Long noncoding RNA#61 synergizes with viral PA-X to augment pyroptosis and attenuate the virulence of highly pathogenic H5N1 influenza virus in mice.

UNLABELLED: Long non-coding RNAs (LncRNAs) play pivotal regulatory roles in various biological processes, notably in immune regulation and viral infection. We previously identified the broad anti-influenza activity for LncRNA#61. Here, we further investigate the mechanism underlying its antiviral effect, both in vitro and in vivo. Using a lipid-nanoparticle-based delivery strategy, LncRNA#61 was successfully delivered into mice and effectively attenuated the replication and virulence of the highly pathogenic H5N1 influenza virus. Integrative transcriptomic analysis revealed that forced expression of LncRNA#61 markedly activated lipid metabolism, cell death, and Ragulator-Rag-mTORC1 pathways. Quantitative reverse transcription PCR analysis and a targeting metabolic assay further confirmed that LncRNA#61 is actively involved in regulating these pathways. Subsequent functional studies demonstrated that LncRNA#61 consistently enhances GSDMD-mediated pyroptosis both in murine LET-1 and canine MDCK cells. Notably, such pyroptosis was found to restrict H5N1 influenza virus replication. Intriguingly, ectopic expression of viral PA-X protein enhanced antiviral activity of LncRNA#61 both in vitro and in vivo. Mechanistically, PA-X interacts with LncRNA#61 and promotes LncRNA#61-mediated pyroptosis via a RagA-dependent reactive oxygen species pathway. Collectively, we here propose a novel model in which viral and host factors cooperate to activate a pro-death antiviral pathway. Our findings not only advance the fundamental knowledge of virus-host interactions but also cross-link cell death, innate immunity, and metabolic regulation, pinpointing novel therapeutic targets against influenza.

IMPORTANCE

A current priority in anti-influenza research is developing broad-spectrum, host-directed therapeutics with low resistance risk. Here, we reveal that LncRNA#61-induced pyroptosis exerts an antiviral effect by restricting H5N1 virus replication both in vitro and in vivo, highlighting a novel cooperative virus-host interaction that enhances antiviral immunity. Key contributions include the following: (i) identifying pyroptosis as a direct executioner mechanism that restricts H5N1 virus infection; (ii) revealing the unexpected role of forced expression of viral PA-X in augmenting antiviral activity of host LncRNA#61; and (iii) deciphering that LncRNA#61 interacts with PA-X and synergistically promotes GSDMD-mediated pyroptosis through a RagA‑ROS signaling cascade. Collectively, our work elucidates a novel antiviral mechanism wherein host LncRNA and viral protein co-opt the RagA-ROS-GSDMD axis to drive pyroptosis and inhibit viral replication. This discovery innovatively establishes a novel connection among viral pathogenesis, host cell death, and cellular metabolism, offering a fresh, integrative perspective for future studies on host-directed antiviral strategies.