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