Abstract
Our previous studies reported that duck Tembusu virus nonstructural protein 2A (NS2A) is a major inhibitor of the IFNβ signaling pathway through competitively binding to STING with TBK1, leading to a reduction in TBK1 phosphorylation. Duck TMUV NS2B3 could cleave and bind STING to subvert the IFNβ signaling pathway. Here, we found that overexpression of duck TMUV NS4B could compete with TBK1 in binding to STING, reducing TBK1 phosphorylation and inhibiting the IFNβ signaling pathway by using the Dual-Glo® Luciferase Assay System and the NanoBiT protein-protein interaction (PPI) assay. We further identified the E2, M3, G4, W5, K10 and D34 residues in NS4B that were important for its interaction with STING and its inhibition of IFNβ induction, which were subsequently introduced into a duck TMUV replicon and an infectious cDNA clone. We found that the NS4B M3A mutant enhanced RNA replication and exhibited significantly higher titer levels than WT at 48-72 hpi but significantly decreased mortality (80%) in duck embryos compared to WT (100%); the NS4B G4A and R36A mutants slightly reduced RNA replication but exhibited the same titer levels as WT. However, the NS4B R36A mutant did not attenuate the virulence in duck embryos, whereas the G4A mutant significantly decreased the mortality (70%) of duck embryos. In addition, the NS4B W5A mutant did not affect viral replication, whereas the D34A mutant slightly reduced RNA replication, and both mutants exhibited significantly lower titer levels than the WT and significantly decreased mortality (90% and 70%, respectively) in duck embryos. Hence, our findings provide new insight into the development of attenuated flaviviruses by targeting the disabling viral strategies used to evade the innate defense mechanisms.
Highlights
The innate immune response of the host is considered the first line of defense against viral infection
We further found that duck TMUV nonstructural protein 2A (NS2A) inhibited IFNb induction by targeting duck stimulator of interferon genes (STING) [13], and duck TMUV NS2B3 could cleave and bind duck STING to subvert the induction of IFNb [14], suggesting that different duck TMUV proteins could commonly target the same cellular components through different strategies to suppress IFNb production
We found that the expression of NS2A, NS2B, and 2KNS4B proteins significantly inhibited the duck TMUV-triggered activation of IFN-b and the ISRE-Luc promoter, suggesting that 2KNS4B may act as an antagonist of IFN induction and alter the phases of the IFN response [13]
Summary
The innate immune response of the host is considered the first line of defense against viral infection. The pattern recognition receptors (PRRs) of the host cell, such as the acid-inducible gene I (RIG-I)-like receptor (RLR) family, recognize the viral nucleic acids, leading to the activation of the transcription factors NF-kB and IFN regulatory factors 3 and 7 (IRF3/7). The activation and nuclear translocation of these transcription factors subsequently induce the production of type I IFNs, which initiate the expression of ISGs that prevent viral infection [1, 2]. The open reading frame (ORF) encodes a unique polyprotein precursor that is subsequently cleaved by cellular and viral proteases into three structural proteins (core, membrane, and envelope) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, 2KNS4B, and NS5) [6,7,8]
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