Hepatitis A is an acute infection caused by Hepatitis A virus (HAV), which is widely distributed throughout the world. The HAV 3C cysteine protease (3C(pro)), an important nonstructural protein, is responsible for most cleavage within the viral polyprotein and is critical for the processes of viral replication. Our group has previously demonstrated that HAV 3C(pro) cleaves human NF-kappa B essential modulator (NEMO), a kinase required in interferon signaling. Based on this finding, we generated four luciferase-based biosensors containing the NEMO sequence (PVLKAQI,ADIYKA) that is cleaved by HAV 3C(pro) and/or the Nostoc punctiforme DnaE intein, to monitor the activity of HAV 3C(pro) in human embryonic kidney cells (HEK-293T). Western blotting showed that HAV 3C(pro) recognized and cleaved the NEMO cleavage sequence incorporated in the four biosensors, whereas only one cyclized luciferasebased biosensor (233-DnaE-HAV, 233DH) showed a measurable and reliable increase in firefly luciferase activity, with very low background, in the presence of HAV 3C(pro). With this biosensor (233DH), we monitored HAV 3C(pro) activity in HEK-293T cells, and tested it against a catalytically deficient mutant HAV 3C(pro) and other virus-encoded proteases. The results showed that the activity of this luciferase biosensor is specifically dependent on HAV 3C(pro). Collectively, our data demonstrate that the luciferase biosensor developed here might provide a rapid, sensitive, and efficient evaluation of HAV 3C(pro) activity, and should extend our better understanding of the biological relevance of HAV 3C(pro). (C) 2017 Elsevier Inc. All rights reserved.