The 5'-end segment-specific noncoding region of influenza A virus regulates both competitive multi-segment RNA transcription and selective genome packaging during infection.

The 3' and 5' termini of the eight RNA segments of the influenza A virus (IAV) genome contain segment-specific or subtype-specific (in HA and NA segments) noncoding regions (ssNCRs), located between the highly conserved terminal promoter sequences and the coding start or stop codons. These ssNCRs vary significantly in both length and sequence among segments and have been implicated in selective genome packaging. Beyond their established role in packaging, we previously demonstrated that the 3'-end H1-ssNCR, together with its adjacent coding region, supports optimal replication of HA viral RNA (vRNA) in a multi-segment context. In this study, we discovered that truncation of the 5'-end H1-ssNCR, unlike its 3' counterpart, resulted in a reduction in HA mRNA levels in a template-competitive manner and impaired incorporation of HA vRNA into progeny virions. Interestingly, upon passaging of a recombinant virus carrying the most severely truncated 5'-end H1-ssNCR, an adaptive mutation occurred seven nucleotides upstream of the truncation site. This mutation restored both HA mRNA expression and HA vRNA packaging, thereby rescuing viral replication. Further analysis revealed that these regulatory effects may be mediated by RNA secondary structures formed around the 5'-end H1-ssNCR. Collectively, our findings reveal, for the first time, a dual regulatory role of the 5'-end segment-specific noncoding regions of influenza A virus RNA segments in modulating multi-segmental RNA transcription and selective genome packaging during infection.IMPORTANCEThe 3' and 5' segment-specific noncoding regions (ssNCRs) of influenza A virus (IAV) have long been recognized as critical signals for selective genome packaging. However, their potential roles in other regulatory processes remain largely unexplored. We previously reported that the 3'-end H1-ssNCR, together with its adjacent coding region, primarily determines optimal HA vRNA accumulation in a multi-segment environment. In this study, we further demonstrate that the 5'-end ssNCR plays a dual role in regulating viral RNA transcription in a template-competitive manner and governing vRNA incorporation into progeny virions. These findings reveal previously underappreciated levels of complexity, highlighting that ssNCRs contribute not only to genome packaging but also to the fine-tuning of viral RNA synthesis in multi-segmented negative-strand RNA viruses.