Circular RNAs (circRNAs) represent a class of covalently closed non-coding RNA molecules that exert vital regulatory effects on host-pathogen interplay. The H9N2 subtype of avian influenza virus (AIV) is a widespread pathogen on a global scale, inflicting considerable economic damage to the poultry sector and harboring potential risks of cross-species transmission to humans. Despite growing evidence suggesting that non-coding RNAs can modulate the replication of influenza viruses, the expression patterns and functions of avian-derived circRNAs during H9N2 AIV infection have been largely unclear. Here, we conducted a systematic investigation into the expression dynamics of circRNAs in DF1 cells infected with H9N2 AIV by high-throughput RNA sequencing technology. A total of 139 differentially expressed circRNAs were identified, with 58 exhibiting upregulation and 81 showing downregulation relative to non-infected control cells. Of note, a circRNA originating from exon 2 of the insulin receptor (INSR) gene displayed consistent upregulation during viral infection. Functional assays verified its contributing role in the replication of H9N2 AIV. Specifically, siRNA-mediated knockdown of circ-INSR significantly suppressed H9N2 AIV replication. This study is the first to identify circ-INSR as a host factor contributing to influenza virus replication. Our results provide a basis for developing circRNA-based strategies against H9N2 avian influenza virus.
ABSTRACT Influenza A viruses (IAVs) pose an ongoing threat to humans and other species because of their zoonotic potential. Accumulating evidence has demonstrated that certain long non-coding RNAs (lncRNAs) exhibit differential expression during viral infection and modulate diverse facets of viral pathogenesis. As key regulatory RNAs, lncRNAs participate in fundamental physiological processes and disease progression via a wide array of functional interactions with DNA, RNA, and proteins. Here, we identified ckATP1A1-AS1 as an antiviral host lncRNA that is induced by IAV infection. Functional analyses demonstrated that ckATP1A1-AS1 overexpression restricted infection by multiple IAV subtypes, whereas ckATP1A1-AS1 knockdown enhanced viral replication. Mechanistically, during IAV infection, the transcription factor JUN transcriptionally activates ckATP1A1-AS1, which further enhances the expression of interferon-β and key interferon-stimulated genes, thereby positively regulating type I interferon immune responses. Furthermore, ckATP1A1-AS1 interacts directly with the viral nucleoprotein, competitively disrupting its binding to importin α5, impairing its oligomerization, and blocking the nuclear import of viral ribonucleoprotein complexes. Consequently, ckATP1A1-AS1 suppresses viral ribonucleoprotein assembly and reduces viral polymerase activity. These findings establish ckATP1A1-AS1 as a key antiviral lncRNA that restricts IAV replication by coordinating innate immune signaling and directly targeting several steps in the viral replication cycle. IMPORTANCE Accumulating evidence indicates that host long non-coding RNAs (lncRNAs) play important roles in regulating virus–host interactions during influenza A virus (IAV) infection. However, the functions and mechanisms of action of most IAV-associated lncRNAs remain unclear. This study identifies the novel chicken antisense lncRNA ckATP1A1-AS1 as a key antiviral factor with a unique dual mechanism: it is transcriptionally activated by transcription factor JUN and, in turn, upregulates the expression of interferon-β and key interferon-stimulated genes to positively regulate the type I interferon immune response. It directly interacts with viral nucleoprotein, competitively disrupting the binding of nucleoprotein to importin α5 and impairing nucleoprotein oligomerization, thereby suppressing viral ribonucleoprotein assembly and reducing viral polymerase activity. Accumulating evidence indicates that host long non-coding RNAs (lncRNAs) play important roles in regulating virus–host interactions during influenza A virus (IAV) infection. However, the functions and mechanisms of action of most IAV-associated lncRNAs remain unclear. This study identifies the novel chicken antisense lncRNA ckATP1A1-AS1 as a key antiviral factor with a unique dual mechanism: it is transcriptionally activated by transcription factor JUN and, in turn, upregulates the expression of interferon-β and key interferon-stimulated genes to positively regulate the type I interferon immune response. It directly interacts with viral nucleoprotein, competitively disrupting the binding of nucleoprotein to importin α5 and impairing nucleoprotein oligomerization, thereby suppressing viral ribonucleoprotein assembly and reducing viral polymerase activity.
Menglu Fan, Zhiyuan Liu, Lu-Lu Deng et al.· Journal of Virology· 0 citations
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