The Processing of eIF4GI by Human Rhinovirus Type 2 2A pro : Relationship to Self-Cleavage and Role of Zinc

Abstract

ABSTRACT The 2A proteinase (2A pro ) of human rhinoviruses (HRVs) is a cysteine protease containing a structurally important zinc ion. In the viral polyprotein, the enzyme cleaves between the C terminus of VP1 and its own N terminus. 2A pro also processes the two isoforms of the cellular protein, eukaryotic initiation factor 4G (eIF4G). We have shown that mature HRV2 2A pro , when translated in vitro in rabbit reticulocyte lysates, efficiently cleaves eIF4GI, although the enzyme was not immediately active upon synthesis. Here, we examine the relationship between self-processing and eIF4GI cleavage. The onset of both reactions first occurred at least 10 min after initiation of protein synthesis. Furthermore, when self-processing was prevented by a specific mutation between VP1 and 2A pro , the VP1-2A pro precursor was essentially unable to cleave eIF4GI, implying that self-processing is a prerequisite for eIF4GI cleavage. 2A pro synthesized in the presence of a potent zinc chelator is inactive; however, upon addition of excess zinc, HRV2 2A pro rapidly gained activity. Finally, the presence of the zinc chelator in the culture medium can protect HeLa cells from HRV infection.