Research on NS1 Proteins of Group 2 Tembusu Virus Inhibiting the Production of Duck Type Ⅰ IFN

【Objective】Tembusu virus (TMUV) disease is an important infectious disease in waterfowl. In previous studies, we found that in the early stages of TMUV infection in ducks, the viral copy number of group 2 TMUV in organs such as the liver, kidney, and brain were significantly higher than those of group 3 TMUV. The study aimed to explore whether group 2 TMUV NS proteins had different effects on the innate immune response of ducks compared with group 3 TMUV.【Method】Taking the group 2 TMUV-JM strain and the group 3 TMUV-GX strain as research subjects, we constructed the eukaryotic expression plasmids of NS proteins of the two strains and compared the effects of the NS proteins on the RIG-Ⅰ-induced activity of the IFN-β (Type Ⅰ) promoter by a dual-Luciferase reporter system. With the eukaryotic expression plasmids of the constructed recombinant chimeric NS1 proteins, we investigated the interaction region between NS1 and the key molecules of RIG-Ⅰ signaling pathway by confocal laser scanning microscopy, co-IP and Western Blotting. By using the reverse genetic system, we constructed NS1 recombinant chimeric TMUV to clarify the inhibitory effect of NS1 on the IFN-β in DEF cells with TMUV infection.【Result】The study results showed that TMUV-JM NS1 could inhibit RIG-Ⅰ-induced activation of the IFN-β promoter, while TMUV-GX NS1 did not exhibit the inhibitory activity. Further studies revealed that the TMUV-JM NS1 inhibited the expression of type Ⅰ IFN via targeting TBK1, and the NS1 255-352 aa was the functional region of the inhibitory effect. It was found that TMUV-JM NS1 did not interact with TBK1 directly, but reduced the phosphorylation level of TBK1 indirectly, thereby suppressed the expression of type Ⅰ IFN.【Conclusion】Based on the study results, we found that the group 2 TMUV NS1 has activity in inhibiting the RIG-Ⅰ signaling pathway, identified the key activity region and targeted molecule of its inhibitory pathway, and clarified that group 2 TMUV NS1 affected duck type Ⅰ IFN production by indirectly inhibiting the phosphorylation of TBK1.