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

Influenza A virus (IAV) noncanonical RNAs are bound by retinoic acid-inducible gene I (RIG-I). However, innate immune activation is infrequent and it is not understood why noncanonical IAV RNAs activate RIG-I in a sequence- or RNA structure-dependent manner. We hypothesized that multiple events need to occur before IAV RNA synthesis activates RIG-I and investigated whether RIG-I activation is stimulated by the noncanonical or aberrant transcription of mini viral RNAs (mvRNA), an RNA that is overexpressed in highly pathogenic IAV infections. We find that mvRNAs can cause noncanonical transcription termination through a truncated 5' polyadenylation signal or a 5' transient RNA structure that interrupts polyadenylation. The resulting capped complementary RNAs stimulate the release of an mvRNA and complement RIG-I activation in trans. Overall, our findings indicate that sequential rounds of noncanonical or aberrant viral replication and transcription are needed for innate immune signaling by IAV RNA synthesis.