Assessing activity of Hepatitis A virus 3C protease using a cyclized luciferase-based biosensor

Biochemical and Biophysical Research Communications
Junwei ZhouShaobo Xiao

Abstract

Hepatitis A is an acute infection caused by Hepatitis A virus (HAV), which is widely distributed throughout the world. The HAV 3C cysteine protease (3Cpro), 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 3Cpro cleaves human NF-κB essential modulator (NEMO), a kinase required in interferon signaling. Based on this finding, we generated four luciferase-based biosensors containing the NEMO sequence (PVLKAQ↓ADIYKA) that is cleaved by HAV 3Cpro and/or the Nostoc punctiforme DnaE intein, to monitor the activity of HAV 3Cpro in human embryonic kidney cells (HEK-293T). Western blotting showed that HAV 3Cpro recognized and cleaved the NEMO cleavage sequence incorporated in the four biosensors, whereas only one cyclized luciferase-based biosensor (233-DnaE-HAV, 233DH) showed a measurable and reliable increase in firefly luciferase activity, with very low background, in the presence of HAV 3Cpro. With this biosensor (233DH), we monitored HAV 3Cpro activity in HEK-293T cells, and tested it against a catalytically deficient mutant HAV 3Cpro and other virus-encoded proteases. The results sh...Continue Reading

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Citations

Feb 9, 2019·Current Protein & Peptide Science·Corina Sarmiento, Julio A Camarero
Nov 7, 2019·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Junwei ZhouShaobo Xiao
Oct 13, 2020·SLAS Discovery·Emery SmithTimothy P Spicer
Nov 11, 2017·ACS Chemical Biology·Aaron C MitchellJennifer R Cochran

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Methods Mentioned

BETA
biosensor
transfection
biosensors

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