Suppression of MazF toxicity by fusing a C‐terminal segment of MazE to MazF, and its activation by sequence specific HIV‐1 and HCV proteases

Abstract

The genomes of human immunodeficiency virus and hepatitis C virus consist of a single‐stranded RNA encoding polyproteins, which are processed to individual functional proteins by virus‐encoded specific proteases. Thus, these proteases are essential for viral infection and have been used as targets for drug development. Here, instead of inhibiting these proteases, we took an opposite approach by using the protease activity to prevent viral infection. We engineered the MazE‐MazF antitoxin‐toxin system of Escherichia coli to fuse a C‐terminal 41‐residue fragment of antitoxin MazE to the N‐terminal end of toxin MazF with a linker having a specific viral protease cleavage site for HIV PR (HIV‐1 protease) and NS3 protease (HCV protease) as well as the cleavage site for factor Xa. These fusion proteins formed a stable dimer (instead of the MazF2‐MazE2‐MazF2 heterohexamer in nature) to inactivate MazF mRNA interferase (endoribonuclease) activity. When the fusion protein was incubated with the corresponding proteases, the MazE fragment was cleaved off to release MazF. MazF thus released functioned as an ACA‐specific mRNA interferase to cleave single‐stranded MS2 phage RNA. The activation of MazF toxicity by proteases of single‐stranded RNA viruses such as HIV‐1 and HCV may provide a novel approach for preventive and therapeutic treatments of infection by HIV‐1, HCV and other RNA viruses.