SPCS2 serves as a critical host factor for JEV replication by regulating viral protein stability and virion assembly

Non-structural protein 2B (NS2B) of flaviviruses plays multiple versatile roles in viral replication through interacting with viral proteins and cellular factors. Identification of NS2B-interacting host factors is essential for a detailed understanding of the viral life cycle, as well as for the development of antiviral therapeutics. To identify the cellular factors that interact with NS2B and are important for JEV replication, JEV-infected cell lysates were co-immunoprecipitated and analyzed by mass spectrometry. Signal peptidase complex subunit 2 (SPCS2) was identified as a novel host factor that interacts with JEV NS2B. SPCS2 was also found to interact with JEV NS5. We revealed that SPCS2 is a host factor required for the replication of JEV by silencing and knocking out endogenous SPCS2. Knockout of SPCS2 markedly impaired intracellular virion assembly and production of infectious JEV particles. Furthermore, we found that SPCS2 depletion promoted the degradation of viral prM, E, and NS1 proteins, but not NS2B protein. Functional loss of SPCS2 does not influence viral attachment, cell entry, RNA replication, protein translation and processing, or even the formation of endoplasmic reticulum membrane-invaginated vesicles during the life cycle of JEV replication. Our findings suggest that SPCS2, a novel host factor that interacts with NS2B and NS5, plays a crucial role in maintaining viral protein stability and in the assembly of JEV virions.IMPORTANCEThe flavivirus non-structural protein, NS2B, participates in viral replication and assembly by interacting with viral proteins and host cellular factors. However, the currently known viral replication, which requires host factors that interact with NS2B, is still very limited. In this study, we identified SPCS2 as a novel NS2B-interacting host factor required for JEV replication using co-immunoprecipitation and mass spectrometry analyses. We revealed that SPCS2 plays essential roles in maintaining the stability of the viral proteins prM, E, and NS1, and in the assembly of infectious JEV particles. This study enriches our understanding of the molecular details underlying host factor involvement in the JEV replication life cycle.