Engineering of a Complex of the DNase Domain of Colicin E9 with the Immunity Protein Im9 Activated by the Protease pS273R of African Swine Fever Virus

African swine fever virus (ASFV) is a large DNA virus that causes a highly lethal disease in pigs and currently has no effective vaccines or antiviral treatments available. We designed a protein switch that combines the DNase domain of colicin E9 (DNase E9) and its inhibitor Im9 with the viral protease cleavage site. The complex is only destroyed in the presence of an ASFV pS273R protease, which releases DNase activity. Several Im9 variants were constructed by inserting the pS273R protease cleavage sequence into different exposed loops. From these, we identified an optimized variant (Im9–1.4) that remains highly stable and tightly bound to DNase E9, suppressing its activity in the absence of protease. Exposure to the ASFV protease results in cleavage of Im9–1.4, rendering it unable to inhibit DNase E9 activity. In vitro assays confirmed that the DNase E9/Im9–1.4 complex becomes catalytically active upon proteolytic digestion with pS273R protease. This virus-triggered ‘kill switch’ is designed to render pig cells nonpermissive to ASFV by aborting infection via viral DNA degradation. Our study offers a generalizable synthetic biology strategy that uses virus-encoded proteases to trigger dormant effectors, exemplified by this protease-sensing DNase. This synthetic restriction system might be used to develop ASFV-resistant pigs.