Flavivirus Attenuation via Targeting the Specific Disulfide Bond of NS1 Protein to Promote Its Degradation

Flaviviruses are an important group of human pathogens that have caused extensive outbreaks worldwide during the last decades. Although live-attenuated vaccines (LAVs) that induce strong and long-lasting cellular and humoral responses represent the most effective approach to combat viral diseases, achieving the ideal balance between vaccine efficacy and safety remains an ongoing challenge for LAVs development. Targeted protein degradation has recently emerged as an attractive strategy for the development of fine-balanced LAVs. However, it mainly depends on pre-fused proteolysis-targeting domain (PTD) in the targeted viral proteins, greatly limiting its wide application, especially in flaviviruses with low tolerance to foreign sequences. In flaviviruses, NS1 protein, a multifunctional nonstructural protein essential for viral replication and virulence, is featured with 6 intra-disulfide bonds formed by 12 highly conserved cysteine residues. Through a systematic Cys-to-Ala mutation screening, we revealed the crucial role of C-terminal disulfides in maintaining NS1 stability, and accomplished the switch of conventional exogenous PTD-dependent degradation to a more straightforward disulfide-targeting degradation machinery. Furthermore, using revertant genetics techniques and serial passaging in Vero cells, we developed a genetically stable West Nile virus (WNV) variant, WNV-C9A-Mut that is replication-competent in Vero cells but producing degradation-prone NS1 protein. In vitro and in vivo experiments indicate that WNV-C9A-Mut is highly attenuated, and a single-dose vaccination elicits robust immune responses, conferring complete protection against WT WNV challenge. Mechanistically, we demonstrated that the labile NS1-C9A undergoes cargo receptor p62-mediated autophagic degradation. Collectively, this study established a rational strategy for LAV design by disrupting a critical disulfide bond that maintains viral protein stability, providing a new perspective to advance TPD application in the vaccine field.