Influenza A virus (IAV) selectively packages eight distinct viral RNA (vRNA) segments into progeny virions through segment-specific packaging signals located in the noncoding regions and their adjacent coding sequences. Emerging evidence indicates that local secondary structures within these regions are involved in intersegment RNA-RNA interactions required for selective genome incorporation. Here, we examined a structured motif in the 3´ coding region of the WSN HA vRNA by introducing synonymous mutations at two cytidines (C1720 and C1726), which were predicted to convert an intrinsic bulge into a stem-loop structure. Although viral protein expression and hemagglutination activity remained comparable to the wild type virus, the mutant virus (WSN-vHA) displayed reduced plaque size, decreased viral titer and a significantly lower PFU/HA ratio. Quantification of vRNAs in released viral particles showed disrupted stoichiometry of segment assembly, and electron microscopy confirmed the presence of empty or vRNP-deficient particles. WSN-vHA also exhibited significantly attenuated replication and pathogenicity in mice. Together, these findings identified two critical nucleotide sites in the WSN HA segment for selective genome incorporation and virion assembly, highlighting the importance of local RNA structural integrity in IAV replication and virulence.