identify RNA structures and synonymous mutations for attenuated vaccine design and antiviral strategies.IMPORTANCEThe biological significance of cis-acting elements (CAEs) within viral coding regions, particularly their impact on infectivity and fitness, remains poorly understood. Here, we characterize an internal ribosome entry site (IRES)-like CAE in Enterovirus A71 (EV-A71), the causative agent of hand, foot, and mouth disease. Through genome-wide screening, we identified novel IRES-like CAEs and demonstrate that EV-A71 translation can proceed independently of the 5'-IRES, with the 2A-2B element specifically mediating this alternative initiation. The 2A-2B CAE and its associated RNA secondary structure are essential for viral infectivity and fitness. Synonymous mutations disrupting local stem-loop structures impair viral RNA binding to the 3D polymerase, resulting in reduced fitness. These findings reveal a noncanonical mechanism for IRES-independent enteroviral protein synthesis, elucidate how RNA structures and synonymous mutations in coding-region CAEs modulate viral replication, and inform attenuated vaccine design and antiviral strategies while revealing the evolutionary implications of these regulatory elements.