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Chronic hepatitis B virus (HBV) infection remains a global public health problem.HBV-encoded X protein (HBx) is a multifunctional regulator that is required to initiate and maintain productive HBV infection,and is involved in HBV-related hepatocellular carcinoma (HCC).Inhibitors that interfere with the functions of HBx could be useful not only for the inhibition of HBV replication but also for the prevention or treatment of HBV-related HCC.To screen molecules that target HBx on a large scale remains a technical challenge due to a lack of sensitive and high-throughput system.In this work,we established an in vitro bioluminescent reporter system for screening HBx-targeting molecules.The system is based on a secretory fusion protein that combines HBx and NanoLuc (HBx-Nluc).The measured activity of NanoLuc in the culture supeatant of HBx-Nluc-expressing cells directly reflects the level of secreted HBx-Nluc.HBx protein-targeting intracellular anti-HBx single-chain variable fragment and RNA-targeting shRNA significantly reduced the secreted NanoLuc activity in HBx-Nluc-expressing cells.This system is simple and sensitive,and compatible with continuous non-disruptive screening,suggesting its potential usefulness for high-throughput screening and evaluating HBx-targeting molecules.