Investigation of hepatitis E ORF1 processing and screening of protease inhibitors against viral and host proteases for therapeutic design

Abstract

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. Like many other single-stranded positive-sense RNA viruses, HEV encodes a non-structural polyprotein that consists of several domains. Proteolytic processing of such polyprotein gives rise to intermediate and mature distinct proteins. The HEV open reading frame 1 (ORF1) has been suggested to be processed by host and viral proteases. This processing is also believed to be essential for viral replication; however, the ORF1 processing mechanism is still unknown. We asked if the HEV ORF1 putative cysteine protease plays a role in this polyprotein processing using an in vitro coupled transcription-translation system. Following, the processing of [ 35S]methionine-labelled ORF1, we analysed the intensity of the subsequent processed products. Combining this approach with different protease inhibitors provided a useful tool to validate our findings and gain insight into ORF1 processing. Overall, we found that ORF1 protein is a direct substrate for the host serine protease, thrombin. We identified the protease inhibitors antipain and pefabloc as potential candidates to inhibit thrombin-mediated processing. Furthermore, utilising a luciferase replicon reporter system, we show an impact of antipain and pefabloc on viral replication, highlighting a potential role for these inhibitors to target HEV replication. Introduction