Biology subjects

Pitre, E.

Publications and source records attributed to Pitre, E..

4 recordsLinked to original sources

bioRxiv2025

A two-step mechanism for RIG-I activation by influenza virus mini viral RNAs

Influenza A virus (IAV) non-canonical replication products can be bound by host pathogen sensors, such as retinoic acid-inducible gene I (RIG-I). However, innate immune activation is infrequent in cell culture infection, in particular by adapted strains. Moreover, it is not understood why non-canonical IAV RNAs activate RIG-I in a sequence- or RNA structure-dependent manner. We therefore hypothesized that multiple errors need to occur before influenza virus RNA synthesis activates innate immune signaling. To test this idea, we investigated whether RIG-I activation is stimulated by the non-canonical or aberrant transcription of mini viral RNAs (mvRNA), a <125 nt long RNA that is overexpressed in pandemic and highly pathogenic IAV infections. Using mvRNA sequences identified in tissue culture and ferret infections, we find that mvRNAs can cause non-canonical transcription termination through a truncated 5' polyadenylation signal or a 5' transient RNA structure that interrupts polyadenylation. The resulting capped complementary RNAs (ccRNA) can stimulate the release of a template mvRNA in vitro. Finally, we find that both mvRNA and ccRNA sequences can be bound by RIG-I in cell culture and that blocking mvRNA transcription with baloxavir reduces IFN promoter activity. Overall, our findings indicate that sequential rounds of non-canonical or aberrant viral replication and transcription are needed before mvRNAs trigger innate immune signaling in a sequence-dependent manner.

microbiology↗

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

Cas13-based amplification-free quantification of aberrant RNAs during influenza virus infection

Influenza A virus RNA synthesis produces full-length and aberrant RNA molecules, which include defective viral genomes (DVG) and mini viral RNAs (mvRNA). Sequencing approaches have shown that aberrant RNA species may be present during infection, and that they can vary in size, segment origin, and sequence. Moreover, a subset of aberrant RNA molecules can bind and activate host pathogen receptor retinoic acid-inducible gene I (RIG-I), leading to innate immune signaling and the expression of type I and III interferons. Understanding the kinetics and distribution of these immunostimulatory aberrant RNA sequences is important for understanding their function in IAV infection. Here, we use an amplification-free LbuCas13a-based detection method to quantify mvRNA amplification dynamics and subcellular distributions. We show that our assay can quantify the copy numbers of specific mvRNA sequences in infected tissue culture cells, ferret upper and lower respiratory tract tissue infected with two different pandemic H1N1 IAV strains, or clinical nasopharyngeal swab extracts of hospitalized patients infected with seasonal H1N1 or H3N2 strains. In addition, we find dynamic differences between immunostimulatory and non-immunostimulatory mvRNAs, as well as among mvRNAs derived from different segments, during IAV infection. Overall, our results reveal a hitherto hidden diversity in the behavior of IAV mvRNAs and suggest that individual aberrant RNAs are not produced stochastically.

microbiology↗

bioRxiv2022

Transient RNA structures cause aberrant influenza virus replication and innate immune activation

During infection, the influenza A virus RNA polymerase produces both full-length and aberrant RNA molecules, such as defective viral genomes (DVG) and mini viral RNAs (mvRNA). Subsequent innate immune activation involves the binding of host pathogen receptor retinoic acid-inducible gene I (RIG-I) to viral RNAs. However, not all influenza A virus RNAs are strong RIG-I agonists. Here we show that potent innate immune activation by mvRNAs is determined by transient RNA structures, called template loops (t-loop) that stall the viral RNA polymerase. The effect of t-loops depends on the formation of an RNA duplex near the template entry and exit channels of the RNA polymerase, and their effect is enhanced by mutation of the template exit path from the RNA polymerase active site. Overall, these findings provide a mechanism that links aberrant viral replication to the activation of the innate immune response.

microbiology↗