Stormy Signatures: Investigating the effect of Leptomycin B on influenza A mini viral RNA trafficking and innate immune activation

Abstract

Influenza A virus (IAV) replicates its genome in the nucleus of a host cell, producing viral RNA (vRNA) transcripts that are packaged and released into neighboring cells. While these vRNA transcripts are typically full-length RNA molecules produced by normal IAV replication, oftentimes the replication machinery may also produce aberrant, shorter RNA fragments, including mini viral RNAs (mvRNAs). Some mvRNAs have a high propensity to act as innate immune agonists, triggering retinoic acid-inducible gene I (RIG-I) activation, downstream innate immune signaling, and cytokine storms, a conserved pathological profile of pandemic-causing IAV strains. While mvRNA-induced RIG-I activation is well-studied, it remains unclear how mvRNAs produced in the nucleus are transported to the cytoplasm where they exert their immunostimulatory effects. Here, we identify a potential trafficking route by treating IAV infected A549 cells with Leptomycin B (LMB), a Chromosomal Maintenance Region 1 (CRM1) inhibitor, and compare their mvRNA production levels and innate immune activation patterns with untreated, infected cells. Infected cells treated with LMB had a steeper distribution skew of mvRNA across the nucleus and cytoplasm, with strong nuclear sequestration of mvRNAs. Although high technical variability limited the statistical power of this observation, the trend seen may point to the role of the CRM1 pathway in mediating mvRNA nuclear export during IAV infection. Luciferase based assays of IFN-β promoter expression revealed a significant fold reduction in IFN-β activity in LMB-treated cells, confirming a potential role of the CRM1 pathway in increasing the accessibility of the cytoplasm, and its innate immune receptors, i.e. RIG-I, to mvRNAs. RPISeq, HDOCK analysis, and ChimeraX predicted CRM1 as a potential direct binding partner for mvRNA, suggesting that mvRNA and its full-length vRNA counterpart are trafficked via mechanistically distinct, yet CRM1-mediated nuclear export routes. Put together, this research provides preliminary insight into IAV mvRNA transport, and paves the way for the development of both, therapeutic agents that optimize the balance between CRM1-pathway inhibition and cytotoxicity, as well as wide-ranging, anti-inflammatory molecules that lower the cytokine storm disease pathology across several viral contexts.