Schweibenz, B. | bioRxiv Science

bioRxiv2024

Influenza A virus transcription generates capped cRNAs that activate RIG-I

During influenza A virus (IAV) infection, host pathogen receptor retinoic acid-inducible gene I (RIG-I) detects the partially complementary, 5'-triphosphorylated ends of the viral genome segments and non-canonical replication products. However, it has also been suggested that innate immune responses may be triggered by viral transcription. In this study, we investigated whether an immunostimulatory RNA is produced during IAV transcription. We show that the IAV RNA polymerase can read though the polyadenylation signal during transcription termination, generating a capped complementary RNA (ccRNA), which contains the 5' cap of an IAV mRNA and the 3' terminus of a cRNA instead of a poly(A) tail. ccRNAs are detectable in vitro and in both ribonucleoprotein reconstitution assays and IAV infections. Mutations that disrupt polyadenylation enhance ccRNA synthesis and increase RIG-I-dependent innate immune activation. Notably, while ccRNA itself is not immunostimulatory, it forms a RIG-I agonist by hybridizing with a complementary negative-sense viral RNA. These findings thus identify a novel non-canonical IAV RNA species and suggest an alternative mechanism for RIG-I activation during IAV infection.

microbiology↗

bioRxiv2023

The NS1 protein of influenza B virus binds 5'-triphosphorylated dsRNA to suppress RIG-I activation and the antiviral innate immune response

Influenza A and B viruses overcome the host antiviral response to cause a contagious and often severe human respiratory disease. Here, integrative structural biology and biochemistry studies on non- structural protein 1 of influenza B virus (NS1B) reveal a previously unrecognized viral mechanism for innate immune evasion. Conserved basic groups of its C-terminal domain (NS1B-CTD) bind 5- triphosphorylated double-stranded RNA (5ppp-dsRNA), the primary pathogen-associated feature that activates the host retinoic acid-inducible gene I protein (RIG-I) to initiate interferon synthesis and the cellular antiviral response. Like RIG-I, NS1B-CTD preferentially binds blunt-end 5ppp-dsRNA. NS1B-CTD also competes with RIG-I for binding 5ppp-dsRNA, and thus suppresses activation of RIG-Is ATPase activity. Although the NS1B N-terminal domain also binds dsRNA, it utilizes a different binding mode and lacks 5ppp-dsRNA end preferences. In cells infected with wild-type influenza B virus, RIG-I activation is inhibited. In contrast, RIG-I activation and the resulting phosphorylation of transcription factor IRF-3 are not inhibited in cells infected with a mutant virus encoding NS1B with a R208A substitution it its CTD that eliminates its 5ppp-dsRNA binding activity. These results reveal a novel mechanism in which NS1B binds 5ppp-dsRNA to inhibit the RIG-I antiviral response during influenza B virus infection, and open the door to new avenues for antiviral drug discovery.

biochemistry↗