A two-step mechanism for RIG-I activation by influenza virus mvRNAs

Influenza A virus (IAV) non-canonical replication products can be bound by various host pathogen sensors, including retinoic acid-inducible gene I (RIG-I). However, it is not clear why non-canonical IAV RNAs activate RIG-I in a sequence- and RNA structure- dependent manner. Insight into this process is critical for understanding the exact molecular steps through which an influenza virus infection activates innate immune signaling and why differences exist in infection outcome. Here we investigated whether non-canonical or aberrant transcription of mini viral RNAs (mvRNA), a <125 nt long RNA whose expression is increased in pandemic and highly pathogenic IAV infections, contributes to RIG-I activation. We find that mvRNAs can cause non-canonical transcription termination through a truncated 5ʹ polyadenylation signal or a 5ʹ transient RNA structure that interrupts polyadenylation. The resulting capped complementary RNAs (ccRNA) can trigger the release of a template mvRNA and activate RIG-I when they form a heteroduplex with a negative sense RNA. Overall, our findings indicate that sequential rounds of non-canonical or aberrant viral replication and transcription explain the sequence-dependent efficiency with which mvRNAs trigger innate immune signaling.