Amelioration of Beta Interferon Inhibition by NS4B Contributes to Attenuating Tembusu Virus Virulence in Ducks

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.

INMI1 Zika Virus NS4B Antagonizes the Interferon Signaling by Suppressing STAT1 Phosphorylation

“…Viruses 2021, 13, 2448 2 of 16 The NS4B antagonism of the IFN system has critical implications in ZIKV immune pathogenesis and virulence [8]. It has been demonstrated that a single mutation in NS4B, associated with a decreased IFNβ production and diminished expression of the IFN stimulated genes (ISGs), causes increased viral replication and neurovirulence in mice, confirming the importance of NS4B as a determinant of pathogenesis [9].…”

“…NS4B is a well-known IFN inhibitor in different flaviviruses, mediating the evasion of both IFN production and signaling [7]. In fact, on one hand, yellow fever virus (YFV), hepatitis C virus (HCV), duck Tembusu virus (TMUV) and bovine viral diarrhea virus (BVDV) NS4B have been reported to block the RIG-I /MDA-5 pathway of IFN production [10][11][12][13][14]. On the other hand, dengue virus (DENV), West Nile virus (WNV) and YFV NS4B were shown to inhibit the IFN signaling pathway by suppressing the phosphorylation and nuclear transport of STAT1/STAT2 [15][16][17][18].…”

New Insights into the Biology of the Emerging Tembusu Virus

“…Moreover, specific mutations in the NS4B protein modifying interactions with TBK1 lead to phenotypic changes decreasing the pathogenicity of TMUV. Thus, NS4B appears to strongly interact with TBK1 and inhibit its recruitment by STING, resulting in a blockage of the signaling pathway [ 117 ]. These studies confirm the importance of TBK1 in the implementation of an efficient antiviral response, as reported by Hua et al [ 109 ], and strategies developed by TMUV to overcome the immune response.…”

The autophagy‐related degradation of MDA5 by Tembusu virus nonstructural 2B disrupts IFNβ production

“…Under the processing of viral and host cell proteases, three structural proteins, namely, capsid protein (C), membrane precursor protein (prM), and envelope protein (E), and 7 nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, 2KNS4B, and NS5), are produced, the 5′ end has a hat structure (m7Gppp Amp), and the 3′ end has no poly‐A tail. A previous study by our and another laboratory investigating TMUV showed that NS1, NS2A, NS2B3, and NS4B could interact differently with MAVS and STING to inhibit IFN production 4–7 . However, in previous studies, RIG‐I has been studied as the main recognition receptor for viral RNA when flaviviruses infect cells.…”

“…A previous study by our and another laboratory investigating TMUV showed that NS1, NS2A, NS2B3, and NS4B could interact differently with MAVS and STING to inhibit IFN production. [4][5][6][7] However, in previous studies, RIG-I has been studied as the main recognition receptor for viral RNA when flaviviruses infect cells. This study may provide ideas for MDA5's weaker recognition of viral RNA than RIG-I.…”