Japanese encephalitis virus NS1 and NS4B synergistically target TLR3 signaling to promote viral replication.

To establish effective infection, viral pathogens employ diverse strategies through encoded proteins to interfere with host antiviral responses. While previous studies have predominantly focused on elucidating the mechanisms by which individual viral proteins regulate type I interferon (IFN) responses, this study presents the first demonstration that Japanese encephalitis virus (JEV)-encoded NS1 and NS4B proteins cooperatively target the TLR3 receptor signaling pathway to suppress IFN production. Here, we first discovered JEV-encoded multifunctional glycoprotein NS1, which inhibits TLR3-mediated IFN-β production by targeting TLR3 and TRIF. Mechanistically, NS1 interacts with these host factors and may induce their degradation via the autophagy pathway. Building on these findings, we further demonstrated that NS1 and NS4B act synergistically to suppress type I IFN production by enhancing TLR3 and TRIF degradation, thereby facilitating JEV replication. Structural simulation of the NS1-NS4B-TLR3 complex unveiled a dynamic mechanism that NS4B binding induces conformational changes in dimerized NS1, which leads to a tighter binding posture between NS1 and TLR3. Notably, NS4B binding expands the interface between NS1 and TLR3 by 1.5-fold that results in enhanced TLR3 degradation and downstream IFN-β suppression. Functional analysis confirmed that the C291/K293/R314 triple mutation in NS1 synergistically impairs TLR3 degradation. Collectively, using JEV as a model, this study reveals a novel mechanism by which two viral components can act synergistically to evade the host antiviral response. Given the common coexistence and functional interplay of viral-encoded proteins in naturally infected cells, this study establishes a framework for investigating cooperative interactions among multiple viral proteins. IMPORTANCE: Viruses evade host immunity through encoded viral proteins, while previous research has predominantly focused on single protein mechanisms. This study reveals a novel cooperative immune evasion strategy, demonstrating for the first time that Japanese encephalitis virus NS1 and NS4B proteins act synergistically to degrade the host's TLR3 and TRIF adaptor, thereby suppressing type I interferon production. Structural simulations show that NS4B induces conformational changes in NS1, enhancing its binding to TLR3 and accelerating its degradation. This work establishes a new paradigm for how multiple viral components can function cooperatively to subvert antiviral defenses. Given that viral proteins naturally coexist and interact, this study provides a crucial framework for investigating complex viral protein interplay, which is fundamental to understanding viral pathogenesis.