Abstract
Hepatitis E Virus (HEV) annually leads to 20 million infections and circa 55,000 deaths, while currently no specific treatment exists. The HEV capsid, helicase, and replicase - needed for viral entry and replication - have been proposed to be processed by the HEV encoded putative cysteine protease (PCP), thus making it an attractive therapeutic target. However, functional characterization of the PCP has been hindered by debate over i) whether the PCP cleaves the polyprotein it resides in (if there is processing at all), and ii) the inability to precisely determine the actual protease domain within the polyprotein sequence that has led to multiple constructs. Previous studies have reported that PCP binds Zn or possibly an Fe-S cluster, metallocofactors that are likely pivotal structural or functional elements, albeit to an unknown capacity. Our work focused on the chemical characterization of the metallocofactor in the PCP and its possible roles in structural stability and proteolytic activity. Additionally, we established that PCP binds an O2-sensitive [4Fe-4S] cluster, which can be therapeutically targeted for degradation by nitroxides such as TEMPOL and DEANONOate. While the exact cellular modus operandum of PCP remains elusive, we are the first group to holistically examine the PCP and its metallocofactor from a diverse set of biochemical approaches and set the stage for better understanding the mechanistic basis of its role in HEV replication.