Structural Basis of Immunostimulatory Potential in Influenza A Mini Viral RNAs: Analysis of Segment 5

Abstract

Influenza A virus replication produces a range of aberrant RNA species, including mini viral RNAs (mvRNAs), which can activate innate immune signaling through the RIG-I pathway. Although mvRNAs are known to stimulate IFN-β expression, it remains unclear why some are strongly immunostimulatory while others appear largely inactive. Clarifying what distinguishes these RNAs could help explain how replication errors shape host antiviral responses. To address this, this study examined a panel of segment 5-derived mvRNAs and asked whether differences in IFN-β activation could be explained by RNA length, predicted structural features, or RNA abundance. mvRNA expression constructs were generated and tested for their ability to activate IFN-β signaling. While very short mvRNAs generally produced weak activation, constructs of similar length often showed markedly different levels of IFN-β induction, indicating that size alone is not sufficient to explain functional differences. Comparisons with predicted RNA folding stability revealed a similarly weak relationship, as mvRNAs with comparable free energy values frequently produced different responses. Primer extension analysis was used to estimate relative mvRNA levels and assess whether these differences could be explained by RNA abundance. Although mvRNA accumulation varied considerably, no clear relationship was observed between abundance and IFN-β activation, with several low-abundance mvRNAs still inducing strong responses. Overall, these findings suggest that mvRNA immunostimulatory potential cannot be explained by any single feature in isolation, but instead reflects the combined influence of multiple properties. By examining a broader range of mvRNAs than typically studied, this work provides a more nuanced view of how variation among mvRNAs may influence the host response to infection.