Influenza A virus circumvents the innate immune response through the sequestration of double-stranded RNA

Double-stranded RNA (dsRNA), which induces an innate immune response against viral infections, is rarely detected in influenza A virus (IAV)-infected cells. Nevertheless, we previously reported that the influenza A viral ribonucleoprotein (vRNP) complex generates looped dsRNAs during RNA synthesis in vitro. This finding suggests that IAV possesses a specific mechanism for sequestering dsRNA within infected cells, thereby enabling viral evasion of the innate immune response. Here, we found that dsRNAs were detected in infected cells lacking the expression of viral non-structural protein 1 (NS1) and nuclear export protein (NEP), both encoded by the same RNA segment. Indeed, the looped dsRNA-vRNP complexes were isolated from IAV-infected cells. Interestingly, NS1 molecules masked the entire looped dsRNA generated by vRNP in vitro, implying a potential role for NS1 in segregating viral dsRNA from cytoplasmic dsRNA sensors. Furthermore, dsRNAs were sequestered within the nucleus of wild-type IAV-infected cells, whereas their translocation to the cytoplasm was observed in NS1-deleted mutant virus-infected cells expressing M1 and NEP. This result indicates the possibility that dsRNA is transported to the cytoplasm in association with vRNP. Notably, the cytoplasmic translocation of dsRNA triggered the nuclear translocation of interferon regulatory factor 3, suggesting the capability of dsRNA in inducing the innate immune response. These findings highlight IAV's distinctive strategy for circumventing innate immunity by sequestration of dsRNAs.IMPORTANCEIt is widely recognized that double-stranded RNA (dsRNA) produced during viral infection triggers an innate immune response. However, the influenza A virus (IAV) has been thought to rarely produce dsRNA within infected cells. Here, we detected dsRNA in the nucleus of IAV-infected cells which lacked the expression of viral non-structural protein 1 (NS1) and nuclear export protein (NEP), both encoded by a single RNA segment. High-speed atomic force microscopy demonstrated that NS1 entirely concealed dsRNA produced by the viral ribonucleoprotein complexes, thereby segregating it from cytoplasmic dsRNA sensors that trigger the innate immune response. Interestingly, cytoplasmic translocation of dsRNA was observed in cells infected with an NS1-deleted mutant virus, where M1 and NEP were expressed, resulting in the nuclear translocation of interferon regulatory factor 3. Collectively, our findings suggest that IAV adeptly sequesters dsRNA to evade the innate immune system.