The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

The Alongshan virus (ALSV), a newly identified tick-borne segmented flavivirus, can infect humans and cause Alongshan fever, making it imperative to understand its pathogenic mechanisms for the development of effective intervention strategies. Our previous research has established that ALSV exhibits sensitivity to interferon-beta (IFN-β) while having evolved the ability to antagonize downstream antiviral responses induced by type I IFN (IFN-I); however, the specific effects and underlying mechanisms by which ALSV modulates IFN-I production remain poorly understood. In the present study, we demonstrated that ALSV infection significantly suppresses host IFN-I production triggered by poly(I:C), a synthetic analog of viral double-stranded RNA that activates innate immune pathways. To unravel the molecular basis of this suppression, we systematically evaluated the impact of individual ALSV viral proteins on Toll-like receptor-mediated IFN-I production, revealing a complex regulatory network wherein distinct viral proteins target specific signaling molecules: specifically, VP2 and VP3 were found to be responsible for inhibiting RIG-I-mediated IFN-I production, while NSP2 and VP1b were identified as key inhibitors of MDA5-mediated IFN-I production, highlighting the virus's strategy of employing multiple proteins to disrupt innate immune signaling. Focusing further on the viral nucleoprotein VP2, we determined that it acts at the upstream signaling level of TANK-binding kinase 1, a critical kinase in the IFN-I signaling cascade. Mechanistically, VP2 directly interacts with RIG-I and mediates its degradation through an autophagy-dependent pathway, thereby impairing the host's ability to detect viral RNA and initiate IFN-I production. These findings not only expand our understanding of the immune evasion mechanisms employed by novel segmented flaviviruses but also offer valuable insights that could facilitate the development of new preventive and therapeutic strategies for the ALSV infection.IMPORTANCEAlongshan virus (ALSV) is an emerging segmented flavivirus that poses a growing threat to human and animal health across Eurasia. Despite its demonstrated capacity to infect humans and suppress interferon (IFN)-mediated antiviral responses, the precise mechanisms of ALSV immune evasion remain largely undefined. This study identifies the viral nucleoprotein VP2 as a key antagonist of host type I IFN (IFN-I) production. By directly interacting with and promoting the autophagy-mediated degradation of RIG-I, VP2 effectively disrupts innate immune recognition and signaling. This finding not only elucidates a previously unknown mechanism of immune suppression by ALSV but also highlights the virus's sophisticated strategy of using multiple proteins to selectively target RIG-I and MDA5 pathways. These insights advance our understanding of segmented flavivirus-host interactions and suggest that restoring RIG-I function may be a promising therapeutic strategy against ALSV infection.