A ribonuclease zymogen activated by the NS3 protease of the hepatitis C virus

Translating proteases as inactive precursors, or zymogens, protects cells from the potentially lethal action of unregulated proteolytic activity. Here, we impose this strategy on bovine pancreatic ribonuclease (RNase A) by creating a zymogen in which quiescent ribonucleolytic activity is activated by the NS3 protease of the hepatitis C virus. Connecting the N-terminus and C-terminus of RNase A with a 14-residue linker was found to diminish its ribonucleolytic activity by both occluding an RNA substrate and dislocating active-site residues, which are devices used by natural zymogens. After cleavage of the linker by the NS3 protease, the ribonucleolytic activity of the RNase A zymogen increased 105-fold. Both before and after activation, the RNase A zymogen displayed high conformational stability and evasion of the endogenous ribonuclease inhibitor protein of the mammalian cytosol. Thus, the creation of ribonuclease zymogens provides a means to control ribonucleolytic activity and has the potential to provide a new class of antiviral chemotherapeutic agents.