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 IAV ribonucleoprotein complex (vRNP) 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 produced 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. Interestingly, NS1 molecules masked the entire looped dsRNA generated by vRNP, implying a potential role for NS1 in segregating viral dsRNA from cytoplasmic dsRNA sensors, including retinoic acid inducible gene-I (RIG-I). Furthermore, dsRNAs were sequestered within the nucleus of infected cells due to the absence of NEP, while their translocation to the cytoplasm occurred only upon NEP expression. Notably, the cytoplasmic translocation of dsRNA triggered the innate immune response in an RIG-I-dependent manner. These findings highlight IAV’s distinctive strategy for circumventing innate immunity by sequestration of dsRNAs. Author summary It 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 confirmed the limited dsRNA production in the nucleus of IAV-infected cells and found that the cells lacked expressions of viral non-structural protein 1 (NS1) and nuclear export protein (NEP), both derived from 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, NEP expression caused cytoplasmic translocation of dsRNA, resulting in the nuclear translocation of interferon regulatory factor 3, which initiates an innate immune response. Collectively, our findings suggest that IAV employs a sophisticated strategy to circumvent the innate immune system, wherein the expressions of NS1 and NEP exert considerable influence.