An lncRNA containing tandem 66-nt repeats recruits PTBP1 to disrupt PB2-NP assembly and restrict influenza virus replication in vivo

Influenza virus replication relies on coordinated assembly of ribonucleoprotein (RNP) complexes in the nucleus, yet how host long noncoding RNAs (lncRNAs) regulate this process remains poorly defined. Here, we identify the lncRNA AL645608.7 as a potent antiviral factor and show that its two splice variants restrict influenza replication. Mechanistically, AL645608.7 contains eight tandem 66-nt repeats that recruit the RNA-binding protein PTBP1, primarily through PTBP1's RRM3 domain. The AL645608.7-PTBP1 assembly then engages key viral RNP components-PB2 and NP-and functionally disrupts the PB2-NP interface, thereby suppressing viral RNA synthesis. Domain-mapping analyses further distinguish the regions responsible for PTBP1 recruitment from those involved in PB2 and NP engagement, demonstrating that distinct structural elements of AL645608.7 coordinate host-protein recruitment and viral RNP interference. Importantly, lipid nanoparticle delivery of AL645608.7 markedly reduces viral burden and improves disease outcomes in mice, providing a proof-of-concept nucleic-acid therapeutic strategy. Together, our findings reveal a repeat-programmed lncRNA mechanism that rewires a host RNA-binding protein to dismantle influenza RNP organization and support the therapeutic potential of lncRNA-based intervention against influenza virus infection. IMPORTANCE: This study identified that influenza virus infection significantly upregulated the long noncoding RNA (lncRNA) AL645608.7. Both of its transcripts, AL645608.7-v1 and AL645608.7-v2, exhibited antiviral activity against influenza virus. Mechanistically, AL645608.7 was shown to directly bind to the host protein PTBP1. Furthermore, the nuclear-localized AL645608.7 recruits PTBP1 via a 66-base repeat motif. Importantly, PTBP1 interacts with both the N- and C-terminal regions of the influenza virus polymerase subunit PB2 as well as the NP, thereby disrupting the NP-PB2 interaction. The AL645608.7-PTBP1 complex amplifies this inhibitory effect, ultimately impairing vRNA synthesis and blocking efficient viral replication. These findings offer novel insights into the functional role of noncoding RNAs in host-influenza virus interactions.